Vitamin B12 alleviates spliceosomopathy via phospholipid remodeling

Vitamin B12 alleviates spliceosomopathy via phospholipid remodeling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Vitamin B 12 alleviates spliceosomopathy via phospholipid remodeling Adam Antebi, Wenming Huang, Jonathan Kölschbach, Anna Loehrke, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8077579/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Spliceosomal dysfunction profoundly impacts cellular metabolism, yet mechanistic links between RNA splicing defects and metabolic rewiring remain limited. Here, we investigate Verheij syndrome (VRJS), a rare disease caused by mutations in the core splicing factor PUF60 . Using a Caenorhabditis elegans model, human cell lines, and patient-derived samples, we demonstrate that RNP-6/PUF60 deficiency disrupts splicing of genes governing one-carbon metabolism and phospholipid remodeling, culminating in impaired S-adenosylmethionine (SAM)/S-adenosylhomocysteine (SAH) cycling and phosphatidylcholine synthesis. These perturbations trigger the integrated stress response and compromise mTORC1 signaling, causing developmental and growth defects. Vitamin B12 (VB12) supplementation restores metabolic balance by reactivating SAM-dependent phospholipid remodelling and mTORC1 activity, effectively rescuing VRJS-like phenotypes. Similar metabolic responses arise from perturbations in other spliceosomal factors such as PRPF19/PRP-19, indicating a conserved mechanism across spliceosomopathies. Interestingly, we identify intron retention of the nhr-114/HNF4 transcription factor as a primary driver of growth defects, and restoring its splicing robustly suppresses these phenotypes. Our findings establish a mechanistic connection between RNA splicing and lipid metabolism, implicating VB12-dependent one-carbon metabolism as a metabolic modulator with broad implications for spliceosome-related diseases, and suggesting VB12 as a potential strategy to mitigate VRJS-related anomalies. Biological sciences/Genetics/RNA splicing Biological sciences/Cell biology/Mechanisms of disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Main Splicing is a fundamental regulatory process in eukaryotic messenger RNA (mRNA) maturation that removes intronic regions from precursor transcripts to produce mature mRNAs encoding proteins of enhanced diversity. This essential activity is catalyzed by the spliceosome, a highly dynamic megadalton complex composed of five small nuclear ribonucleoprotein particles (snRNPs) and numerous associated splicing factors 1 . Dysregulation of splicing impairs proper cellular function and is associated with various pathological conditions, including cancer 2 and ageing 3 . Pathogenic mutations in genes encoding core spliceosomal components disrupt splicing, leading to various genetic diseases with overlapping phenotypes 4 . PUF60 (also known as FIR or Hfp) encodes an essential splicing factor containing two central RNA recognition motifs and a C-terminal U2AF-homology motif 5 . It operates early in spliceosome assembly by promoting the recruitment of precursor transcripts to the U2 snRNP complex and facilitating the splicing of weak 3’ acceptor sites 5 , 6 . Heterozygous de novo variants causing PUF60 deficiency result in Verheij syndrome (OMIM #615583) 7 , 8 , a congenital disorder associated with growth retardation, short stature, and recurrent infections 8 – 14 . We have previously reported on a phenotypic spectrum of PUF60 -related disorders ranging from neurodevelopmental delay to multisystem involvement 15 . However, the exact pathomechanisms of VRJS remain elusive, and no targeted therapies currently exist. Vitamin B 12 (also known as cobalamin) is a complex water-soluble organic compound essential for several biological functions in animals 16 . It serves as a cofactor for two metabolically important enzymes: methionine synthase (MS), which converts homocysteine to methionine within the methionine cycle, and methylmalonyl-CoA mutase, which converts L-methylmalonyl-CoA to succinyl-CoA in propionate metabolism. These two pathways are linked through homocysteine as a shared intermediate. Consequently, VB12 deficiency profoundly disrupts metabolic homeostasis and instigates various disorders in humans, including growth delay, hypotonia, anemia and cognitive impairment 16 . In Caenorhabditis elegans, rnp-6 (RNA-binding domain-containing protein 6) encodes the sole ortholog of human PUF60 17 . Depletion of rnp-6 by RNAi-mediated knockdown causes severe developmental defects, including growth retardation, smaller body size, dysregulated immune function, and neuronal abnormalities reminiscent of VRJS in humans 18 – 21 . Previously, we identified a viable hypomorphic allele, rnp-6(G281D) , that enhances abiotic stress resistance and extends lifespan on the standard B-type Escherichia coli OP50 diet 22 , 23 . In this study, we unexpectedly found that K12-type E. coli can fully rescue growth defects observed in rnp-6(G281D) mutants. Through unbiased genetic screens in E. coli and C. elegans , we discovered that E. coli K12 exerts its benefits through VB12-dependent methionine metabolism. VB12 restores rnp-6 mutant growth by driving synthesis of methionine (Met), S-adenosylmethionine (SAM) and phosphatidylcholine (PC), while inhibition of enzymes required for the Met/SAM cycle abrogates such rescue. Metabolomic and lipidomic analyses reveal that rnp-6 mutants maintained on OP50 harbor deficiencies in methylation capacity and phosphatidylcholine lipids, which are replenished by provisioning K12-type E. coli or VB12. Mechanistically, we deduce that aberrant splicing of nhr-114 ( homolog of human HNF4 ), is a major proximal cause of metabolic dysregulation and developmental defects in the rnp-6 mutant. We further demonstrate that altered Met/SAM/PC metabolism in rnp-6(G281D) modulates the integrated stress response (ISR) and mTOR signaling. Finally, we provide evidence that PUF60 deficiency induces alternative splicing of genes related to methionine and phospholipid metabolism, and PUF60 pathogenic mutations induce metabolic and lipidomic changes in VRJS patient plasma. Notably, this metabolic dysregulation is not limited to PUF60/RNP-6: depletion of PRPF19/prp-19 phenocopies rnp-6 loss, suggesting conserved metabolic vulnerability across spliceosomopathies. Together, our findings identify dysregulated S-adenosylmethionine/S-adenosylhomocysteine balance and phospholipid metabolism as central contributors of rnp-6 -dependent spliceosomal pathology, and highlight VB12 supplementation as a potential strategy to restore metabolic homeostasis. Results A K12-type E. coli diet alleviates growth defects in a C. elegans Verheij syndrome model Previously, we identified a hypomorphic mutation in rnp-6 that extends lifespan in C. elegans 22 . Further characterization showed that this mutant exhibits moderate but significant developmental defects, including smaller body size and slower growth rate when raised on the standard B-type E. coli OP50 diet (O) at 20°C (Fig. 1 a–c). These phenotypes are analogous to the growth delay and small stature seen in VRJS patients 7 . In the course of our studies, we discovered that rnp-6(G281D) developmental phenotypes were remarkably restored by feeding worms with the E. coli K12 strains HT115 (H) or BW25113 (B) (Fig. 1 a–c). These K12-type E. coli strains also suppressed other previously observed rnp-6(G281D) traits, including cold tolerance (Figure S1 a) and extended lifespan (Fig. 1 d). Of note, dietary mixtures of O + B containing as little as ~ 10% BW25113 sufficed to rescue growth defects (Figure S1 b). Rescue did not require active bacterial metabolism, as UV-killed BW25113 also effectively restored body size (Fig. S1 c). These results imply that BW25113-derived nutrients or metabolites compensate for RNP-6(G281D) dysfunction. To explore whether K12-type E. coli restored RNP-6 protein 20 directly or acted downstream, we measured steady-state RNP-6 levels. However, neither HT115 nor BW25113 diets restored RNP-6 protein levels (Figure S1 d–e), indicating that the rescue occurs via pathways downstream of RNP-6. To further elucidate the impact of K12-type E. coli , we performed RNA sequencing (RNA-seq) on wild-type and rnp-6(G281D) worms raised on either OP50 or BW25113 (Figure S1 f). Consistent with prior work, rnp-6(G281D) mutants grown on OP50 exhibited profound transcriptomic changes, with 1,541 genes and 473 genes showing differential expression (adjusted p-value 0.5; Table S1 ) and differential splicing (adjusted p-value 5%; Table S2, Figure S1 g and i), respectively. About 11% of differentially spliced genes also showed altered expression (Table S3, Figure S1 j), indicating distinct layers of regulation by rnp-6 . Feeding with BW25113 significantly changed the expression of 1319 genes in rnp-6 mutants and restored 573 genes toward wild-type levels (Fig. 1 e, Figure S1 h, Table S4). WormCat 2.0 24 gene set analysis of the BW25113-restored genes revealed significant enrichment in stress response, proteolysis/proteasome, and transmembrane transport pathways (Fig. 1 f). In contrast, BW25113 had minimal effect on splicing profiles, with no significant restoration observed (Table S2, Figure S1 k). Validation of two established rnp-6- dependent splicing targets, tcer-1 and tos-1 , confirmed this observation (Figure S1 l–m). Together, these findings suggest that K12-type E. coli alleviates rnp-6(G281D) phenotypes through mechanisms downstream of, or independent from, splicing activity. Analysis of our RNA-seq data revealed that the expression of Y41C4A.32 , an ortholog of human COPI coat complex subunit beta 2 ( COPB2 ), was among the strongest upregulated by rnp-6(G281D) mutation, and conversely suppressed by E. coli K12 (Fig. 1 e). This gene, previously annotated as Y41C4A.11 , has been commonly used to assess innate immune and endoplasmic reticulum (ER) stress responses 25 – 28 . rnp-6(G281D) did not cause significant splicing changes in Y41C4A.32 mRNA (Table S2). Quantitative RT-PCR confirmed a ~ 40-fold increase in Y41C4A.32 transcript levels on OP50 diet, which was almost completely reversed by BW25113 (Fig. 1 g). We thus purposed Y41C4A.32 as a robust marker for dissecting bacteria-host interactions. To this end, we generated an endogenous C-terminal mNeonGreen fusion via CRISPR/Cas9 genome editing. Consistent with our RNA-seq and RT-qPCR results, Y41C4A.32::mNeonGreen expression increased by ~ 10-fold in rnp-6(G281D) mutants compared to wild-type controls on OP50, but was substantially suppressed by BW25113 (Fig. 1 h). Expression of transgenic wild-type rnp-6 fully normalized Y41C4A.32 levels in rnp-6(G281D) mutants, confirming Y41C4A.32 expression as a faithful readout of activity downstream of RNP-6 (Figure S1 n–o). Complementary genetic screens identify vitamin B 12 as a key alleviator of rnp-6(G281D) phenotypes Utilizing the Y41C4A.32 reporter strain, we performed a two-way genetic screen to identify K12-type E. coli -derived nutrients/metabolites and their corresponding host effectors (Fig. 2 a). We took advantage of the Keio E. coli mutant library, which encompasses two independent single-gene knockout mutants for 3,985 nonessential genes in the BW25113 background 29 . We posited that deletion of genes crucial for the rescue metabolite production or transport would specifically de-repress (i.e., reactivate) Y41C4A.32 reporter expression in rnp-6 mutants maintained on E. coli K12 diet. After two rounds of screening a total of 7970 Keio strains, we confirmed nine bacterial mutants that enhanced Y41C4A.32 expression relative to the parental strain (Fig. 2 b). These candidate genes are involved in various biological processes, including cytoskeleton formation ( yfgA ), membrane transport ( tonB , btuF and znuB ), and purine metabolism ( purA ) (Table S5). Notably, both BtuF and TonB are core components of the E. coli cobalamin uptake system 30 (Fig. 2 c), suggesting that VB12 or related metabolites mediate the rescue effect. Subsequent phenotypic analysis revealed that deletion of btuF or tonB in BW25113 also mimics OP50 E. coli effects, resulting in smaller body size of rnp-6 mutants (Figure S2a–b). In contrast, deletion of other putative VB12 transporters ( btuB , btuC and btuD ) had negligible effects (Figure S2a–b), indicating btuF and tonB act as rate-limiting components in VB12-associated rescue. E. coli strains cannot synthesize VB12 and largely rely on environmental scavenging 31 . Previous studies have shown that OP50 accumulates less VB12 than E. coli K12 32,33 , providing a plausible explanation for the strain-specific rescue of rnp-6(G281D) . Accordingly, we hypothesized that VB12 supplementation of OP50 would mimic the beneficial effects of a K12-type E. coli diet. Indeed, adding 1 nM VB12 throughout development to rnp-6(G281D) mutants fed on OP50 suppressed Y41C4A.32 reporter expression and restored body size to levels comparable with BW25113-fed worms (Fig. 2 e–f, Figure S2c–d). VB12 rescued rnp-6(G281D) phenotypes even when cultured on UV-killed OP50, demonstrating a direct effect on the worm, independent of bacterial metabolism (Figure S2e–f). VB12 also alleviated other rnp-6(G281D) phenotypes, including slower growth rate, cold tolerance, and reduced fecundity (Figure S2g–i), confirming its role as the active metabolite mediating rescue. Surprisingly, VB12 supplementation did not reverse the rnp-6(G281D) longevity phenotype (Fig. 2 g), indicating that E. coli K12-derived and VB12-mediated effects on lifespan are separable. To determine the window of action, we supplemented worms with VB12 at different developmental stages. We found that VB12 treatment before the L4 larval stage fully restored normal development, matching the efficacy of continuous treatment (Figure S2j–k). Even a brief 24-hour treatment from the young adult stage onwards significantly increased body size and suppressed reporter expression (Figure S2j–k), underscoring VB12’s potent effect on rnp-6(G281D) development. Given that rnp-6(G281D) is a specific hypomorphic allele, we investigated whether VB12’s benefits extend to broader forms of RNP-6 insufficiency that mimic the partial loss-of-function features of PUF60 pathogenic variants in Verheij syndrome. To this end, we engineered an inducible RNP-6 loss-of-function strain using the auxin-inducible degradation (AID) system (Figure S2l) 34 . Consistent with previous data showing that rnp-6 null mutants are embryonic lethal and complete knockdown in early life triggers larval arrest 35 – 37 , increasing auxin (K-NAA) doses yielded graded phenotypes and recapitulated VRJS-like defects in our system: 0.5 µM K-NAA reduced RNP-6 levels and body size (Figure S2m–n), whereas doses at 4 µM or above caused larval arrest (Figure S2n–o). Of note, partial depletion (0.5 µM K-NAA) activated the Y41C4A.32 reporter and impaired growth (Fig. 2 h). Under these graded conditions of RNP-6 deficiency, VB12 supplementation restored body size and suppressed reporter induction (Fig. 2 h–j). These results support the notion that both rnp-6(G281D) and the AID-mediated partial depletion model pathogenic PUF60 haploinsufficiency, pointing to VB12 as a potential metabolic intervention. Vitamin B 12 rescues rnp-6(G281D) mutant defects through methionine metabolism Meanwhile, we performed EMS mutagenesis screens to identify host factors mediating rescue by K12-type E. coli (Figure S3a). As with the bacterial screen, we reasoned that mutations disrupting key host genes would de-repress Y41C4A.32 expression in rnp-6(G281D) mutants fed BW25113. After screening ~ 20,000 haploid genomes, we isolated 84 mutants that suppressed the rescue efficacy of BW25113 on reporter expression (Fig. 3 a). Through Hawaiian SNP mapping and whole-genome sequencing 38 , we identified causative genes for 20 mutants. Excitingly, three of these genes, pmp-5 , mtrr-1 , and mthf-1 , are directly involved in the VB12-dependent methionine synthesis cycle (Fig. 3 b–c). pmp-5 encodes the ortholog of human ABCD4 vitamin B 12 transporter 39 , mtrr-1 encodes the MTRR methionine synthase reductase 40 , and mthf-1 encodes the MTHFR methylene-tetrahydrofolate reductase 40 . All three genes have been implicated in the “vitamin B 12 -mechanism-II” transcriptional response that senses and compensates for perturbed Met/SAM cycle activity 41 . Hence, convergent E. coli and C. elegans genetic screens strongly implicate VB12-dependent methionine metabolism as a critical modulator of rnp-6(G281D) phenotypes. To further validate this, we directly tested whether VB12-dependent enzymes were required for the rescue effect of BW25113 diet and VB12. Vitamin B 12 serves as a known cofactor for two enzymes: methionine synthase ( metr-1 ), which catalyzes the conversion of homocysteine to methionine, and L-methylmalonyl-CoA mutase ( mmcm-1 ), which converts L-methylmalonyl-CoA to succinyl-CoA 40 . We found that the deletion of metr-1 , but not mmcm-1 , completely abolished the benefits of BW25113 and VB12 on rnp-6(G281D) mutants (Fig. 3 d–e). As expected, supplementation with the metr-1 downstream product, methionine, was sufficient to bypass the requirement for metr-1 (Fig. 3 f–g). Altogether, these findings suggest that rnp-6 mutants grown on OP50 are deficient in methionine metabolism and/or downstream pathways, which can be restored by stimulating VB12-dependent methionine production. However, additional mutation of methionine biosynthesis pathway components reinstates methionine deficiency, which is rescuable by (exogenous) methionine itself. We next asked how pathways downstream of methionine production interact with rnp-6 mutant phenotypes. An important product of the methionine cycle is SAM, the principal methyl donor produced by SAM synthetase ( sams-1 ) 42 . We found that deletion of sams-1 fully abolished the rescue by BW25113, VB12 and methionine in rnp-6(G281D) mutants (Fig. 3 h–i), indicating a crucial role for SAM or SAM-dependent metabolites. Among other functions, SAM is essential for the de novo synthesis of PC 43,44 , a key component of cellular membranes 45 . PC can also be synthesized in a SAM-independent manner via the Kennedy pathway, which utilizes dietary choline 46 (Fig. 3 b). Consistent with this framework, choline supplementation reduced Y41C4A.32 expression, rescued body size defects, and importantly, bypassed the requirement for sams-1 in rnp-6(G281D) mutants (Fig. 3 j–k). These findings support a model in which SAM-dependent phosphatidylcholine synthesis constitutes a major downstream mechanism by which K12-type E. coli and VB12 rescue rnp-6 mutant phenotypes. rnp-6(G281D) mutation disrupts methylation potential and phosphatidylcholine metabolism Our genetic data suggest that rnp-6(G281D) perturbs the methionine cycle and/or PC metabolism when grown on OP50. To examine this directly, we performed metabolomic and lipidomic analyses on rnp-6(G281D) and wild-type worms grown on OP50, OP50 + VB12, and BW25113 (Figure S4a). Mass spectrometry (MS)-based metabolomics identified 115 metabolites spanning amino acid, nucleotide, glucose, and polyamine metabolism (Table S6). Using MetaboAnalyst 47 , we observed that rnp-6(G281D) grown on OP50 significantly altered the abundance of 56 metabolites (FDR 0.5) (Fig. 4 a-b). These metabolites were enriched in TCA cycle (succinate, citrate, malate), pyrimidine metabolism (CTP, UTP, CDP), purine metabolism (AMP, IMP, GMP), as well as the one-carbon pool by folate (SAM and SAH) (Figure S4b). Notably, the levels of SAM, SAH, and the SAM/SAH ratio were significantly decreased in rnp-6(G281D) , while levels of methionine, homocysteine or Met/Hcy ratio remained unchanged (Fig. 4 c). This pattern suggests that rnp-6(G281D) may impact cellular methylation potential 48 (i.e., SAM/SAH ratio) rather than steady-state concentrations of these metabolites. Both BW25113 E. coli and VB12 supplementation significantly increased methylation potential (Fig. 4 d), confirming the central role of SAM-mediated methylation deficiency in rnp-6 mutant phenotypes. Interestingly, several other metabolites were also rescued by BW25113 and VB12 (Fig. 4 b, S4c, Table S6). These included intermediates in TCA cycle and gluconeogenesis (malate, phosphoenolpyruvate) (Figure S4d–e), amino and nucleotide sugar metabolites (UDP-N-acetyl-alpha-D-glucosamine, UDP-N-acetyl-D-galactosamine) (Figure S4f–g), tryptophan metabolism components (kynurenine, tryptophan) (Figure S4h–i), and mitochondrial β-oxidation-related acylcarnitines (propionylcarnitine, butyrylcarnitine) (Figure S4j–k). Further, the oxidized-to-reduced glutathione ratio, a marker for cellular oxidative stress, was significantly elevated in rnp-6 mutants and suppressed by BW25113 and VB12 (Figure S4l-m), indicating perturbed redox balance. MS-based lipidomics on the same sample set detected 18 lipid classes encompassing 735 species, of which the most abundant classes were phosphatidylethanolamine (PE), phosphatidylcholine (PC), and triacylglycerol (TG) (Table S7). The rnp-6 mutation broadly altered lipid composition (Fig. 4 e), significantly changing the abundance of 339 lipids across 14 classes (FDR 0.5) (Fig. 4 f-g). Notably, we found that PC were dramatically decreased, while PE were increased (Fig. 4 g), resulting in a significant elevation of the PE/PC ratio (Figure S5b). This pattern is consistent with impaired SAM-dependent PC synthesis in rnp-6(G281D) mutants. A detailed examination with individual lipids showed that TGs exhibited a biphasic response: of the 100 significantly altered TG lipids, 42% were downregulated, whereas 58% were upregulated (Fig. 4 f). More interestingly, the upregulated TGs contained longer and more unsaturated fatty acid chains (58 carbons, 8 double bonds on average) than downregulated TGs (50 carbons, 2 double bonds on average) (Fig. 4 h). Given the link between membrane phospholipid unsaturation and cold adaptation 49 , the observed lipid changes likely contribute to the enhanced cold tolerance observed in rnp-6 mutants. BW25113 feeding and VB12 supplementation exerted similar effects on the lipidome (Fig. 4 e), restoring the levels of 269 and 277 lipids, respectively. 252 lipids were shared between the two treatments (Fig. 4 f, S5a). The relative abundance of 12 lipid classes and the PE/PC ratio were significantly rescued (Fig. 4 g, Figure S5b–c). These findings demonstrate that disrupted lipid metabolism is a major driver of rnp-6 mutant defects and suggest that K12-type E. coli and VB12 rescue rnp-6 defects mainly through lipid remodeling. Supporting these metabolomic findings, transcriptomic analysis revealed dysregulation of 78 lipid-metabolism genes in rnp-6(G281D) mutants on OP50 (Figure S5d), including peroxisomal acyl-CoA oxidases ( acox-1.2, 1.3, 1.5, 3 ), fatty acid CoA synthetases ( acs-1, 2, 7, 9 ), desaturases ( fat-2, 5, 6, 7 ), short-chain dehydrogenases ( dhs-2, 4, 19, 23, 26, 31 ), lipid-binding proteins ( lbp-5, 6, 7 ), and glycerophospholipid remodeling enzymes ( ckc-1, ckb-2, eppl-1, acl-12 ). Feeding BW25113 significantly restored the expression of more than half (41/78) of these genes, including fat-2, 5, 6, 7 ; acox-1.2, 1.3, 1.5 ; acs-1,2 ; lbp-5,6 ; and ckc-1, ckb-2, acl-12 (Figure S5d). Previous studies have shown that amino acid starvation (e.g., methionine restriction), or lipid dysregulation (e.g., PE/PC imbalance), can trigger the ISR and ER stress-related pathways 25 , 50 – 53 . Reflecting this, we detected robust phosphorylation of eukaryotic initiation factor 2α (eIF2α), an established marker of ISR activation 54 , in rnp-6(G281D) mutants grown on OP50 (Figure S5e–f). Consistently, the bZIP transcription factor GCN4/ATF-4, a central ISR effector 55 , 56 , was markedly elevated in rnp-6(G281D) on OP50 and effectively suppressed upon VB12 supplementation (Figure S5g–h), indicating restoration of ISR activity. In contrast, expression of hsp-4 , a canonical ER-stress marker, and cpl-1* , an ER-associated degradation substrate 57 , 58 , was not induced by rnp-6 mutation (Figure S4i–l). Collectively, these findings unveil a novel link from splicing dysfunction to ISR activation, rather than canonical ER stress, in rnp-6 mutants. rnp-6(G281D) mutation causes aberrant splicing of methionine metabolism-related genes Building on our findings of SAM and PC metabolic disruption, we sought to understand the proximal molecular mechanisms. Since RNP-6 primarily regulates pre-mRNA splicing, we focused on the aberrant splicing landscape mentioned above (Figure S1 j, Table S2). Among 638 significant splicing alterations, intron retention (IR) and cassette exon skipping (SE) predominated (Fig. 5 a). Nearly all IR events (315 out of 330 events) increased, whereas most SE events (217 out of 227 events) decreased (Fig. 5 b), confirming widespread disruption of canonical splicing in rnp-6(G281D) . Gene set enrichment analysis revealed an overrepresentation of metabolic pathway genes (Fig. 5 c), notably those involved in lipid ( eppl-1, haao-1, cka-1, pcyt-2.1 ) and one-carbon metabolism ( metr-1, cbl-1 ) (Figure S6a). We also detected an enrichment of transcription factors, including atfs-1 , nhr-68 , and nhr-114 , which are involved in methionine and lipid metabolism 41 , 59 , 60 . Together, these findings implicate aberrant splicing of metabolism-related genes as a driver of rnp-6 mutant defects. metabolism 41 , 59 , 60 . To pinpoint critical splicing targets, we filtered for metabolism-related genes and transcription factors with robust splicing changes (adjusted p-value 0.1). This yielded 64 events, primarily involving increased intron retention or exon skipping (49/64; Table S8). Given that intron retention or exon skipping typically impairs gene function 61 , 62 , we conducted a targeted RNAi screen of 41 genes representing these events. In particular, we searched for candidates that phenocopied rnp-6(G281D) , that is, decreased body size and/or enhanced Y41C4A.32 reporter expression in the wild-type background. In total we identified nine RNAi clones that decreased body size (> 5%) and twelve that increased Y41C4A.32 reporter expression (> 10%) (Fig. 5 d–e; Table S9). Of those candidates, six affected both phenotypes, including nhr-114 , cbl-1 , and eppl-1 (Fig. 5 d-e). Interestingly, these genes converge on the same metabolic network: nhr-114 encodes an ortholog of hepatocyte nuclear factor 4 (HNF4), a key transcriptional factor regulating methionine metabolism and lipid homeostasis in C. elegans 41 , 60 , 63 ; cbl-1 encodes a cystathionine-beta-lyase, which converts cystathionine to homocysteine, feeding into the methionine cycle; eppl-1 encodes an ortholog of ETNPPL ethanolamine-phosphate-phospho-lyase, which affects phosphoethanolamine metabolism. Among these candidates, nhr-114 showed the strongest effect on both phenotypes (Fig. 5 d–e, S6b–d). Deletion of nhr-114 causes polyunsaturated fatty acid accumulation and PC depletion, both of which are reversible by VB12 or choline supplementation 60 , 64 . RT-PCR analyses confirmed that the rnp-6(G281D) mutation increased retention of nhr-114 intron 4 by approximately 20% (Fig. 5 f–g). Intron 4 harbors a relatively weak 3’ splice site, and its inclusion introduces a premature stop codon (Fig. 5 k), likely triggering nonsense-mediated mRNA decay and reducing protein expression. Consistently, both nhr-114 mRNA and protein levels were significantly diminished in rnp-6(G281D) mutants compared to wild-type controls (Fig. 5 h-j). Of note, a gain-of-function mutation with rbm-39 22 , a splicing factor known to interact with rnp-6 , markedly suppressed intron 4 retention in rnp-6(G281D) mutants (Figure S6d), supporting the notion that nhr-114 intron 4 is a direct splicing target of the RNP-6/RBM-39 complex. To further elucidate the functional relationship between nhr-114 and rnp-6 , we reanalyzed publicly available RNA-seq data of the nhr-114 null mutant 60 and compared it with the rnp-6(G281D) transcriptome. As reported 60 , 63 , nhr-114 deletion caused widespread transcriptional changes, altering the expression of 4,235 protein-coding genes (DEseq2, adjusted p-value 0.5) (Table S10). 483 of these genes were also significantly altered in rnp-6(G281D) (overlap significance, p < 5e-12) (Figure S6e–f; Table S10). In particular, genes related to lipid metabolism were significantly enriched (Figure S6g), including peroxisomal acyl-CoA oxidases ( acox-1.2, 1.3 ), fatty acid desaturases ( fat-2, 5, 6, 7 ), lipid binding proteins ( lbp-5, 6, 7 ), and phospholipid-remodeling genes ( ckb-2, acl-12 ) (Table S10). Using a less stringent cutoff (adjusted p-value 0.1), we also observed overlap of one-carbon cycle genes ( ahcy-1, dao-3 ) (Table S10). Less than 4% (18/483) of the overlapped differentially expressed genes displayed aberrant splicing in rnp-6 mutants (overlap significance, p < 0.102) (Table S10), supporting separable splicing and transcriptional contributions. Strikingly, ~ 60% (288/483) of the shared genes were rescued by VB12 supplementation in the nhr-114 mutant and by BW25113 feeding in the rnp-6 mutant (Figure S6h, Table S10), suggesting common downstream pathways. These findings strongly implicate intron retention-induced nhr-114 loss-of-function as a key mediator of rnp-6(G281D) transcriptomic changes and suggest that splicing and transcriptional programs converge on the Met/SAM/PC axis to cause the observed phenotypes. To directly test this hypothesis, we generated two nhr-114 alleles manipulating intron 4 retention: nhr-114(i4+) , with three thymine-to-adenine substitutions at the 3’ splice site to enhance intron retention, and nhr-114(i4–) , with complete intron 4 deletion (Fig. 5 k). RT-PCR verified the intended splicing alterations (Figure S6i-j). As predicted, nhr-114(i4+) phenocopied rnp-6(G281D) , reducing body size, robustly activating the Y41C4A.32 reporter, and increasing cold tolerance in the wild-type background, while not further elevating Y41C4A.32 expression in rnp-6(G281D) mutants (Fig. 5 m–o). Conversely, the nhr-114(i4–) allele alone produced no overt phenotype in wild-type animals (Fig. 5 m–o), but significantly suppressed multiple rnp-6(G281D) phenotypes, including Y41C4A.32 reporter expression, cold tolerance, and body size defects (Fig. 5 m–o). Taken together, these results provide compelling evidence that intron 4 retention of nhr-114 is a major driver of rnp-6 mutant pathology. Next, we asked whether the rnp-6 mutation-associated metabolic changes hold true for other splicing factors. Pre-mRNA processing factor 19 ( PRPF19/prp-19 ) encodes a key component of the spliceosome and is an essential splicing factor whose mutations are associated with developmental delay and neurological defects in humans 65 . To determine whether prp-19 deficiency disrupts metabolism, we utilized public RNA-seq datasets 66 to perform transcriptional and splicing analysis. prp-19 disruption altered the expression of 2776 genes (adjusted p-value 1) (Figure S7a, Table S11). Similar to the rnp-6 mutant, nhr-114 and Y41C4A.32 mRNA levels were significantly downregulated and upregulated, respectively (Figure S7a). Gene set enrichment analysis identified metabolism as one of the most enriched categories, with the majority of metabolic genes downregulated, including those involved in one-carbon (1CC) (e.g. cbl-1, ahcy-1, mmcm-1 ) and phospholipid metabolism (e.g. ckb-4 and pcyt-2.1 ) (Figure S7b-c). 1184 splicing events were significantly altered (adjusted p-value 5%) (Figure S7d, Table S12). In line with the DEGs, the metabolism category was enriched among the DSGs, including genes related to 1CC metabolism (e.g. cblc-1 , metr-1 and sams-1 ) and phospholipid metabolism (e.g. ckc-1 and pcyt-2.1 ) (Figure S7e-f). Of note, prp-19 depletion also significantly increased nhr-114 intron retention (Figure S7f), suggesting that aberrant nhr-114 splicing may represent a shared mechanism underlying distinct spliceosomeopathies. RNAi-mediated knockdown of prp-19 significantly induced Y41C4A.32::mNG reporter expression and reduced body size, whereas boosting 1CC metabolism through methionine supplementation rescued both phenotypes (Figure S7g-i). These findings together suggest that splicing factor inhibition may converge on shared downstream pathways, triggering similar metabolic responses. Dysregulated metabolism may thus represent a common feature of spliceosome-related diseases. Lastly, we explored the physiological regulation of nhr-114 splicing. Dietary restriction (DR) decreases SAM levels in flies and mice 67 – 69 , and DR-induced longevity is mediated by sams-1 in C. elegans 70 , 71 . Further, nutrient-sensing pathways have been shown to regulate splicing and metabolism 72 . We hypothesized that nutrient availability modulates SAM metabolism partially through nhr-114 splicing. We therefore investigated C. elegans adult reproductive diapause (ARD), a state induced by long-term fasting and associated with downregulation of RNA processing complexes 73 , 74 . Under ARD conditions nhr-114 intron 4 retention was significantly increased by ~ 10% relative to fed controls (Figure S7k), accompanied by reduced levels of nhr-114 mRNA (Figure S7l). These findings suggest that nutrient status modulates nhr-114 splicing and the associated SAM/phospholipid metabolic network. Vitamin B 12 activates mTOR signaling to rescue rnp-6(G281D) mutant defects mTOR signaling orchestrates key processes of development, growth, and metabolism 75 . Previously, we showed that rnp-6(G281D) mutation inhibits mTORC1 activity when worms are fed OP50 22 . Here, we asked whether VB12 supplementation could restore mTORC1 function in these mutants. To this end, we (i) measured phosphorylation of AMPK, a cellular energy sensor inversely related to mTORC1 activity, and (ii) monitored nuclear localization of HLH-30, the C. elegans ortholog of TFEB, whose nuclear translocation is suppressed by active mTORC1 signaling 76 – 78 . Consistent with mTORC1 inhibition, rnp-6(G281D) mutants on OP50 exhibited elevated AMPK phosphorylation (Fig. 6 a–b) and increased HLH-30 nuclear localization (Fig. 6 c–d). Remarkably, VB12 supplementation reversed both phenotypes, indicating a reactivation of mTORC1 signaling. Next, we wondered whether mTORC1 activity is required for rescue of rnp-6(G281D) growth defects by VB12/Met/SAM/PC. Loss-of-function mutation of raga-1 , a Rag GTPase essential for mTORC1 activation, further reduced the body size of rnp-6 mutants and completely abolished the beneficial effects of K12-type E. coli , as well as VB12, methionine, and choline supplementation on body size in rnp-6(G281D) mutants (Fig. 6 e). A raga-1 single-copy insertion 78 rescued body size in the raga-1(lof);rnp-6 double mutant on OP50 and fully reinstated the rescue of body size upon VB12, methionine or choline supplementation (Fig. 6 e). Altogether, these findings demonstrate that intact mTORC1 signaling is critical for mediating the rescue effect of VB12 and related metabolic interventions in rnp-6(G281D) mutants. PUF60 deficiency impairs 1CC and phospholipid metabolism in human cells Since RNP-6 deficiency induces aberrant splicing of 1CC and phospholipid metabolism genes in C. elegans , we wondered whether PUF60 deficiency has a similar effect in human cells. We mined public datasets in which the human lung adenocarcinoma cell line PC9 was subjected to PUF60 siRNA 79 . Based on our findings in worms, we reasoned that splicing defects would remain unchanged even when cells were cultured in the presence of VB12 and methionine-rich medium. We re-analyzed the data and identified 2599 splicing events (adjusted p-value 0.1) corresponding to 1730 genes (Figure S8a, Table S13). KEGG analysis of the aberrant spliced genes revealed the significant enrichment of metabolic pathways (Fig. 7 a, Table S14). These include genes related to propanoate metabolism ( e.g. , ethylmalonyl-CoA decarboxylase 1 ECHDC1/C32E8.9 , methylmalonyl-CoA epimerase MCEE/mce-1 ), choline metabolism ( e.g. , choline kinase alpha CHKA/cka-2 ), glycerophospholipid metabolism (diacylglycerol kinase DGKQ/dgk-1 , phosphocholine cytidylyltransferase PCYT1A/pcyt-1 , phosphatidate phosphatase LPIN2/lpin-1 ), cysteine and methionine metabolism (methionine synthase MTR/metr-1 , kynurenine aminotransferase 1 KYAT1/nkat-1 ) and VB12 transport ( ABCD4/pmp-5 ) (Fig. 7 b). These results together imply that PUF60 deficiency might dysregulate 1CC and phospholipid metabolism through altered splicing. Last, we sought to understand if the PUF60 pathogenic mutation also affects metabolism in VRJS patients. To this end, we collected blood samples from 8 Verheij syndrome patients (6 males and 2 females) and 3 age-matched non- PUF60 mutation controls (1 male and 2 females) and performed metabolomic and lipidomic analyses with plasma (Figure S8b). In total 103 metabolites and 653 lipid species belonging to 15 lipid groups were annotated (Table S15-16). Although variable, the VRJS samples clustered differently from controls in PCA plots (Figure S8c). Out of the 103 metabolites, 30 were significantly altered (p 0.2) (Fig. 7 c). Strikingly, tryptophan and methionine were among the most significantly changed metabolites, decreased by ~ 90% and ~ 85% in VRJS, respectively (Figure S8d-e). A handful of metabolites involved in TCA cycle, such as citrate, malate and fumarate, were upregulated in VRJS patients (Figure S8f-h). We were unable to quantify SAM or SAH as they were not detected in our plasma samples, due to low abundance. Levels of TG and LPE were significantly decreased, while the abundance of PC-O, hexose ceramide, ceramide and sphingomyelin lipid were significantly increased (P < 0.05) (Fig. 7 c). Although the levels of PC remained unchanged, the levels of PE and the ratio of PC/PE were slightly increased (Fig. 7 d). When we checked individual lipids, 94 were significantly changed (p 0.2) (Fig. 7 e). Consistent with the group analysis, most of the significantly altered PE and LPE were downregulated, while PC-O, sphingomyelin, ceramide and hexose ceramide were upregulated (Fig. 7 e). Interestingly, we found that, similarly to C. elegans , the significantly upregulated TG lipids contained longer and more unsaturated fatty acid chains (59 carbons, 11 double bonds on average) than downregulated TGs (48 carbons, 2.5 double bonds on average) (Fig. 7 f). Taken together, our results from human cell lines and patient plasma provide evidence that PUF60 deficiency might dysregulate sulfur amino acid and phospholipid metabolism. Discussion Verheij syndrome remains an exceptionally rare and poorly understood genetic disorder with no targeted treatments to date. Albeit relatively simple, C. elegans rnp-6 hypomorphic mutants recapitulate multiple features of VRJS such as smaller body size, growth delay, immune alterations, and neurological defects 7 , 9 , 10 , 12 , 14 , 15 , 80 , highlighting the nematode as a powerful system for dissecting the molecular and cellular mechanisms of PUF60 insufficiency-associated pathologies. Leveraging the C. elegans rnp-6 mutant as an in vivo disease model, we found that VRJS-associated growth defects arise primarily from SAM/SAH and PE/PC imbalance. The metabolic impairments are cumulatively driven by aberrant splicing of one-carbon and phospholipid metabolism-related genes (such as metr-1 , cbl-1 , eppl-1 ) and nhr-114 , a pivotal transcription factor of one-carbon and phospholipid metabolism. Reconstitution of SAM/SAH and PE/PC balance through supplementation of VB12, methionine, choline, or VB12-enriched bacteria can robustly rescue development (Fig. 7 g). These interventions mechanistically converge on reactivating mTORC1 signaling, a key driver of development, establishing a direct link between splicing factor dysfunction, metabolic rewiring, and nutrient-sensing signaling networks. To our knowledge, this is the first study to integrate the aforementioned metabolic dysregulation with spliceosomopathies, and to identify vitamin B 12 as a candidate therapeutic for VRJS-like pathologies. rnp-6 mutation disrupts expression and splicing of diverse metabolism-related genes (Table S1 and S2). By combining targeted RNAi screening, genetic epistasis analysis, and transcriptomic profiling, we identify aberrant intron retention of nhr-114 as a critical node linking RNP-6 perturbation to downstream transcriptional and metabolic imbalances, thereby driving defects in rnp-6 mutants. As nhr-114 / HNF4 homologs govern pivotal aspects of methionine and lipid homeostasis across species 41 , 60 , 63 , 81 , 82 , our findings offer a mechanistic bridge connecting spliceosomal dysfunction with metabolic dysregulation. Moreover, the novel observation that nhr-114 splicing is dynamically regulated by both genetic perturbation and physiological states (such as fasting-induced diapause) further broadens the connection between nutrient-responsive control of RNA processing and cellular metabolism. VB12 is an essential micronutrient critical for human health, particularly in preventing anemia and neurological dysfunction 16 . Clinically, VB12 supplements address malabsorption disorders such as pernicious anemia and gastrointestinal disorders 83 . Beyond these classic roles, emerging studies demonstrate that VB12 enhances somatic cell reprogramming and tissue repair in mammalian models 84 . In C. elegans , VB12 deficiency induces severe phenotypes, including cognitive impairment, growth retardation, infertility, and shortened lifespan 85 , 86 . Supplementation of VB12 mitigates amyloid-beta and dithiothreitol-induced toxicity in C. elegans 87 , 88 , highlighting its essential role in metabolic homeostasis. Our work extends these insights by demonstrating that VB12 supplementation rescues RNP-6 deficiency-associated spliceosomopathy, confirming the critical function of VB12 under both physiological and disease conditions. Beyond the central role of SAM/SAH balance and phospholipid metabolism, our data also implicate additional metabolic pathways and organelles as contributors to VRJS-like defect manifestations. For example, metabolites related to the tricarboxylic acid (TCA) cycle (e.g., malate, phosphoenolpyruvate) and mitochondrial β-oxidation (propionylcarnitine and butyrylcarnitine) are reduced in rnp-6 mutants, implying mitochondrial dysfunction. Supporting this, the mitochondrial stress reporter hsp-6 is strongly induced in rnp-6(G281D) mutants and fully suppressed by the K12-type E. coli strain BW25113 (Figure S9a–b). This is also consistent with previous reports that link VB12 supplementation to improved mitochondrial function 89 , 90 . Moreover, crucial metabolites in the de novo NAD + synthesis pathway, such as kynurenine and tryptophan, are reduced in rnp-6 mutants and restored by BW25113 E. coli or VB12, hinting at dysregulated NAD + metabolism in rnp-6(G281D) pathology. Additionally, metabolites associated with the glucosamine pathway appear dysregulated. A unifying notion is that rnp-6 mutation induces a catabolic state of lower methylation capacity, mitochondrial function, and glucose metabolism, while VB12 reverses these features and re-establishes a more anabolic state, consistent with the activation of mTOR signaling. Of particular interest is the induction of the integrated stress response in rnp-6 mutants. The ISR is a conserved pathway for eukaryotic cells that limits protein synthesis and secretion to cope with diverse stresses 54 . The impact of the splicing machinery on ISR has been little explored, though a recent investigation shows that aberrant splicing of protein translation genes induces the ISR and inflammation-related gene expression in acute myeloid leukemia patients 91 . Our results provide direct evidence that splicing factor dysfunction triggers the ISR in C. elegans , in this case through phospholipid remodeling. Further research should help elucidate the detailed mechanisms. Our study demonstrates that VB12 supplementation activates mTORC1 signaling to rescue growth and metabolic defects in the rnp-6 mutant, revealing a novel mechanistic link between micronutrient status and spliceosomal dysfunction. Previous work showed that methylcobalamin, a VB12 analog, activates mTOR via Akt to promote neuronal outgrowth, supporting VB12’s capacity to stimulate mTOR in a cellular context 92 . In contrast, recent studies report VB12 inhibits mTOR phosphorylation to induce autophagy in disease models 93 . Our data uniquely position VB12-induced mTORC1 activation as an anabolic rescue mechanism critical for correcting spliceosomal metabolic defects. Although mTOR activation typically limits longevity in C. elegans , the lack of lifespan limitation by VB12 may reflect tissue-specific effects of mTORC1, which restricts lifespan in neurons but promotes growth in somatic tissues 23 – 25 . We speculate that VB12 preferentially activates mTORC1 in non-neuronal tissues to support development without affecting longevity. Dissecting the tissue-specific roles of RNP-6/NHR-114 signaling, SAM/SAH–PC/PE metabolism, and mTOR within this metabolic network will be important to elucidate. While K12-type E. coli (BW25113 and HT115) and VB12 exert largely overlapping effects in rnp-6(G281D) mutants, the bacterial diet may also regulate physiology through VB12-independent mechanisms. For instance, K12-type E. coli suppresses rnp-6(G281D) -associated longevity (Fig. 1 d), whereas VB12 treatment shows little effect (Fig. 2 g), suggesting bacterial metabolites or lipids beyond VB12 contribute to lifespan regulation 94 . Together, our findings highlight the pleiotropic and context-dependent modulation of mTOR by VB12 and advance current paradigms by identifying mTORC1 as a promising therapeutic target in spliceosomal diseases. Our analysis of splicing changes in the PC9 cell line suggests that RNP-6/PUF60 might play conserved roles in regulating methionine and phospholipid metabolism, as we observed enrichment of aberrantly spliced genes in choline, propanoate, and methionine/cysteine metabolism. Though we did not find (significant) splicing changes in HNF4 (functional homolog of nhr-114) , we did see splicing changes in NR1H3 (Table S13) a related nuclear receptor that also regulates lipid metabolism 95 , suggesting that RNP-6 and PUF60 may affect similar and different splicing targets, dependent on cell type. It is also interesting to note that mTOR signaling pathway is significantly enriched in the PUF60-dependent splicing genes, including NPRL2, NPRL3, RPS6KB2, RPS6KA3 and TSC1 (Table S14). The aberrant splicing with mTOR complex core components might contribute to mTORC1 inhibition under PUF60 deficiency conditions 22 . By performing metabolic and lipidomic assays in VRJS plasma, we provide evidence that the PUF60 pathogenic mutation might dysregulate methionine/cysteine and phospholipid metabolism. Of note, some metabolites (such as cysteine, tryptophan, PEP, pantothenate, adenine and long chain TGs), were changed in the same direction in rnp-6 mutants and VRJS patients, while other metabolites (such as citrate, malate and fumarate) and lipids (such as PE, hexose ceramide, sphingomyelin lipid and PC/PE ratio) were changed in the opposite direction. Despite these discrepancies, these findings suggest that similar pathways are dysregulated, while the directionality could reflect different metabolic/lipidomic profiles of plasma versus tissues 96 , 97 . Moreover, as the plasma samples were obtained from subjects of different ages and genders, this may also partially contribute to the observed changes. It will be important to validate these results in plasma and patient-derived cells (such as fibroblasts) with a larger cohort (age and gender matched) and test the effect of VB12 supplementation. Splicing is a highly coordinated process that involves hundreds of splicing factors 1 . RNP-6/PUF60 functionally and physically interacts with many other splicing factors acting at various stages of spliceosome assembly 98 . Mutations in several of these splicing factors, including PRP-19/PRPF19 65 , UAF-1/U2AF2 65,99 , PRP-8/PRPF8 100 , RSP-7/SRSF11 101 and RBM-5/RBM10 102 , have been linked to VRJS-like neurodevelopmental defects in humans, suggesting convergent pathomechanisms. Our data on prp-19 and other splicing factors (Figure S7) indicate that dysregulation of the SAM/SAH balance and phospholipid metabolism is not restricted to PUF60/RNP-6-associated VRJS. The metabolic imbalance identified here may represent a shared downstream pathway regulated by distinct spliceosomal components. It will be of great interest to comprehensively test this hypothesis across human spliceosomopathies and evaluate the therapeutic potential of VB12 supplementation. Furthermore, given the neurological manifestations in VRJS and other spliceosomopathies 21 , 22 , assessing VB12’s effects on neuronal functions constitutes a critical direction for future research. In summary, our results illuminate a previously unappreciated metabolic bottleneck underlying RNP-6/PUF60 deficiency and warrant the need for further investigation of vitamin B 12 supplementation as a therapeutic strategy for Verheij syndrome. Methods C. elegans strains and maintenance Worms were maintained at 20°C following standard procedures 103 . Detailed information for strains used in this study are found in Supplementary Table S17. All mutant strains obtained from CGC and Sunybiotech ( www.sunybiotech.com ) were outcrossed with our N2 at least twice. Detailed information regarding CRISPR gene editing can be provided upon request. For all experiments, worm synchronization was done by egg laying. Compound supplementation assay For supplementation of methionine (L-methionine, Sigma-Aldrich: M9625) and choline (choline chloride, Thermo Fisher: A15828.22), methionine/choline was added to NGM medium before pouring to a final concentration of 10 mM. OP50 was seeded on these plates two days before worms lay eggs. For vitamin B 12 supplementation, VB12 (Cyanocobalamin, Sigma-Aldrich: V6629) was mixed with OP50 bacteria and seeded on NGM plates. Worms were cultured on these plates from the egg stage onwards, unless noted otherwise. For the OP50-BW25113 mixture assay, OP50 and BW25113 were cultured to log phase, washed with M9 and concentrated to the same OD600 value. Then OP50 and BW25113 was mixed at different ratios and seeded on peptone-free NGM plates to avoid bacterial growth. Worm images were taken at the young adult stage. For the tunicamycin response assay, tunicamycin (Sigma-Aldrich: T7765) stock solution was added on top of seeded NGM plates (final concentration 2 ug/ml) with synchronized young adult stage worms. After overnight treatment (~ 16 h), worms were anesthetized with sodium azide (50 mM) and imaged. Worm imaging Analysis of worm reporters Y41C4A.32::mNeonGreen and worm size were performed on a Leica stereo microscope (Leica M165 FC, LAS X) with Leica DFC3000G CCD camera. Analysis of mTOR reporter HLH-30::mNeonGreen was performed on a Zeiss Axioplan2 microscope (Axio Vision SE64, Rel.4.9.1) with a Zeiss AxioCam 506 CCD camera. Fiji software (Version 2.0.0/1.52p) 104 was used for quantifying fluorescent intensity and worm area. For HLH-30::mNeonGreen images, the nuclei of hypodermal cells were selected for quantification. To reduce bias, worms were randomly picked under a dissection microscope and imaged. At least 20 worms per genotype were picked for imaging and all the experiments were independently carried out at least three times unless otherwise indicated. Developmental rate assay Worms were synchronized by short-term (1h) egg lay on the indicated plates. After 48 hours of growth, ~ 15 worms from each condition were randomly singled to 3 cm NGM plates. After 12 hours, each worm was checked every hour until it laid the first egg. Experiments were repeated at least three times. Brood size assay Worms were synchronized by short term (1h) egg lay on the indicated plates. After 48 hours of growth, ~ 15 L4-stage worms of each condition were singled to 3 cm NGM plates. Worms were transferred every day until egg laying stopped. The total number of hatched larval worms was counted. Experiments were repeated at least three times. Cold tolerance assay Worms were synchronized and grown on the indicated plates. When the worms reached the young adult stage, they were transferred to a 2°C incubator for 24 hours. Worms were recovered at room temperature for 4 hours and the number of alive and dead worms was scored. Cold survival ratio was measured as the ratio of the number of live worms to the number of total worms. At least 60 worms from each genotype were used in the assay for each replicate. Experiments were repeated at least three times. Lifespan assay All lifespans were performed at 20°C with the indicated diet or treatment condition for the whole life. Worms were allowed to grow to the young adult stage on standard NGM plates. ~150 young adults were transferred to NGM plates supplemented with 10 µM of FUdR. Survival was monitored every other day. Worms that did not respond to gentle touch by a worm pick were scored as dead and were removed from the plates. Animals that crawled off the plate or had ruptured vulva phenotypes were censored. All lifespan experiments were blinded and performed at least three times. Graphpad Prism (9.0.0) was used to plot survival curves. Survival curves were compared, and p-values were calculated using the log-rank (Mantel-Cox) analysis method. Complete lifespan data are found in Supplementary Table S18. RNA interference RNAi experiments were performed as previously described 23 . E. coli HT115 (high VB12) and E. coli OP50(xu363) (low VB12) bacterial strains were used in this study. HT115 bacteria came from the Vidal or Ahringer library. OP50(xu363) competent bacteria were transformed with dsRNA expression plasmids, which were extracted from the respective HT115 bacterial strains. The RNAi bacteria were grown in LB medium supplemented with 100 µg/mL ampicillin at 37 ̊C to reach log phase, washed with fresh medium, and concentrated 5-fold. Bacteria was then spread on RNAi plates, which are NGM plates containing 100 µg/mL ampicillin and 0.4 mM isopropyl ß-D-1-thiogalactopyranoside (IPTG). dsRNA-expressing bacteria were grown on plates at room temperature for two days. RNAi was initiated by letting the animals feed on the desired RNAi bacteria. Luciferase (L4440::luc, i.e., luci) RNAi vector was used as a non-targeting control in all experiments. Keio library screen The rnp-6(dh1127);Y41C4A.32(syb2725) reporter strain was used for screening. The E. coli Keio knockout collection comprised two independent deletion strains for each of the 3985 nonessential genes, yielding a total of 7970 strains. All strains were tested as follows: briefly, Keio library bacteria were inoculated in 96-well plates overnight in LB (+ kanamycin) medium and seeded on 3 cm NGM plates. After two days of growth, ~ 30 synchronized worm eggs (generated by bleaching) were seeded on the plates. The worms were scored after three days. For the primary screen, the plates were checked manually under a Leica stereo microscope (Leica M165 FC, LAS X). Mutant bacterial strains that resulted in more than 3 worms showing a bright mNeonGreen signal were marked and selected for further validation. In the confirmation screen, OP50 and the parental BW25113 strain were used as positive and negative control, respectively. Images of worms were captured for fluorescence intensity quantification. Bacterial hits were confirmed by Sanger sequencing. EMS mutagenesis screen EMS mutagenesis was performed as described previously 22 . Briefly, ~ 1,000 synchronized L4 larvae worms of rnp-6(dh1127);Y41C4A.32(syb2725) reporter strain were exposed to 0.15% ethyl methanesulfonate in M9 buffer for 4h at room temperature, and then washed and transferred to normal NGM plates for recovery. After overnight growth, P0 adult animals were transferred to new plates seeded with OP50 for egg laying. After 3 days of growing, adult F1 worms were bleached, and eggs were seeded onto NGM plates seeded with BW25113 bacteria. After 3- and 4-day growth, the plates were scored, and F2 worms that showed a bright fluorescence signal were singled to individual NGM plates. To exclude the false positive hits, the reporter fluorescence intensity of mutants growing on OP50 or BW25113 was measured, and the rescue efficiency (RE) of BW25113 diet was calculated. The mutants with RE value less than 0.5 were selected for further characterizations. The rnp-6(dh1127);Y41C4A.32(syb2725) animals were used as negative control in all the assays. To map causative mutations, Hawaiian-SNP mapping and whole genome sequencing were used as previously described 105 . In brief, EMS mutants were crossed with Hawaiian strain (CB4856) males. The non-fluorescent F1 worms were picked, raised until adulthood, and allowed to lay eggs on NGM plates seeded with BW25113. Fluorescent F2 adult worms were singled. After 5-days of growth, worms were then pooled together, and their genomic DNA purified using Gentra Puregene Kit (Qiagen). The pooled DNA was sequenced on an Illumina HiSeq platform (paired-end 150 nucleotide). MiModD pipeline ( http://www.celegans.de/en/mimodd ) was used to narrow down the causative mutations. The WBcel235/ce11 C. elegans assembly was used as a reference genome. Causative mutations were confirmed by multiple outcrosses. AID degradation experiments The AID inducible-degradation system was designed following previously described protocols 106 . Degron and GFP were inserted at the N-terminus of endogenous RNP-6. The transgenic line was crossed with TIR1-expression strain to generate the inducible-degradation strain AA5498 ( rnp-6 ( syb6010 [degron::3XFLAG::GFP::RNP-6]);Si57 [Peft-3::TIR1::mRuby::unc-54 3'UTR + Cbr-unc-119(+)]). To induce RNP-6 degradation, strain AA5498 was grown on plates supplemented with different concentrations of Potassium Naphthaleneacetic Acid (K-NAA). RNA extraction and cDNA synthesis C. elegans were lysed with QIAzol Lysis Reagent. RNA was extracted using chloroform extraction. Samples were then purified using RNeasy Mini Kit (Qiagen). Purity and concentration of the RNA samples were assessed using a NanoDrop 2000c (peqLab). cDNA synthesis was performed using iScript cDNA synthesis kit (Bio-Rad). Standard manufacturer protocols were followed for all mentioned commercial kits. RNA-Seq and bioinformatic analysis 1 µg of total RNA was used per sample for library preparation. The protocol of Illumina Tru-Seq stranded RiboZero was used for RNA preparation. After purification and validation (2200 TapeStation; Agilent Technologies), libraries were pooled for quantification using the KAPA Library Quantification kit (Peqlab) and the 7900HT Sequence Detection System (Applied Biosystems). The libraries were then sequenced with Illumina HiSeq4000 sequencing system using the paired-end 2×100 bp sequencing protocol. For data analysis, Wormbase genome (WBcel235_89) was used for alignment of the reads. Kallisto was used to map raw reads to reference transcriptome and quantify transcript abundance 107 . DESeq2 was used for each pairwise comparison 108 . For the splicing analysis, SAJR 109 and IRFinder 110 pipelines were used. The significant events from different pipelines were combined, and the unique events were kept for further analysis. Row Z-score heatmaps were generated by using the iHeatmap function from FLASKI (Version 2.0.0) (DOI: 10.5281/zenodo.5254193 ). Adjusted p-value or q-value < 0.05 is considered to be significant for gene expression and splicing. Gene enrichment visualization was performed with WormCat 2.0 24 . Alternative splicing PCR assay (RT-PCR) A new batch of samples (different from the samples used for RNAseq) was used for RNA preparation and cDNA synthesis. Phusion Polymerase (Thermo Fisher) was used to amplify the tos-1, tcer-1 and nhr-114 segments. PCR reactions were cycled 30 times with an annealing temperature of 53°C. RT-PCR products were visualized with ChemiDoc Imager (BioRad, ChemiDoc MP, Image Lab 6.1) after staining with Roti-GelStain (Carl Roth). Sequences of the primers used in the RT–PCR assays are found in Supplementary Table S17. Quantitative reverse transcription PCR (RT–qPCR) Power SYBR Green master mix (Applied Biosystems) was used for RT-qPCR experiments. A JANUS automated workstation (PerkinElmer) was used for pipetting the reagents and cDNA samples into a 384 well plate. Thermal cycling was performed using a ViiA7 384 Real-Time PCR System machine (Applied Biosystems). act-1 and cdc-42 were used for internal normalization. Relative expression levels were calculated using the comparative CT method. Sequences of the primers used in the RT–qPCR assays are provided in Supplementary Table S17. Western blot For C. elegans samples, animals were first washed with M9 buffer. Worm pellets were resuspended in 4% SDS buffer (4% SDS in 0.1 M Tris/HCl pH 8 with 1 mM EDTA) supplemented with cOmplete Protease Inhibitor (Roche) and PhosSTOP (Roche) and snap frozen in liquid nitrogen. Thawed samples were lysed using Bioruptor Sonication System (Diagenode), centrifuged (20,000g, 10 min) and protein concentrations were measured with Pierce BCA kit. Protein samples were then heated to 95 ̊C for 10 min in Laemmli buffer with 0.8% 2-mercaptoethanol in order to denature proteins. 10 ug protein samples were loaded on 4–15% Mini PROTEAN TGXTM Precast Protein Gels (Bio-Rad), and electrophoresis was performed at a constant voltage of 200V for around 40 min. After separation, the proteins were transferred to PVDF membranes using Trans-Blot TurboTM Transfer System (BioRad). 5% bovine serum albumin (BSA) or 5% milk in Tris-buffered Saline and Tween20 (TBST) were used for blocking of the membranes. After antibody incubations (anti-HA 1:1000, anti-Phospho-AMPKα (Thr172) 1:2000, anti-beta Actin 1:5000, Anti-Mouse HRP 1:5000, Anti-Rabbit HRP 1:5000 and Anti-Rat HRP 1:5000) and washing with TBST buffer, imaging of the membranes was performed with ChemiDoc Imager (BioRad, ChemiDoc MP, Image Lab 6.1). Western Lightning Plus Enhanced Chemiluminescence Substrate (PerkinElmer) was used as the chemiluminescence reagent. A list of antibodies is provided in Supplementary Table S17. Metabolomics and lipidomics sample preparation Worms were synchronized by egg laying and collected when they reached the young adult stage. For each sample, ~ 2000 worms were washed three times with ddH₂O, snap-frozen in liquid nitrogen, and stored at − 80°C before use. For the metabolite/lipid extraction, 500 µL extraction buffer (MTBE:MeOH:H2O, 50:30:20) supplemented with internal standards was added to each sample. Worm pellets were homogenized with ~ 100 µL 1 mm zirconia beads using a TissueLyser (Qiagen) at 50 Hz and 4°C for 20 min. After initial homogenization, 500 µL extraction buffer was added to each sample and homogenization resumed for 5 min. Worm lysates were centrifuged at 21000 x g and 4°C for 10 min. Supernatant was transferred to new tubes. Residual buffer was removed before drying protein pellets under a fume hood overnight. Protein concentration was determined using a BCA kit (ThermoFisher). The cleared supernatant was mixed with 200 µL MTBE (Sigma) and 150 µL H2O, and incubated at 15°C for 10 min. Samples were centrifuged at 15°C and 16000 x g for 10 min to obtain phase separation (top lipid phase, bottom polar metabolites phase). 650 µL of the lipid phase was transferred to new tubes and dried in a SpeedVac concentrator at 20°C and 1000 rpm for ~ 2 h. 600 µL polar phase solution was transferred to new tubes and dried in a Speed Vac concentrator at 20°C and 1000 rpm for ~ 6 h. Samples were stored at − 80°C until further processing. Semi-targeted liquid chromatography-high-resolution mass spectrometry-based (LC-HRS-MS) analysis of amine-containing metabolites Amine-containing compounds were analyzed using a Q-Exactive Plus high-resolution mass spectrometer coupled to a Vanquish UHPLC chromatography system (Thermo Fisher Scientific). As previously described, dried extracts were reconstituted in 150 µL LC-MS-grade water at 4°C and 1500 rpm shaking for 10 min. After centrifugation, 50 µL supernatant was mixed with 25 µL 100 mM sodium carbonate, followed by 25 µL 2% (v/v) benzoyl chloride in acetonitrile (UPLC/MS-grade, Biosolve) 111 . Samples were mixed and stored at 20°C until analysis. 1 µL of derivatized sample was injected onto a 100 × 2.1 mm HSS T3 UPLC column (Waters) at 40°C, 400 µL/min, using a binary buffer system: buffer A (10 mM ammonium formate, 0.15% formic acid in water) and buffer B (acetonitrile). LC gradient: 0% B (0 min); 0–15% B (0–4.1 min); 15–17% B (4.1–4.5 min); 17–55% B (4.5–11 min); 55–70% B (11–11.5 min); 70–100% B (11.5–13 min); 100% B (13–14 min); 100–0% B (14–14.1 min); 0% B (14.1–19 min). MS acquisition was performed in positive ionization mode (m/z 100–1000), with the following source settings: 3.5 kV spray voltage, capillary temperature 300°C, sheath gas 60 AU, aux gas 20 AU at 330°C, sweep gas 2 AU, RF lens 60. Raw mass spectra files were converted to mzML using MSConvert (v3.0.22060) 112 , and analyzed in El Maven (v0.12.0) 113 . Area of the protonated [M + nBz + H] + mass peaks of every required compound was extracted and integrated (mass accuracy of < 5 ppm, retention time tolerance of < 0.05 min compared to reference compounds). Data were normalized to internal standards and protein content. Anion-exchange chromatography mass spectrometry (AEX-MS) for the analysis of anionic metabolites Extracted metabolites were reconstituted in 150 µL UPLC/MS grade water (Biosolve) of which 100 µL was transferred to polypropylene autosampler vials (Chromatography Accessories Trott). Analysis was performed as previously described, using a Dionex Integrion ion chromatography system (Thermo Fisher Scientific) 4 . 5 µL of extract was injected (push-full mode, overfill factor 1) onto a Dionex IonPac AS11-HC column (2 × 250 mm, 4 µm particle size) equipped with a AG11-HC guard column (2 × 50 mm, 4 µm) at 30°C and autosampler at 6°C. Metabolites were separated at 380 µL/min using a KOH cartridge (Eluent Generator, Thermo Scientific) with the following gradient: 0–3 min, 10 mM; 3–12 min, 10–50 mM; 12–19 min, 50–100 mM; 19–22 min, 100 mM; 22–23 min, 100–10 mM; re-equilibration at 10 mM (3 min). Eluting metabolites were detected in negative ion mode (m/z 77–770) on a Q-Exactive HF MS. Source settings: 3.2 kV spray voltage, capillary 300°C, sheath gas 50 AU, aux gas 14 AU at 380°C, sweep gas 3 AU, S-lens 40. Raw mass spectra files were converted to mzML using MSConvert (v3.0.22060) 3 , and analyzed in El Maven (v0.12.0) 2 . Area of the deprotonated [M-H + ] −1 or doubly deprotonated [M-2H] −2 isotopologues mass peaks of every required compound was extracted and integrated (mass accuracy of < 5 ppm, retention time tolerance of < 0.05 min compared to reference compounds). Data were normalized to internal standards and protein content. Liquid Chromatography-High Resolution Mass Spectrometry-based (LC-HRMS) analysis of lipids Stored (-80°C) lipid extracts were reconstituted in 150 µL UPLC-grade acetonitrile:isopropanol (70:30, v/v). After vortexing, samples were incubated for 10 min at 4°C with continuous shaking. Samples were clarified by centrifugation at 16000 x g, 4°C, for 5 min. Supernatants were transferred to 2 mL glass vials equipped with 300 µL glass inserts (Chromatography Zubehör Trott). Aliquots of 20 µL from each sample were pooled to generate quality control (QC) samples, which were injected after every 10th sample or after each replicate group. Samples and QCs were maintained at 6°C in a Vanquish UHPLC system (Thermo Fisher Scientific) fitted with a quaternary pump and coupled to a TimsTOF Pro 2 HRMS with a heated ESI (VIP-HESI) source (Bruker Daltonics). For each run, 1 µL sample was injected onto a 100 x 2.1 mm CSH C18 UPLC column (1.7 µm, Waters). The chromatographic gradient was performed at 400 µL/min using buffer A (10 mM ammonium formate, 0.1% formic acid in acetonitrile:water, 60:40, v/v) and buffer B (10 mM ammonium formate, 0.1% formic acid in isopropanol:acetonitrile, 90:10, v/v) as follows: 0–0.5 min, 45–48% B; 0.5–1 min, 48–55% B; 1–1.8 min, 55–60% B; 1.8–10 min, 60–85% B; 10–11 min, 85–99% B; 11–11.5 min, 99% B; 11.7–15 min, re-equilibration at 45% B (total run time: 15 min/sample). Prior to each batch, mass and mobility calibrations were performed using a 1:1 mixture of 10 mM sodium formate and ESI-L Low Concentration Tuning Mix (Agilent). Data were acquired in data-dependent PASEF mode, primarily in positive ionization mode (source settings: 4.5 kV capillary, 500 V end plate offset, nebulizer 2 bar, dry gas 8 L/min at 230°C, sheath gas 4 L/min at 400°C). For MS/MS, isolation width was set to 2 mD and collision energy to 30 eV. Pooled QC samples were additionally injected in negative mode (collision energy 40 eV, -3.5 kV capillary) for further fatty acid annotation. Samples were analyzed in randomized order. QC samples were analyzed after every 10th injection in both positive and negative ionization modes to ensure data quality and stability. Raw spectra were processed in MetaboScape (v2024) to extract features and annotate lipid species, using pooled QC samples for validation. Lipids were only included in downstream analysis if their abundance in QC samples was at least threefold higher than in extraction blanks. Human sample handling and preparation The peripheral blood samples of VRJS patients were collected in the Center for Rare Diseases, University Hospital of Cologne, Germany. All patients and/or custodians gave informed consent according to local institutional review board approval (20-1711, Medical Faculty at University of Cologne). The plasma was isolated with standard protocol and processed for metabolomic and lipidomic analysis in the Metabolomic Core Facility of the Max Planck Institute for Biology of Ageing. Statistics & Reproducibility In all figures, the numbers of independent replicates and the total number of animals analyzed are indicated in each panel. All statistical analyses were performed in GraphPad Prism (Version 9.0.0 (86)). Asterisks denote corresponding statistical significance *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data distribution was assumed to be normal but this was not formally tested. No statistical method was used to predetermine sample size but our sample sizes are similar to those reported in previous publications 23 , 74 ,114,115 . Missing or outlier data points were excluded from the analyses. At least three independent experiments for each assay were performed to verify the reproducibility of the findings, unless indicated otherwise. For worm experiments, samples preparation and data collection were randomized. For lifespan experiments, all the genotypes were blinded before the assays. For cold tolerance, developmental rate, body size, brood size, Western blot, and imaging experiments, the genotypes were not blinded before assay, as mutant worms have obvious phenotypes that revealed the sample identity (body size and developmental rate). However, worms were randomly picked and assigned to the different treatment conditions in a random order. For RNA-seq experiments, the genotypes were not blinded before collecting samples. Once the RNA samples were ready, they were processed at the Cologne Center for Genomics (CCG) in a blinded manner. Declarations Data availability The RNA-seq datasets generated in this study are available in the GEO datasets with the accession number GSE307065 as of the date of publication. The prp-19 RNAseq data was obtained under accession number GSE191294 66 . The list of nhr-114 target genes was as defined 60 , available under the accession number GSE211747. The siPUF60-treated RNAseq data was obtained under accession number OEP004324 79 in NODE (The National Omics Data Encyclopedia) database. Acknowledgements We thank the Caenorhabditis Genetics Center (CGC). Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We thank Lianfeng Wu (Westlake University) and William Mair (Harvard T.H. Chan School of Public Health) for kindly providing some C. elegans strains. We thank the Bioinformatics, Imaging, Metabolomics, and Proteomics core facilities of the Max Planck Institute for Biology of Ageing for their technical support. We would also like to thank the members of the Antebi lab, especially Dr. Kreuz, Dr. Tabrez and Dr. Kawamura for valuable comments on the manuscript. We also thank Dr. Filipe Cabreiro for his valuable feedback on the manuscript. We thank the PostDoc Seed funding 2023 (W.H.) from CECAD Cluster for Excellence, University of Cologne (Gefördert durch die Deutsche Forschungsgemeinschaft (DFG) im Rahmen der Exzellenzstrategie des Bundes und der Länder - EXC 2030 – 390661388), Cologne Graduate School for Ageing Research (J.K.) and the Max Planck Society, Germany (A.A.) for funding this project. Author contributions W.H., J.K. and A.A. conceived and designed the study. W.H., J.K. and A.L. performed the investigation in C. elegans . K.C. performed the western blot experiment. W.H. and J.K. analyzed the data. 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1","display":"","copyAsset":false,"role":"figure","size":325268,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA bacterial \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eK12 diet mitigates growth defects in an \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ernp-6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e/VRJS \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. elegans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e model.\u003c/strong\u003e \u003cstrong\u003ea, b\u003c/strong\u003e, Body size (area) of worms fed OP50 (O), HT115 (H) and BW25113 (B) (\u003cem\u003en\u003c/em\u003e = 7, scale bar: 500 μm). \u003cstrong\u003ec\u003c/strong\u003e, Assessment of K12 diet on worm growth rate (\u003cem\u003en\u003c/em\u003e = 3–6). \u003cstrong\u003ed\u003c/strong\u003e, Lifespan analysis, with survival curves illustrating one representative replicate (\u003cem\u003en\u003c/em\u003e = 3). Details of worm numbers and statistical analyses for each experimental repeat are available in Supplementary Table S18. \u003cstrong\u003ee,\u003c/strong\u003e Transcriptomic changes induced by BW25113 \u003cem\u003eE. coli\u003c/em\u003e; 1541 differentially expressed genes (DEGs) regulated by \u003cem\u003ernp-6(G281D)\u003c/em\u003erelative to wild-type on OP50 (O) are shown. Blue circles indicate genes that are significantly restored by the BW25113 diet; the black arrow highlights \u003cem\u003eY41C4A.32\u003c/em\u003eexpression (red circle). \u003cstrong\u003ef\u003c/strong\u003e, WormCat gene set enrichment analysis of BW25113-responsive genes. \u003cstrong\u003eg\u003c/strong\u003e, RT-qPCR quantification of \u003cem\u003eY41C4A.32\u003c/em\u003eexpression (\u003cem\u003en\u003c/em\u003e = 4; F.C., fold change). \u003cstrong\u003eh\u003c/strong\u003e, Representative images of Y41C4A.32::mNeonGreen (mNG) reporter expression across the indicated diets and genotypes (n = 3, scale bar: 500 μm). Data are presented as mean ± s.e.m. unless otherwise indicated; “\u003cem\u003en\u003c/em\u003e” denotes experimental replicates, with animal counts summarized in each respective bar. Statistical analyses were conducted using the log-rank (Mantel-Cox) test for lifespan (\u003cstrong\u003ed\u003c/strong\u003e) and one-way ANOVA with Dunnett’s multiple comparisons for body size, developmental progression, and gene expression (\u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003e c\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e). Significance is represented as *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ns: not significant. All the source data for figures are provided in supplementary tables.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8077579/v1/7d50444647d981d4f18f74d0.png"},{"id":96058574,"identity":"e6000bb6-1b80-45b8-8c71-e48272c89476","added_by":"auto","created_at":"2025-11-17 08:09:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":271035,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA complementary two-way genetic screen identifies vitamin B\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e12\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e as an alleviator of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ernp-6(G281D)\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant defects.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Workflow outlining the genetic screening approach. \u003cstrong\u003eb\u003c/strong\u003e, Identification of positive hits from the Keio library \u003cem\u003eE. coli\u003c/em\u003e BW25113 deletion strains. \u003cstrong\u003ec\u003c/strong\u003e, Schematic of key genes involved in VB12 uptake in \u003cem\u003eE. coli\u003c/em\u003e. OM, outer membrane. IM, inner membrane. \u003cstrong\u003ed\u003c/strong\u003e, Representative images of Y41C4A.32 reporter expression in worms fed bacterial deletion mutants (\u003cem\u003en\u003c/em\u003e = 4, scale bar: 500 μm). \u003cstrong\u003ee–f\u003c/strong\u003e, Effects of VB12 (1 nM) supplementation on \u003cem\u003ernp-6(G281D) \u003c/em\u003emutant phenotypes, shown for reporter expression (\u003cstrong\u003ee\u003c/strong\u003e) and body size (\u003cstrong\u003ef\u003c/strong\u003e) (\u003cem\u003en\u003c/em\u003e = 4). \u003cstrong\u003eg\u003c/strong\u003e, Lifespan analysis following VB12 supplementation (OV, 1 nM), with survival curves illustrating one representative replicate (\u003cem\u003en\u003c/em\u003e = 4). Details of worm numbers and statistical analyses for each experimental repeat are available in Supplementary Table S18. \u003cstrong\u003eh–j\u003c/strong\u003e, Impact of VB12 supplementation on RNP-6-depleted worms on OP50 (0.5 μM K-NAA, 1 nM VB12) (\u003cem\u003en\u003c/em\u003e = 3, scale bar: 500 μm). Statistical analyses were performed using log-rank (Mantel-Cox) survival test (\u003cstrong\u003eg\u003c/strong\u003e) and one-way ANOVA with Dunnett’s multiple comparisons (\u003cstrong\u003ee–f\u003c/strong\u003e, \u003cstrong\u003ei–j\u003c/strong\u003e). Significance is represented as *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u0026nbsp; Images of \u003cstrong\u003ea\u003c/strong\u003e and \u003cstrong\u003ec\u003c/strong\u003e were generated with BioRender with permision.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8077579/v1/af044e4ecd1c3d6bc6b5b1e8.png"},{"id":96058582,"identity":"fe48b430-bee8-4113-b8a7-60ed3e5fe847","added_by":"auto","created_at":"2025-11-17 08:09:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":329743,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVitamin B\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e12\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e alleviates \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ernp-6(G281D)\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant defects through methionine metabolism pathways.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Summary of hits from the \u003cem\u003eC. elegans\u003c/em\u003e EMS mutagenesis screen; shown are mutants exhibiting BW25113 rescue efficacy below 50%. \u003cstrong\u003eb\u003c/strong\u003e, Schematic illustrating VB12-dependent metabolic pathways in \u003cem\u003eC. elegans\u003c/em\u003e, with genes denoted by an asterisk indicating VB12 dependency. \u003cstrong\u003ec\u003c/strong\u003e, Representative images of Y41C4A.32 reporter expression in \u003cem\u003eC. elegans\u003c/em\u003e mutants of EMS candidate genes (\u003cem\u003en\u003c/em\u003e = 2, scale bar: 500 μm). \u003cstrong\u003ed–e\u003c/strong\u003e, Effects of \u003cem\u003emmcm-1\u003c/em\u003e and \u003cem\u003emetr-1\u003c/em\u003e deletion on reporter expression (\u003cstrong\u003ed\u003c/strong\u003e) and body size (\u003cstrong\u003ee\u003c/strong\u003e) in \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants under varying treatment conditions (\u003cem\u003en\u003c/em\u003e = 5). \u003cstrong\u003ef–g\u003c/strong\u003e, Effect of methionine supplementation (10 mM) on reporter expression (\u003cstrong\u003ef\u003c/strong\u003e) and body size (\u003cstrong\u003eg\u003c/strong\u003e) in \u003cem\u003ernp-6;metr-1\u003c/em\u003e double mutants (\u003cem\u003en\u003c/em\u003e = 4). \u003cstrong\u003eh–i\u003c/strong\u003e, Effect of \u003cem\u003esams-1\u003c/em\u003e deletion on reporter expression (\u003cstrong\u003eh\u003c/strong\u003e) and body size (\u003cstrong\u003ei\u003c/strong\u003e) in \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants under different treatment conditions (\u003cem\u003en\u003c/em\u003e = 4–5). \u003cstrong\u003ej–k\u003c/strong\u003e, Effect of choline supplementation (10 mM) on reporter expression (\u003cstrong\u003ej\u003c/strong\u003e) and body size (\u003cstrong\u003ek\u003c/strong\u003e) in \u003cem\u003ernp-6;sams-1\u003c/em\u003e double mutants (\u003cem\u003en\u003c/em\u003e = 4). Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons. Significance is represented as *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, n.s.: not significant.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8077579/v1/7404d8311aa5e09e5b140cc7.png"},{"id":96247043,"identity":"e8f75877-e37d-444c-89ab-06127b84534e","added_by":"auto","created_at":"2025-11-19 07:27:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":448063,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVitamin B\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e12\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e restores methylation potential and phosphatidylcholine metabolism in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ernp-6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutants.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Principal component analysis of metabolite profiles in wild-type and \u003cem\u003ernp-6\u003c/em\u003e mutants under different conditions (\u003cem\u003en\u003c/em\u003e = 5 per condition). \u003cstrong\u003eb\u003c/strong\u003e, Heat map of significantly altered metabolites. “*” denotes metabolites significantly restored by both BW25113 and VB12; “#” denotes metabolites significantly restored by BW25113 diet; “$” indicates metabolites significantly restored by VB12 supplementation (1 nM). The full names of all the metabolites can be found in Table S6. \u003cstrong\u003ec\u003c/strong\u003e, Relative abundance (normalized peak area) and ratio of methionine cycle-related metabolites in \u003cem\u003ernp-6\u003c/em\u003e mutants. \u003cstrong\u003ed\u003c/strong\u003e, Ratio of S-adenosylmethionine/S-adenosylhomocysteine (SAM/SAH) in \u003cem\u003ernp-6\u003c/em\u003e mutants across conditions. \u003cstrong\u003ee\u003c/strong\u003e, Principal component analysis of lipid profiles in wild-type and \u003cem\u003ernp-6\u003c/em\u003e mutants under different conditions (n = 5 per condition). \u003cstrong\u003ef\u003c/strong\u003e, Heat map illustration of significantly altered lipid species. \u003cstrong\u003eg\u003c/strong\u003e, Heat map illustration of lipid groups. The black square labels the significantly altered lipid groups in \u003cem\u003ernp-6\u003c/em\u003e mutants on OP50. “*” indicates lipid groups that are significantly rescued in by both BW25113 diet and VB12 supplementation. \u003cstrong\u003eh\u003c/strong\u003e, Distribution of the relative abundance of triacylglycerol (TG) lipid species with different fatty acyl chain lengths (nC) and numbers of double bonds (nD). TG, Triacylglycerol; Cer;O2, Dihydroceramide; LPE, Lysophosphatidylethanolamine; PG, Phosphatidylglycerol; PC, Phosphatidylcholine; LPC, Lysophosphatidylcholine; PC-O, Ether-linked Phosphatidylcholine; SM;O2, sphingomyelin; PS, Phosphatidylserine; PE-O, Ether-linked Phosphatidylethanolamine; PI, Phosphatidylinositol; PE, Phosphatidylethanolamine; DG, Diacylglycerol; LPE-O, ether-linked lysophosphatidylethanolamine; CL, Cardiolipin. DG-O, Ether-linked Diacylglycero. Statistical significance was determined by unpaired t-test (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e) and one-way ANOVA with Dunnett’s multiple comparisons (\u003cstrong\u003ed\u003c/strong\u003e). Significance is represented as *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, n.s.: not significant.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8077579/v1/b3343fb55c7b14ab07b10c9c.png"},{"id":96058576,"identity":"67fe574d-959b-49ed-a813-56475a2fa947","added_by":"auto","created_at":"2025-11-17 08:09:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":314438,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ernp-6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant defects are primarily caused by aberrant splicing of transcription factor \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003enhr-114\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Pie chart displaying differentially spliced events in \u003cem\u003ernp-6\u003c/em\u003e mutants. A3SS, alternative 3’ splice site; A5SS, alternative 5’ splice site; AFE, alternative first exon; ALE, alternative last exon; IR, intron retention; SE, cassette exon/exon skipping.\u003cstrong\u003e b\u003c/strong\u003e, Scatter plot showing changes in cassette exon (SE) and intron retention (IR) events. \u003cstrong\u003ec\u003c/strong\u003e, Gene set enrichment analysis of differentially spliced genes using WormCat 2.0. \u003cstrong\u003ed–e\u003c/strong\u003e, Targeted RNAi screen identifying genes mimicking \u003cem\u003ernp-6\u003c/em\u003e mutant effects on Y41C4A.32 reporter expression (\u003cstrong\u003ed\u003c/strong\u003e) and body size (\u003cstrong\u003ee\u003c/strong\u003e). \u003cem\u003eluci\u003c/em\u003e and \u003cem\u003ernp-6i\u003c/em\u003ewere used as controls. The \u003cem\u003emthf-1(W601stop);Y41C4A.32::mNG\u003c/em\u003e strain was used to mitigate suppression effects of HT115 bacteria. \u003cstrong\u003ef\u003c/strong\u003e, Genome browser view (top) and RT-PCR analysis (bottom) of \u003cem\u003enhr-114\u003c/em\u003e intron retention (IR, intron retention; IS, intron skipping). \u003cstrong\u003eg\u003c/strong\u003e, Quantification of RT-PCR from \u003cstrong\u003ef\u003c/strong\u003e (\u003cem\u003en\u003c/em\u003e = 4). \u003cstrong\u003eh\u003c/strong\u003e, FPKM of \u003cem\u003enhr-114\u003c/em\u003e from RNA sequencing. \u003cstrong\u003ei–j\u003c/strong\u003e, Representative images and quantification of GFP::NHR-114 intensity in \u003cem\u003ernp-6\u003c/em\u003e mutant background (mean ± s.d.; scale bar: 10 μm). \u003cstrong\u003ek\u003c/strong\u003e, Schematic of CRISPR-Cas9 genome editing of \u003cem\u003enhr-114\u003c/em\u003e. The consensus sequence of intron 3 splice site is labelled in green; the red dotted line and nucleotides indicate edited residues, and bold letters in \"\u003cem\u003ei4+\u003c/em\u003e\" represent a premature stop codon if intron 4 is retained. \u003cstrong\u003el–n\u003c/strong\u003e, Representative images and quantification of Y41C4A.32::mNG fluorescence intensity and body size in different \u003cem\u003enhr-114\u003c/em\u003emutants (\u003cem\u003en\u003c/em\u003e = 4, scale bar: 500 μm). \u003cstrong\u003eo\u003c/strong\u003e, Effects of \u003cem\u003enhr-114\u003c/em\u003eintron 4 editing on cold tolerance (\u003cem\u003en\u003c/em\u003e = 3). Statistical significance was determined using unpaired t-test (\u003cstrong\u003eg\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003ej\u003c/strong\u003e) and one-way ANOVA with Dunnett’s multiple comparisons (\u003cstrong\u003em\u003c/strong\u003e–\u003cstrong\u003eo\u003c/strong\u003e). Significance is represented as *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8077579/v1/015e1a91fbfcf9b84219f477.png"},{"id":96058487,"identity":"d6e53c78-d572-490c-98ee-c088a6c6cef7","added_by":"auto","created_at":"2025-11-17 08:09:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":208013,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVitamin B\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e12\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e restored mTOR signaling in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ernp-6(G281D)\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Western blotting of AMPK phosphorylation in \u003cem\u003ernp-6\u003c/em\u003e mutants. \u003cstrong\u003eb\u003c/strong\u003e, Quantification of Western blotting experiments in a. n=3. \u003cstrong\u003ec\u003c/strong\u003e, Representative images of HLH-30::mNG nuclear localization in \u003cem\u003ernp-6\u003c/em\u003e mutants (hypodermis). Scale bar, 20 mm. \u003cstrong\u003ed\u003c/strong\u003e, Quantification of nuclear HLH-30::mNG fluorescent intensity. The results from three biological replicates were merged. \u003cstrong\u003ee\u003c/strong\u003e, The effects of \u003cem\u003eraga-1\u003c/em\u003e deletion and \u003cem\u003eraga-1\u003c/em\u003e single-copy insertion on worm body size in \u003cem\u003ernp-6\u003c/em\u003e mutants. n=2-7. One-way ANOVA with Dunnett’s multiple comparison test were used for statistical analysis. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, n.s., not significant.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8077579/v1/a76ea0d688011c50ad06e95e.png"},{"id":96058620,"identity":"1ab5c7d1-3b1c-4cdc-b566-bde73a420684","added_by":"auto","created_at":"2025-11-17 08:10:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":308712,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePUF60 deficiency dysregulates 1CC and phospholipid metabolism. a\u003c/strong\u003e, KEGG pathway analysis of PUF60-dependent splicing events in PC9 lung cancer cell line. GPL, glycerophospholipid. \u003cstrong\u003eb\u003c/strong\u003e, Heatmap of aberrant spliced genes related to methionine and phospholipid metabolism. \u003cstrong\u003ec\u003c/strong\u003e, Heatmap illustration of significantly altered metabolites and lipid groups. “*” and “#” indicate the metabolite/lipids that show the same and opposite direction of changes, respectively, between VRJS plasma samples and worm samples. d, Quantification of the relative level of PE and PC in VRJS plasma. \u003cstrong\u003ee\u003c/strong\u003e, Heatmap illustration of altered lipids in VRJS plasma. \u003cstrong\u003ef\u003c/strong\u003e, Distribution of the relative abundance of triacylglycerol (TG) lipid species with different fatty acyl chain lengths (nC) and numbers of double bonds (nD). \u003cstrong\u003eg\u003c/strong\u003e, Model depicting the pathomechanisms underlying RNP-6/PUF60 deficiency. RNP-6 loss-of-function (LOF) causes aberrant splicing of metabolism-related genes (MRGs) as well as metabolism-related transcriptional factors such as \u003cem\u003enhr-114, \u003c/em\u003ewhich in turn alter the expression of MRGs. Altered MRGs collectively cause an imbalance of SAM/SAH and PE/PC metabolic axis, which triggers stress responses (including ISR and mitochondrial stress) and delays growth through inhibiting mTOR activity. VB12 supplementation restores SAM/SAH balance and PE/PC metabolism, suppressing stress response and rescuing normal development. “*” indicates the splicing targets of \u003cem\u003ernp-6\u003c/em\u003e; “#” indicates the transcriptional targets of \u003cem\u003enhr-114\u003c/em\u003e. In humans, PUF60 loss-of-function causes aberrant splicing of 1CC and phospholipid metabolism-related genes and is associated with altered levels of methionine and lipid levels in patient plasma. Dysregulated metabolism might drive some pathologies in VRJS patients.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8077579/v1/331a4141af540bff1fd9ec4a.png"},{"id":96256099,"identity":"2b024c0d-03f8-4585-9eb2-7d92bb58bd00","added_by":"auto","created_at":"2025-11-19 07:49:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3990784,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8077579/v1/9dc641c8-f85b-4919-91d8-13bf65e1c424.pdf"},{"id":96058608,"identity":"b2cc57b5-a32c-43b5-9c65-3ecc0f9156a0","added_by":"auto","created_at":"2025-11-17 08:09:59","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4341315,"visible":true,"origin":"","legend":"Supplementary_Table S1-S18","description":"","filename":"HuangSupplementaryTable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8077579/v1/a64a3c70edbff08f7c16b22c.xlsx"},{"id":96058588,"identity":"0f8a2b87-8df9-4f0e-8b65-4fbbb9a6f517","added_by":"auto","created_at":"2025-11-17 08:09:54","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":8956952,"visible":true,"origin":"","legend":"Supplementary Figure S1-S9","description":"","filename":"HuangSupplementaryfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-8077579/v1/56a91a92de5d9951f6ab51c5.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"\u003cp\u003eVitamin B\u003csub\u003e12\u003c/sub\u003e alleviates spliceosomopathy via phospholipid remodeling\u003c/p\u003e","fulltext":[{"header":"Main","content":"\u003cp\u003eSplicing is a fundamental regulatory process in eukaryotic messenger RNA (mRNA) maturation that removes intronic regions from precursor transcripts to produce mature mRNAs encoding proteins of enhanced diversity. This essential activity is catalyzed by the spliceosome, a highly dynamic megadalton complex composed of five small nuclear ribonucleoprotein particles (snRNPs) and numerous associated splicing factors\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Dysregulation of splicing impairs proper cellular function and is associated with various pathological conditions, including cancer\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and ageing\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Pathogenic mutations in genes encoding core spliceosomal components disrupt splicing, leading to various genetic diseases with overlapping phenotypes\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ePUF60\u003c/em\u003e (also known as FIR or Hfp) encodes an essential splicing factor containing two central RNA recognition motifs and a C-terminal U2AF-homology motif\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. It operates early in spliceosome assembly by promoting the recruitment of precursor transcripts to the U2 snRNP complex and facilitating the splicing of weak 3\u0026rsquo; acceptor sites\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Heterozygous \u003cem\u003ede novo\u003c/em\u003e variants causing PUF60 deficiency result in Verheij syndrome (OMIM #615583)\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, a congenital disorder associated with growth retardation, short stature, and recurrent infections\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12 CR13\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. We have previously reported on a phenotypic spectrum of \u003cem\u003ePUF60\u003c/em\u003e-related disorders ranging from neurodevelopmental delay to multisystem involvement\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, the exact pathomechanisms of VRJS remain elusive, and no targeted therapies currently exist.\u003c/p\u003e\u003cp\u003eVitamin B\u003csub\u003e12\u003c/sub\u003e (also known as cobalamin) is a complex water-soluble organic compound essential for several biological functions in animals\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. It serves as a cofactor for two metabolically important enzymes: methionine synthase (MS), which converts homocysteine to methionine within the methionine cycle, and methylmalonyl-CoA mutase, which converts L-methylmalonyl-CoA to succinyl-CoA in propionate metabolism. These two pathways are linked through homocysteine as a shared intermediate. Consequently, VB12 deficiency profoundly disrupts metabolic homeostasis and instigates various disorders in humans, including growth delay, hypotonia, anemia and cognitive impairment\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn \u003cem\u003eCaenorhabditis elegans, rnp-6\u003c/em\u003e (RNA-binding domain-containing protein 6) encodes the sole ortholog of human \u003cem\u003ePUF60\u003c/em\u003e\u003csup\u003e17\u003c/sup\u003e. Depletion of \u003cem\u003ernp-6\u003c/em\u003e by RNAi-mediated knockdown causes severe developmental defects, including growth retardation, smaller body size, dysregulated immune function, and neuronal abnormalities reminiscent of VRJS in humans\u003csup\u003e\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Previously, we identified a viable hypomorphic allele, \u003cem\u003ernp-6(G281D)\u003c/em\u003e, that enhances abiotic stress resistance and extends lifespan on the standard B-type \u003cem\u003eEscherichia coli\u003c/em\u003e OP50 diet\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In this study, we unexpectedly found that K12-type \u003cem\u003eE. coli\u003c/em\u003e can fully rescue growth defects observed in \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants. Through unbiased genetic screens in \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eC. elegans\u003c/em\u003e, we discovered that \u003cem\u003eE. coli\u003c/em\u003e K12 exerts its benefits through VB12-dependent methionine metabolism. VB12 restores \u003cem\u003ernp-6\u003c/em\u003e mutant growth by driving synthesis of methionine (Met), S-adenosylmethionine (SAM) and phosphatidylcholine (PC), while inhibition of enzymes required for the Met/SAM cycle abrogates such rescue. Metabolomic and lipidomic analyses reveal that \u003cem\u003ernp-6\u003c/em\u003e mutants maintained on OP50 harbor deficiencies in methylation capacity and phosphatidylcholine lipids, which are replenished by provisioning K12-type \u003cem\u003eE. coli\u003c/em\u003e or VB12. Mechanistically, we deduce that aberrant splicing of \u003cem\u003enhr-114 (\u003c/em\u003ehomolog of human \u003cem\u003eHNF4\u003c/em\u003e), is a major proximal cause of metabolic dysregulation and developmental defects in the \u003cem\u003ernp-6\u003c/em\u003e mutant. We further demonstrate that altered Met/SAM/PC metabolism in \u003cem\u003ernp-6(G281D)\u003c/em\u003e modulates the integrated stress response (ISR) and mTOR signaling. Finally, we provide evidence that PUF60 deficiency induces alternative splicing of genes related to methionine and phospholipid metabolism, and \u003cem\u003ePUF60\u003c/em\u003e pathogenic mutations induce metabolic and lipidomic changes in VRJS patient plasma. Notably, this metabolic dysregulation is not limited to PUF60/RNP-6: depletion of \u003cem\u003ePRPF19/prp-19\u003c/em\u003e phenocopies \u003cem\u003ernp-6\u003c/em\u003e loss, suggesting conserved metabolic vulnerability across spliceosomopathies. Together, our findings identify dysregulated S-adenosylmethionine/S-adenosylhomocysteine balance and phospholipid metabolism as central contributors of \u003cem\u003ernp-6\u003c/em\u003e-dependent spliceosomal pathology, and highlight VB12 supplementation as a potential strategy to restore metabolic homeostasis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eA K12-type\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003ediet alleviates growth defects in a\u003c/b\u003e \u003cb\u003eC. elegans\u003c/b\u003e \u003cb\u003eVerheij syndrome model\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePreviously, we identified a hypomorphic mutation in \u003cem\u003ernp-6\u003c/em\u003e that extends lifespan in \u003cem\u003eC. elegans\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Further characterization showed that this mutant exhibits moderate but significant developmental defects, including smaller body size and slower growth rate when raised on the standard B-type \u003cem\u003eE. coli\u003c/em\u003e OP50 diet (O) at 20\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea\u0026ndash;c). These phenotypes are analogous to the growth delay and small stature seen in VRJS patients\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In the course of our studies, we discovered that \u003cem\u003ernp-6(G281D)\u003c/em\u003e developmental phenotypes were remarkably restored by feeding worms with the \u003cem\u003eE. coli\u003c/em\u003e K12 strains HT115 (H) or BW25113 (B) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea\u0026ndash;c). These K12-type \u003cem\u003eE. coli\u003c/em\u003e strains also suppressed other previously observed \u003cem\u003ernp-6(G281D)\u003c/em\u003e traits, including cold tolerance (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea) and extended lifespan (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Of note, dietary mixtures of O\u0026thinsp;+\u0026thinsp;B containing as little as ~\u0026thinsp;10% BW25113 sufficed to rescue growth defects (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb). Rescue did not require active bacterial metabolism, as UV-killed BW25113 also effectively restored body size (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec). These results imply that BW25113-derived nutrients or metabolites compensate for RNP-6(G281D) dysfunction. To explore whether K12-type \u003cem\u003eE. coli\u003c/em\u003e restored RNP-6 protein\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e directly or acted downstream, we measured steady-state RNP-6 levels. However, neither HT115 nor BW25113 diets restored RNP-6 protein levels (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ed\u0026ndash;e), indicating that the rescue occurs via pathways downstream of RNP-6.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further elucidate the impact of K12-type \u003cem\u003eE. coli\u003c/em\u003e, we performed RNA sequencing (RNA-seq) on wild-type and \u003cem\u003ernp-6(G281D)\u003c/em\u003e worms raised on either OP50 or BW25113 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ef). Consistent with prior work, \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants grown on OP50 exhibited profound transcriptomic changes, with 1,541 genes and 473 genes showing differential expression (adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, |log\u003csub\u003e2\u003c/sub\u003eFC| \u0026gt;0.5; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and differential splicing (adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ΔPSI\u0026thinsp;\u0026gt;\u0026thinsp;5%; Table S2, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eg and i), respectively. About 11% of differentially spliced genes also showed altered expression (Table S3, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ej), indicating distinct layers of regulation by \u003cem\u003ernp-6\u003c/em\u003e. Feeding with BW25113 significantly changed the expression of 1319 genes in \u003cem\u003ernp-6\u003c/em\u003e mutants and restored 573 genes toward wild-type levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eh, Table S4). WormCat 2.0\u003csup\u003e24\u003c/sup\u003e gene set analysis of the BW25113-restored genes revealed significant enrichment in stress response, proteolysis/proteasome, and transmembrane transport pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). In contrast, BW25113 had minimal effect on splicing profiles, with no significant restoration observed (Table S2, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ek). Validation of two established \u003cem\u003ernp-6-\u003c/em\u003edependent splicing targets, \u003cem\u003etcer-1\u003c/em\u003e and \u003cem\u003etos-1\u003c/em\u003e, confirmed this observation (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003el\u0026ndash;m). Together, these findings suggest that K12-type \u003cem\u003eE. coli\u003c/em\u003e alleviates \u003cem\u003ernp-6(G281D)\u003c/em\u003e phenotypes through mechanisms downstream of, or independent from, splicing activity.\u003c/p\u003e\u003cp\u003eAnalysis of our RNA-seq data revealed that the expression of \u003cem\u003eY41C4A.32\u003c/em\u003e, an ortholog of human COPI coat complex subunit beta 2 (\u003cem\u003eCOPB2\u003c/em\u003e), was among the strongest upregulated by \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutation, and conversely suppressed by \u003cem\u003eE. coli\u003c/em\u003e K12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). This gene, previously annotated as \u003cem\u003eY41C4A.11\u003c/em\u003e, has been commonly used to assess innate immune and endoplasmic reticulum (ER) stress responses\u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ernp-6(G281D)\u003c/em\u003e did not cause significant splicing changes in \u003cem\u003eY41C4A.32\u003c/em\u003e mRNA (Table S2). Quantitative RT-PCR confirmed a\u0026thinsp;~\u0026thinsp;40-fold increase in \u003cem\u003eY41C4A.32\u003c/em\u003e transcript levels on OP50 diet, which was almost completely reversed by BW25113 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). We thus purposed \u003cem\u003eY41C4A.32\u003c/em\u003e as a robust marker for dissecting bacteria-host interactions. To this end, we generated an endogenous C-terminal mNeonGreen fusion via CRISPR/Cas9 genome editing. Consistent with our RNA-seq and RT-qPCR results, \u003cem\u003eY41C4A.32::mNeonGreen\u003c/em\u003e expression increased by ~\u0026thinsp;10-fold in \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants compared to wild-type controls on OP50, but was substantially suppressed by BW25113 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). Expression of transgenic wild-type \u003cem\u003ernp-6\u003c/em\u003e fully normalized \u003cem\u003eY41C4A.32\u003c/em\u003e levels in \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants, confirming \u003cem\u003eY41C4A.32\u003c/em\u003e expression as a faithful readout of activity downstream of RNP-6 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003en\u0026ndash;o).\u003c/p\u003e\u003cp\u003e\u003cb\u003eComplementary genetic screens identify vitamin B\u003c/b\u003e\u003csub\u003e\u003cb\u003e12\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eas a key alleviator of\u003c/b\u003e \u003cb\u003ernp-6(G281D)\u003c/b\u003e \u003cb\u003ephenotypes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUtilizing the \u003cem\u003eY41C4A.32\u003c/em\u003e reporter strain, we performed a two-way genetic screen to identify K12-type \u003cem\u003eE. coli\u003c/em\u003e-derived nutrients/metabolites and their corresponding host effectors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). We took advantage of the Keio \u003cem\u003eE. coli\u003c/em\u003e mutant library, which encompasses two independent single-gene knockout mutants for 3,985 nonessential genes in the BW25113 background\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. We posited that deletion of genes crucial for the rescue metabolite production or transport would specifically de-repress (i.e., reactivate) \u003cem\u003eY41C4A.32\u003c/em\u003e reporter expression in \u003cem\u003ernp-6\u003c/em\u003e mutants maintained on \u003cem\u003eE. coli\u003c/em\u003e K12 diet. After two rounds of screening a total of 7970 Keio strains, we confirmed nine bacterial mutants that enhanced \u003cem\u003eY41C4A.32\u003c/em\u003e expression relative to the parental strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). These candidate genes are involved in various biological processes, including cytoskeleton formation (\u003cem\u003eyfgA\u003c/em\u003e), membrane transport (\u003cem\u003etonB\u003c/em\u003e, \u003cem\u003ebtuF\u003c/em\u003e and \u003cem\u003eznuB\u003c/em\u003e), and purine metabolism (\u003cem\u003epurA\u003c/em\u003e) (Table S5). Notably, both BtuF and TonB are core components of the \u003cem\u003eE. coli\u003c/em\u003e cobalamin uptake system\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), suggesting that VB12 or related metabolites mediate the rescue effect. Subsequent phenotypic analysis revealed that deletion of \u003cem\u003ebtuF\u003c/em\u003e or \u003cem\u003etonB\u003c/em\u003e in BW25113 also mimics OP50 \u003cem\u003eE. coli\u003c/em\u003e effects, resulting in smaller body size of \u003cem\u003ernp-6\u003c/em\u003e mutants (Figure S2a\u0026ndash;b). In contrast, deletion of other putative VB12 transporters (\u003cem\u003ebtuB\u003c/em\u003e, \u003cem\u003ebtuC\u003c/em\u003e and \u003cem\u003ebtuD\u003c/em\u003e) had negligible effects (Figure S2a\u0026ndash;b), indicating \u003cem\u003ebtuF\u003c/em\u003e and \u003cem\u003etonB\u003c/em\u003e act as rate-limiting components in VB12-associated rescue.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e strains cannot synthesize VB12 and largely rely on environmental scavenging\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that OP50 accumulates less VB12 than \u003cem\u003eE. coli\u003c/em\u003e K12\u003csup\u003e32,33\u003c/sup\u003e, providing a plausible explanation for the strain-specific rescue of \u003cem\u003ernp-6(G281D)\u003c/em\u003e. Accordingly, we hypothesized that VB12 supplementation of OP50 would mimic the beneficial effects of a K12-type \u003cem\u003eE. coli\u003c/em\u003e diet. Indeed, adding 1 nM VB12 throughout development to \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants fed on OP50 suppressed \u003cem\u003eY41C4A.32\u003c/em\u003e reporter expression and restored body size to levels comparable with BW25113-fed worms (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee\u0026ndash;f, Figure S2c\u0026ndash;d). VB12 rescued \u003cem\u003ernp-6(G281D)\u003c/em\u003e phenotypes even when cultured on UV-killed OP50, demonstrating a direct effect on the worm, independent of bacterial metabolism (Figure S2e\u0026ndash;f). VB12 also alleviated other \u003cem\u003ernp-6(G281D)\u003c/em\u003e phenotypes, including slower growth rate, cold tolerance, and reduced fecundity (Figure S2g\u0026ndash;i), confirming its role as the active metabolite mediating rescue. Surprisingly, VB12 supplementation did not reverse the \u003cem\u003ernp-6(G281D)\u003c/em\u003e longevity phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg), indicating that \u003cem\u003eE. coli\u003c/em\u003e K12-derived and VB12-mediated effects on lifespan are separable. To determine the window of action, we supplemented worms with VB12 at different developmental stages. We found that VB12 treatment before the L4 larval stage fully restored normal development, matching the efficacy of continuous treatment (Figure S2j\u0026ndash;k). Even a brief 24-hour treatment from the young adult stage onwards significantly increased body size and suppressed reporter expression (Figure S2j\u0026ndash;k), underscoring VB12\u0026rsquo;s potent effect on \u003cem\u003ernp-6(G281D)\u003c/em\u003e development.\u003c/p\u003e\u003cp\u003eGiven that \u003cem\u003ernp-6(G281D)\u003c/em\u003e is a specific hypomorphic allele, we investigated whether VB12\u0026rsquo;s benefits extend to broader forms of RNP-6 insufficiency that mimic the partial loss-of-function features of \u003cem\u003ePUF60\u003c/em\u003e pathogenic variants in Verheij syndrome. To this end, we engineered an inducible RNP-6 loss-of-function strain using the auxin-inducible degradation (AID) system (Figure S2l)\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Consistent with previous data showing that \u003cem\u003ernp-6\u003c/em\u003e null mutants are embryonic lethal and complete knockdown in early life triggers larval arrest\u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, increasing auxin (K-NAA) doses yielded graded phenotypes and recapitulated VRJS-like defects in our system: 0.5 \u0026micro;M K-NAA reduced RNP-6 levels and body size (Figure S2m\u0026ndash;n), whereas doses at 4 \u0026micro;M or above caused larval arrest (Figure S2n\u0026ndash;o). Of note, partial depletion (0.5 \u0026micro;M K-NAA) activated the \u003cem\u003eY41C4A.32\u003c/em\u003e reporter and impaired growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). Under these graded conditions of RNP-6 deficiency, VB12 supplementation restored body size and suppressed reporter induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh\u0026ndash;j). These results support the notion that both \u003cem\u003ernp-6(G281D)\u003c/em\u003e and the AID-mediated partial depletion model pathogenic PUF60 haploinsufficiency, pointing to VB12 as a potential metabolic intervention.\u003c/p\u003e\u003cp\u003e\u003cb\u003eVitamin B\u003c/b\u003e\u003csub\u003e\u003cb\u003e12\u003c/b\u003e\u003c/sub\u003e \u003cb\u003erescues\u003c/b\u003e \u003cb\u003ernp-6(G281D)\u003c/b\u003e \u003cb\u003emutant defects through methionine metabolism\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMeanwhile, we performed EMS mutagenesis screens to identify host factors mediating rescue by K12-type \u003cem\u003eE. coli\u003c/em\u003e (Figure S3a). As with the bacterial screen, we reasoned that mutations disrupting key host genes would de-repress \u003cem\u003eY41C4A.32\u003c/em\u003e expression in \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants fed BW25113. After screening\u0026thinsp;~\u0026thinsp;20,000 haploid genomes, we isolated 84 mutants that suppressed the rescue efficacy of BW25113 on reporter expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Through Hawaiian SNP mapping and whole-genome sequencing\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, we identified causative genes for 20 mutants. Excitingly, three of these genes, \u003cem\u003epmp-5\u003c/em\u003e, \u003cem\u003emtrr-1\u003c/em\u003e, and \u003cem\u003emthf-1\u003c/em\u003e, are directly involved in the VB12-dependent methionine synthesis cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u0026ndash;c). \u003cem\u003epmp-5\u003c/em\u003e encodes the ortholog of human ABCD4 vitamin B\u003csub\u003e12\u003c/sub\u003e transporter\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003emtrr-1\u003c/em\u003e encodes the MTRR methionine synthase reductase\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003emthf-1\u003c/em\u003e encodes the MTHFR methylene-tetrahydrofolate reductase\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. All three genes have been implicated in the \u0026ldquo;vitamin B\u003csub\u003e12\u003c/sub\u003e-mechanism-II\u0026rdquo; transcriptional response that senses and compensates for perturbed Met/SAM cycle activity\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Hence, convergent \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eC. elegans\u003c/em\u003e genetic screens strongly implicate VB12-dependent methionine metabolism as a critical modulator of \u003cem\u003ernp-6(G281D)\u003c/em\u003e phenotypes. To further validate this, we directly tested whether VB12-dependent enzymes were required for the rescue effect of BW25113 diet and VB12. Vitamin B\u003csub\u003e12\u003c/sub\u003e serves as a known cofactor for two enzymes: methionine synthase (\u003cem\u003emetr-1\u003c/em\u003e), which catalyzes the conversion of homocysteine to methionine, and L-methylmalonyl-CoA mutase (\u003cem\u003emmcm-1\u003c/em\u003e), which converts L-methylmalonyl-CoA to succinyl-CoA\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. We found that the deletion of \u003cem\u003emetr-1\u003c/em\u003e, but not \u003cem\u003emmcm-1\u003c/em\u003e, completely abolished the benefits of BW25113 and VB12 on \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed\u0026ndash;e). As expected, supplementation with the \u003cem\u003emetr-1\u003c/em\u003e downstream product, methionine, was sufficient to bypass the requirement for \u003cem\u003emetr-1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef\u0026ndash;g). Altogether, these findings suggest that \u003cem\u003ernp-6\u003c/em\u003e mutants grown on OP50 are deficient in methionine metabolism and/or downstream pathways, which can be restored by stimulating VB12-dependent methionine production. However, additional mutation of methionine biosynthesis pathway components reinstates methionine deficiency, which is rescuable by (exogenous) methionine itself.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next asked how pathways downstream of methionine production interact with \u003cem\u003ernp-6\u003c/em\u003e mutant phenotypes. An important product of the methionine cycle is SAM, the principal methyl donor produced by SAM synthetase (\u003cem\u003esams-1\u003c/em\u003e)\u003csup\u003e42\u003c/sup\u003e. We found that deletion of \u003cem\u003esams-1\u003c/em\u003e fully abolished the rescue by BW25113, VB12 and methionine in \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh\u0026ndash;i), indicating a crucial role for SAM or SAM-dependent metabolites. Among other functions, SAM is essential for the \u003cem\u003ede novo\u003c/em\u003e synthesis of PC\u003csup\u003e43,44\u003c/sup\u003e, a key component of cellular membranes\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. PC can also be synthesized in a SAM-independent manner via the Kennedy pathway, which utilizes dietary choline\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Consistent with this framework, choline supplementation reduced \u003cem\u003eY41C4A.32\u003c/em\u003e expression, rescued body size defects, and importantly, bypassed the requirement for \u003cem\u003esams-1\u003c/em\u003e in \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej\u0026ndash;k). These findings support a model in which SAM-dependent phosphatidylcholine synthesis constitutes a major downstream mechanism by which K12-type \u003cem\u003eE. coli\u003c/em\u003e and VB12 rescue \u003cem\u003ernp-6\u003c/em\u003e mutant phenotypes.\u003c/p\u003e\u003cp\u003e\u003cb\u003ernp-6(G281D)\u003c/b\u003e \u003cb\u003emutation disrupts methylation potential and phosphatidylcholine metabolism\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur genetic data suggest that \u003cem\u003ernp-6(G281D)\u003c/em\u003e perturbs the methionine cycle and/or PC metabolism when grown on OP50. To examine this directly, we performed metabolomic and lipidomic analyses on \u003cem\u003ernp-6(G281D)\u003c/em\u003e and wild-type worms grown on OP50, OP50\u0026thinsp;+\u0026thinsp;VB12, and BW25113 (Figure S4a). Mass spectrometry (MS)-based metabolomics identified 115 metabolites spanning amino acid, nucleotide, glucose, and polyamine metabolism (Table S6). Using MetaboAnalyst\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, we observed that \u003cem\u003ernp-6(G281D)\u003c/em\u003e grown on OP50 significantly altered the abundance of 56 metabolites (FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05, |log\u003csub\u003e2\u003c/sub\u003eFC| \u0026gt;0.5) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-b). These metabolites were enriched in TCA cycle (succinate, citrate, malate), pyrimidine metabolism (CTP, UTP, CDP), purine metabolism (AMP, IMP, GMP), as well as the one-carbon pool by folate (SAM and SAH) (Figure S4b). Notably, the levels of SAM, SAH, and the SAM/SAH ratio were significantly decreased in \u003cem\u003ernp-6(G281D)\u003c/em\u003e, while levels of methionine, homocysteine or Met/Hcy ratio remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). This pattern suggests that \u003cem\u003ernp-6(G281D)\u003c/em\u003e may impact cellular methylation potential\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e (i.e., SAM/SAH ratio) rather than steady-state concentrations of these metabolites. Both BW25113 \u003cem\u003eE. coli\u003c/em\u003e and VB12 supplementation significantly increased methylation potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), confirming the central role of SAM-mediated methylation deficiency in \u003cem\u003ernp-6\u003c/em\u003e mutant phenotypes. Interestingly, several other metabolites were also rescued by BW25113 and VB12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, S4c, Table S6). These included intermediates in TCA cycle and gluconeogenesis (malate, phosphoenolpyruvate) (Figure S4d\u0026ndash;e), amino and nucleotide sugar metabolites (UDP-N-acetyl-alpha-D-glucosamine, UDP-N-acetyl-D-galactosamine) (Figure S4f\u0026ndash;g), tryptophan metabolism components (kynurenine, tryptophan) (Figure S4h\u0026ndash;i), and mitochondrial β-oxidation-related acylcarnitines (propionylcarnitine, butyrylcarnitine) (Figure S4j\u0026ndash;k). Further, the oxidized-to-reduced glutathione ratio, a marker for cellular oxidative stress, was significantly elevated in \u003cem\u003ernp-6\u003c/em\u003e mutants and suppressed by BW25113 and VB12 (Figure S4l-m), indicating perturbed redox balance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMS-based lipidomics on the same sample set detected 18 lipid classes encompassing 735 species, of which the most abundant classes were phosphatidylethanolamine (PE), phosphatidylcholine (PC), and triacylglycerol (TG) (Table S7). The \u003cem\u003ernp-6\u003c/em\u003e mutation broadly altered lipid composition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee), significantly changing the abundance of 339 lipids across 14 classes (FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05, |log\u003csub\u003e2\u003c/sub\u003eFC| \u0026gt;0.5) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef-g). Notably, we found that PC were dramatically decreased, while PE were increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg), resulting in a significant elevation of the PE/PC ratio (Figure S5b). This pattern is consistent with impaired SAM-dependent PC synthesis in \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants. A detailed examination with individual lipids showed that TGs exhibited a biphasic response: of the 100 significantly altered TG lipids, 42% were downregulated, whereas 58% were upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). More interestingly, the upregulated TGs contained longer and more unsaturated fatty acid chains (58 carbons, 8 double bonds on average) than downregulated TGs (50 carbons, 2 double bonds on average) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). Given the link between membrane phospholipid unsaturation and cold adaptation\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, the observed lipid changes likely contribute to the enhanced cold tolerance observed in \u003cem\u003ernp-6\u003c/em\u003e mutants. BW25113 feeding and VB12 supplementation exerted similar effects on the lipidome (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee), restoring the levels of 269 and 277 lipids, respectively. 252 lipids were shared between the two treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef, S5a). The relative abundance of 12 lipid classes and the PE/PC ratio were significantly rescued (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg, Figure S5b\u0026ndash;c). These findings demonstrate that disrupted lipid metabolism is a major driver of \u003cem\u003ernp-6\u003c/em\u003e mutant defects and suggest that K12-type \u003cem\u003eE. coli\u003c/em\u003e and VB12 rescue \u003cem\u003ernp-6\u003c/em\u003e defects mainly through lipid remodeling. Supporting these metabolomic findings, transcriptomic analysis revealed dysregulation of 78 lipid-metabolism genes in \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants on OP50 (Figure S5d), including peroxisomal acyl-CoA oxidases (\u003cem\u003eacox-1.2, 1.3, 1.5, 3\u003c/em\u003e), fatty acid CoA synthetases (\u003cem\u003eacs-1, 2, 7, 9\u003c/em\u003e), desaturases (\u003cem\u003efat-2, 5, 6, 7\u003c/em\u003e), short-chain dehydrogenases (\u003cem\u003edhs-2, 4, 19, 23, 26, 31\u003c/em\u003e), lipid-binding proteins (\u003cem\u003elbp-5, 6, 7\u003c/em\u003e), and glycerophospholipid remodeling enzymes (\u003cem\u003eckc-1, ckb-2, eppl-1, acl-12\u003c/em\u003e). Feeding BW25113 significantly restored the expression of more than half (41/78) of these genes, including \u003cem\u003efat-2, 5, 6, 7\u003c/em\u003e; \u003cem\u003eacox-1.2, 1.3, 1.5\u003c/em\u003e; \u003cem\u003eacs-1,2\u003c/em\u003e; \u003cem\u003elbp-5,6\u003c/em\u003e; \u003cem\u003eand ckc-1, ckb-2, acl-12\u003c/em\u003e (Figure S5d).\u003c/p\u003e\u003cp\u003ePrevious studies have shown that amino acid starvation (e.g., methionine restriction), or lipid dysregulation (e.g., PE/PC imbalance), can trigger the ISR and ER stress-related pathways\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan additionalcitationids=\"CR51 CR52\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Reflecting this, we detected robust phosphorylation of eukaryotic initiation factor 2α (eIF2α), an established marker of ISR activation\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, in \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants grown on OP50 (Figure S5e\u0026ndash;f). Consistently, the bZIP transcription factor GCN4/ATF-4, a central ISR effector\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, was markedly elevated in \u003cem\u003ernp-6(G281D)\u003c/em\u003e on OP50 and effectively suppressed upon VB12 supplementation (Figure S5g\u0026ndash;h), indicating restoration of ISR activity. In contrast, expression of \u003cem\u003ehsp-4\u003c/em\u003e, a canonical ER-stress marker, and \u003cem\u003ecpl-1*\u003c/em\u003e, an ER-associated degradation substrate\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, was not induced by \u003cem\u003ernp-6\u003c/em\u003e mutation (Figure S4i\u0026ndash;l). Collectively, these findings unveil a novel link from splicing dysfunction to ISR activation, rather than canonical ER stress, in \u003cem\u003ernp-6\u003c/em\u003e mutants.\u003c/p\u003e\u003cp\u003e\u003cb\u003ernp-6(G281D)\u003c/b\u003e \u003cb\u003emutation causes aberrant splicing of methionine metabolism-related genes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBuilding on our findings of SAM and PC metabolic disruption, we sought to understand the proximal molecular mechanisms. Since RNP-6 primarily regulates pre-mRNA splicing, we focused on the aberrant splicing landscape mentioned above (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ej, Table S2). Among 638 significant splicing alterations, intron retention (IR) and cassette exon skipping (SE) predominated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Nearly all IR events (315 out of 330 events) increased, whereas most SE events (217 out of 227 events) decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), confirming widespread disruption of canonical splicing in \u003cem\u003ernp-6(G281D)\u003c/em\u003e. Gene set enrichment analysis revealed an overrepresentation of metabolic pathway genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), notably those involved in lipid (\u003cem\u003eeppl-1, haao-1, cka-1, pcyt-2.1\u003c/em\u003e) and one-carbon metabolism (\u003cem\u003emetr-1, cbl-1\u003c/em\u003e) (Figure S6a). We also detected an enrichment of transcription factors, including \u003cem\u003eatfs-1\u003c/em\u003e, \u003cem\u003enhr-68\u003c/em\u003e, and \u003cem\u003enhr-114\u003c/em\u003e, which are involved in methionine and lipid metabolism\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Together, these findings implicate aberrant splicing of metabolism-related genes as a driver of \u003cem\u003ernp-6\u003c/em\u003e mutant defects. metabolism\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo pinpoint critical splicing targets, we filtered for metabolism-related genes and transcription factors with robust splicing changes (adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ∆PSI\u0026thinsp;\u0026gt;\u0026thinsp;0.1). This yielded 64 events, primarily involving increased intron retention or exon skipping (49/64; Table S8). Given that intron retention or exon skipping typically impairs gene function\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e, we conducted a targeted RNAi screen of 41 genes representing these events. In particular, we searched for candidates that phenocopied \u003cem\u003ernp-6(G281D)\u003c/em\u003e, that is, decreased body size and/or enhanced \u003cem\u003eY41C4A.32\u003c/em\u003e reporter expression in the wild-type background. In total we identified nine RNAi clones that decreased body size (\u0026gt;\u0026thinsp;5%) and twelve that increased \u003cem\u003eY41C4A.32\u003c/em\u003e reporter expression (\u0026gt;\u0026thinsp;10%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed\u0026ndash;e; Table S9). Of those candidates, six affected both phenotypes, including \u003cem\u003enhr-114\u003c/em\u003e, \u003cem\u003ecbl-1\u003c/em\u003e, and \u003cem\u003eeppl-1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-e). Interestingly, these genes converge on the same metabolic network: \u003cem\u003enhr-114\u003c/em\u003e encodes an ortholog of hepatocyte nuclear factor 4 (HNF4), a key transcriptional factor regulating methionine metabolism and lipid homeostasis in \u003cem\u003eC. elegans\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e; \u003cem\u003ecbl-1\u003c/em\u003e encodes a cystathionine-beta-lyase, which converts cystathionine to homocysteine, feeding into the methionine cycle; \u003cem\u003eeppl-1\u003c/em\u003e encodes an ortholog of ETNPPL ethanolamine-phosphate-phospho-lyase, which affects phosphoethanolamine metabolism.\u003c/p\u003e\u003cp\u003eAmong these candidates, \u003cem\u003enhr-114\u003c/em\u003e showed the strongest effect on both phenotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed\u0026ndash;e, S6b\u0026ndash;d). Deletion of \u003cem\u003enhr-114\u003c/em\u003e causes polyunsaturated fatty acid accumulation and PC depletion, both of which are reversible by VB12 or choline supplementation\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. RT-PCR analyses confirmed that the \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutation increased retention of \u003cem\u003enhr-114\u003c/em\u003e intron 4 by approximately 20% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef\u0026ndash;g). Intron 4 harbors a relatively weak 3\u0026rsquo; splice site, and its inclusion introduces a premature stop codon (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek), likely triggering nonsense-mediated mRNA decay and reducing protein expression. Consistently, both \u003cem\u003enhr-114\u003c/em\u003e mRNA and protein levels were significantly diminished in \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants compared to wild-type controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh-j). Of note, a gain-of-function mutation with \u003cem\u003erbm-39\u003c/em\u003e\u003csup\u003e\u003cem\u003e22\u003c/em\u003e\u003c/sup\u003e, a splicing factor known to interact with \u003cem\u003ernp-6\u003c/em\u003e, markedly suppressed intron 4 retention in \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants (Figure S6d), supporting the notion that \u003cem\u003enhr-114\u003c/em\u003e intron 4 is a direct splicing target of the RNP-6/RBM-39 complex.\u003c/p\u003e\u003cp\u003eTo further elucidate the functional relationship between \u003cem\u003enhr-114\u003c/em\u003e and \u003cem\u003ernp-6\u003c/em\u003e, we reanalyzed publicly available RNA-seq data of the \u003cem\u003enhr-114\u003c/em\u003e null mutant\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e and compared it with the \u003cem\u003ernp-6(G281D)\u003c/em\u003e transcriptome. As reported\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003enhr-114\u003c/em\u003e deletion caused widespread transcriptional changes, altering the expression of 4,235 protein-coding genes (DEseq2, adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, |log\u003csub\u003e2\u003c/sub\u003eFC| \u0026gt;0.5) (Table S10). 483 of these genes were also significantly altered in \u003cem\u003ernp-6(G281D)\u003c/em\u003e (overlap significance, p\u0026thinsp;\u0026lt;\u0026thinsp;5e-12) (Figure S6e\u0026ndash;f; Table S10). In particular, genes related to lipid metabolism were significantly enriched (Figure S6g), including peroxisomal acyl-CoA oxidases (\u003cem\u003eacox-1.2, 1.3\u003c/em\u003e), fatty acid desaturases (\u003cem\u003efat-2, 5, 6, 7\u003c/em\u003e), lipid binding proteins (\u003cem\u003elbp-5, 6, 7\u003c/em\u003e), and phospholipid-remodeling genes (\u003cem\u003eckb-2, acl-12\u003c/em\u003e) (Table S10). Using a less stringent cutoff (adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, |log\u003csub\u003e2\u003c/sub\u003eFC| \u0026gt;0.1), we also observed overlap of one-carbon cycle genes (\u003cem\u003eahcy-1, dao-3\u003c/em\u003e) (Table S10). Less than 4% (18/483) of the overlapped differentially expressed genes displayed aberrant splicing in \u003cem\u003ernp-6\u003c/em\u003e mutants (overlap significance, p\u0026thinsp;\u0026lt;\u0026thinsp;0.102) (Table S10), supporting separable splicing and transcriptional contributions. Strikingly, ~\u0026thinsp;60% (288/483) of the shared genes were rescued by VB12 supplementation in the \u003cem\u003enhr-114\u003c/em\u003e mutant and by BW25113 feeding in the \u003cem\u003ernp-6\u003c/em\u003e mutant (Figure S6h, Table S10), suggesting common downstream pathways. These findings strongly implicate intron retention-induced \u003cem\u003enhr-114\u003c/em\u003e loss-of-function as a key mediator of \u003cem\u003ernp-6(G281D)\u003c/em\u003e transcriptomic changes and suggest that splicing and transcriptional programs converge on the Met/SAM/PC axis to cause the observed phenotypes.\u003c/p\u003e\u003cp\u003eTo directly test this hypothesis, we generated two \u003cem\u003enhr-114\u003c/em\u003e alleles manipulating intron 4 retention: \u003cem\u003enhr-114(i4+)\u003c/em\u003e, with three thymine-to-adenine substitutions at the 3\u0026rsquo; splice site to enhance intron retention, and \u003cem\u003enhr-114(i4\u0026ndash;)\u003c/em\u003e, with complete intron 4 deletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek). RT-PCR verified the intended splicing alterations (Figure S6i-j). As predicted, \u003cem\u003enhr-114(i4+)\u003c/em\u003e phenocopied \u003cem\u003ernp-6(G281D)\u003c/em\u003e, reducing body size, robustly activating the \u003cem\u003eY41C4A.32\u003c/em\u003e reporter, and increasing cold tolerance in the wild-type background, while not further elevating \u003cem\u003eY41C4A.32\u003c/em\u003e expression in \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003em\u0026ndash;o). Conversely, the \u003cem\u003enhr-114(i4\u0026ndash;)\u003c/em\u003e allele alone produced no overt phenotype in wild-type animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003em\u0026ndash;o), but significantly suppressed multiple \u003cem\u003ernp-6(G281D)\u003c/em\u003e phenotypes, including \u003cem\u003eY41C4A.32\u003c/em\u003e reporter expression, cold tolerance, and body size defects (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003em\u0026ndash;o). Taken together, these results provide compelling evidence that intron 4 retention of \u003cem\u003enhr-114\u003c/em\u003e is a major driver of \u003cem\u003ernp-6\u003c/em\u003e mutant pathology.\u003c/p\u003e\u003cp\u003eNext, we asked whether the \u003cem\u003ernp-6\u003c/em\u003e mutation-associated metabolic changes hold true for other splicing factors. Pre-mRNA processing factor 19 (\u003cem\u003ePRPF19/prp-19\u003c/em\u003e) encodes a key component of the spliceosome and is an essential splicing factor whose mutations are associated with developmental delay and neurological defects in humans\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. To determine whether \u003cem\u003eprp-19\u003c/em\u003e deficiency disrupts metabolism, we utilized public RNA-seq datasets\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e to perform transcriptional and splicing analysis. \u003cem\u003eprp-19\u003c/em\u003e disruption altered the expression of 2776 genes (adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, log\u003csub\u003e2\u003c/sub\u003eFC\u0026thinsp;\u0026gt;\u0026thinsp;1) (Figure S7a, Table S11). Similar to the \u003cem\u003ernp-6\u003c/em\u003e mutant, \u003cem\u003enhr-114\u003c/em\u003e and \u003cem\u003eY41C4A.32\u003c/em\u003e mRNA levels were significantly downregulated and upregulated, respectively (Figure S7a). Gene set enrichment analysis identified metabolism as one of the most enriched categories, with the majority of metabolic genes downregulated, including those involved in one-carbon (1CC) (e.g. \u003cem\u003ecbl-1, ahcy-1, mmcm-1\u003c/em\u003e) and phospholipid metabolism (e.g. \u003cem\u003eckb-4\u003c/em\u003e and \u003cem\u003epcyt-2.1\u003c/em\u003e) (Figure S7b-c). 1184 splicing events were significantly altered (adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ΔPSI\u0026thinsp;\u0026gt;\u0026thinsp;5%) (Figure S7d, Table S12). In line with the DEGs, the metabolism category was enriched among the DSGs, including genes related to 1CC metabolism (e.g. \u003cem\u003ecblc-1\u003c/em\u003e, \u003cem\u003emetr-1\u003c/em\u003e and \u003cem\u003esams-1\u003c/em\u003e) and phospholipid metabolism (e.g. \u003cem\u003eckc-1\u003c/em\u003e and \u003cem\u003epcyt-2.1\u003c/em\u003e) (Figure S7e-f). Of note, \u003cem\u003eprp-19\u003c/em\u003e depletion also significantly increased \u003cem\u003enhr-114\u003c/em\u003e intron retention (Figure S7f), suggesting that aberrant \u003cem\u003enhr-114\u003c/em\u003e splicing may represent a shared mechanism underlying distinct spliceosomeopathies. RNAi-mediated knockdown of \u003cem\u003eprp-19\u003c/em\u003e significantly induced \u003cem\u003eY41C4A.32::mNG\u003c/em\u003e reporter expression and reduced body size, whereas boosting 1CC metabolism through methionine supplementation rescued both phenotypes (Figure S7g-i). These findings together suggest that splicing factor inhibition may converge on shared downstream pathways, triggering similar metabolic responses. Dysregulated metabolism may thus represent a common feature of spliceosome-related diseases.\u003c/p\u003e\u003cp\u003eLastly, we explored the physiological regulation of \u003cem\u003enhr-114\u003c/em\u003e splicing. Dietary restriction (DR) decreases SAM levels in flies and mice\u003csup\u003e\u003cspan additionalcitationids=\"CR68\" citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e, and DR-induced longevity is mediated by \u003cem\u003esams-1\u003c/em\u003e in \u003cem\u003eC. elegans\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e,\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. Further, nutrient-sensing pathways have been shown to regulate splicing and metabolism\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. We hypothesized that nutrient availability modulates SAM metabolism partially through \u003cem\u003enhr-114\u003c/em\u003e splicing. We therefore investigated \u003cem\u003eC. elegans\u003c/em\u003e adult reproductive diapause (ARD), a state induced by long-term fasting and associated with downregulation of RNA processing complexes\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e,\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. Under ARD conditions \u003cem\u003enhr-114\u003c/em\u003e intron 4 retention was significantly increased by ~\u0026thinsp;10% relative to fed controls (Figure S7k), accompanied by reduced levels of \u003cem\u003enhr-114\u003c/em\u003e mRNA (Figure S7l). These findings suggest that nutrient status modulates \u003cem\u003enhr-114\u003c/em\u003e splicing and the associated SAM/phospholipid metabolic network.\u003c/p\u003e\u003cp\u003e\u003cb\u003eVitamin B\u003c/b\u003e\u003csub\u003e\u003cb\u003e12\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eactivates mTOR signaling to rescue\u003c/b\u003e \u003cb\u003ernp-6(G281D)\u003c/b\u003e \u003cb\u003emutant defects\u003c/b\u003e\u003c/p\u003e\u003cp\u003emTOR signaling orchestrates key processes of development, growth, and metabolism\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. Previously, we showed that \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutation inhibits mTORC1 activity when worms are fed OP50\u003csup\u003e22\u003c/sup\u003e. Here, we asked whether VB12 supplementation could restore mTORC1 function in these mutants. To this end, we (i) measured phosphorylation of AMPK, a cellular energy sensor inversely related to mTORC1 activity, and (ii) monitored nuclear localization of HLH-30, the \u003cem\u003eC. elegans\u003c/em\u003e ortholog of TFEB, whose nuclear translocation is suppressed by active mTORC1 signaling\u003csup\u003e\u003cspan additionalcitationids=\"CR77\" citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. Consistent with mTORC1 inhibition, \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants on OP50 exhibited elevated AMPK phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea\u0026ndash;b) and increased HLH-30 nuclear localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec\u0026ndash;d). Remarkably, VB12 supplementation reversed both phenotypes, indicating a reactivation of mTORC1 signaling.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, we wondered whether mTORC1 activity is required for rescue of \u003cem\u003ernp-6(G281D)\u003c/em\u003e growth defects by VB12/Met/SAM/PC. Loss-of-function mutation of \u003cem\u003eraga-1\u003c/em\u003e, a Rag GTPase essential for mTORC1 activation, further reduced the body size of \u003cem\u003ernp-6\u003c/em\u003e mutants and completely abolished the beneficial effects of K12-type \u003cem\u003eE. coli\u003c/em\u003e, as well as VB12, methionine, and choline supplementation on body size in \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). A \u003cem\u003eraga-1\u003c/em\u003e single-copy insertion\u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e rescued body size in the \u003cem\u003eraga-1(lof);rnp-6\u003c/em\u003e double mutant on OP50 and fully reinstated the rescue of body size upon VB12, methionine or choline supplementation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Altogether, these findings demonstrate that intact mTORC1 signaling is critical for mediating the rescue effect of VB12 and related metabolic interventions in \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePUF60 deficiency impairs 1CC and phospholipid metabolism in human cells\u003c/h2\u003e\u003cp\u003eSince RNP-6 deficiency induces aberrant splicing of 1CC and phospholipid metabolism genes in \u003cem\u003eC. elegans\u003c/em\u003e, we wondered whether PUF60 deficiency has a similar effect in human cells. We mined public datasets in which the human lung adenocarcinoma cell line PC9 was subjected to PUF60 siRNA\u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. Based on our findings in worms, we reasoned that splicing defects would remain unchanged even when cells were cultured in the presence of VB12 and methionine-rich medium. We re-analyzed the data and identified 2599 splicing events (adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Δpsi\u0026thinsp;\u0026gt;\u0026thinsp;0.1) corresponding to 1730 genes (Figure S8a, Table S13). KEGG analysis of the aberrant spliced genes revealed the significant enrichment of metabolic pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, Table S14). These include genes related to propanoate metabolism (\u003cem\u003ee.g.\u003c/em\u003e, ethylmalonyl-CoA decarboxylase 1 \u003cem\u003eECHDC1/C32E8.9\u003c/em\u003e, methylmalonyl-CoA epimerase \u003cem\u003eMCEE/mce-1\u003c/em\u003e), choline metabolism (\u003cem\u003ee.g.\u003c/em\u003e, choline kinase alpha \u003cem\u003eCHKA/cka-2\u003c/em\u003e), glycerophospholipid metabolism (diacylglycerol kinase \u003cem\u003eDGKQ/dgk-1\u003c/em\u003e, phosphocholine cytidylyltransferase \u003cem\u003ePCYT1A/pcyt-1\u003c/em\u003e, phosphatidate phosphatase \u003cem\u003eLPIN2/lpin-1\u003c/em\u003e), cysteine and methionine metabolism (methionine synthase \u003cem\u003eMTR/metr-1\u003c/em\u003e, kynurenine aminotransferase 1 \u003cem\u003eKYAT1/nkat-1\u003c/em\u003e) and VB12 transport (\u003cem\u003eABCD4/pmp-5\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). These results together imply that PUF60 deficiency might dysregulate 1CC and phospholipid metabolism through altered splicing.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eLast, we sought to understand if the \u003cem\u003ePUF60\u003c/em\u003e pathogenic mutation also affects metabolism in VRJS patients. To this end, we collected blood samples from 8 Verheij syndrome patients (6 males and 2 females) and 3 age-matched non-\u003cem\u003ePUF60\u003c/em\u003e mutation controls (1 male and 2 females) and performed metabolomic and lipidomic analyses with plasma (Figure S8b). In total 103 metabolites and 653 lipid species belonging to 15 lipid groups were annotated (Table S15-16). Although variable, the VRJS samples clustered differently from controls in PCA plots (Figure S8c). Out of the 103 metabolites, 30 were significantly altered (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, log2FC\u0026thinsp;\u0026gt;\u0026thinsp;0.2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). Strikingly, tryptophan and methionine were among the most significantly changed metabolites, decreased by ~\u0026thinsp;90% and ~\u0026thinsp;85% in VRJS, respectively (Figure S8d-e). A handful of metabolites involved in TCA cycle, such as citrate, malate and fumarate, were upregulated in VRJS patients (Figure S8f-h). We were unable to quantify SAM or SAH as they were not detected in our plasma samples, due to low abundance. Levels of TG and LPE were significantly decreased, while the abundance of PC-O, hexose ceramide, ceramide and sphingomyelin lipid were significantly increased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). Although the levels of PC remained unchanged, the levels of PE and the ratio of PC/PE were slightly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). When we checked individual lipids, 94 were significantly changed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, log2FC\u0026thinsp;\u0026gt;\u0026thinsp;0.2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). Consistent with the group analysis, most of the significantly altered PE and LPE were downregulated, while PC-O, sphingomyelin, ceramide and hexose ceramide were upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). Interestingly, we found that, similarly to \u003cem\u003eC. elegans\u003c/em\u003e, the significantly upregulated TG lipids contained longer and more unsaturated fatty acid chains (59 carbons, 11 double bonds on average) than downregulated TGs (48 carbons, 2.5 double bonds on average) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef). Taken together, our results from human cell lines and patient plasma provide evidence that PUF60 deficiency might dysregulate sulfur amino acid and phospholipid metabolism.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eVerheij syndrome remains an exceptionally rare and poorly understood genetic disorder with no targeted treatments to date. Albeit relatively simple, \u003cem\u003eC. elegans rnp-6\u003c/em\u003e hypomorphic mutants recapitulate multiple features of VRJS such as smaller body size, growth delay, immune alterations, and neurological defects\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e, highlighting the nematode as a powerful system for dissecting the molecular and cellular mechanisms of PUF60 insufficiency-associated pathologies. Leveraging the \u003cem\u003eC. elegans rnp-6\u003c/em\u003e mutant as an \u003cem\u003ein vivo\u003c/em\u003e disease model, we found that VRJS-associated growth defects arise primarily from SAM/SAH and PE/PC imbalance. The metabolic impairments are cumulatively driven by aberrant splicing of one-carbon and phospholipid metabolism-related genes (such as \u003cem\u003emetr-1\u003c/em\u003e, \u003cem\u003ecbl-1\u003c/em\u003e, \u003cem\u003eeppl-1\u003c/em\u003e) and \u003cem\u003enhr-114\u003c/em\u003e, a pivotal transcription factor of one-carbon and phospholipid metabolism. Reconstitution of SAM/SAH and PE/PC balance through supplementation of VB12, methionine, choline, or VB12-enriched bacteria can robustly rescue development (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg). These interventions mechanistically converge on reactivating mTORC1 signaling, a key driver of development, establishing a direct link between splicing factor dysfunction, metabolic rewiring, and nutrient-sensing signaling networks. To our knowledge, this is the first study to integrate the aforementioned metabolic dysregulation with spliceosomopathies, and to identify vitamin B\u003csub\u003e12\u003c/sub\u003e as a candidate therapeutic for VRJS-like pathologies.\u003c/p\u003e\u003cp\u003e\u003cem\u003ernp-6\u003c/em\u003e mutation disrupts expression and splicing of diverse metabolism-related genes (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2). By combining targeted RNAi screening, genetic epistasis analysis, and transcriptomic profiling, we identify aberrant intron retention of \u003cem\u003enhr-114\u003c/em\u003e as a critical node linking RNP-6 perturbation to downstream transcriptional and metabolic imbalances, thereby driving defects in \u003cem\u003ernp-6\u003c/em\u003e mutants. As \u003cem\u003enhr-114\u003c/em\u003e/\u003cem\u003eHNF4\u003c/em\u003e homologs govern pivotal aspects of methionine and lipid homeostasis across species\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e,\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e, our findings offer a mechanistic bridge connecting spliceosomal dysfunction with metabolic dysregulation. Moreover, the novel observation that \u003cem\u003enhr-114\u003c/em\u003e splicing is dynamically regulated by both genetic perturbation and physiological states (such as fasting-induced diapause) further broadens the connection between nutrient-responsive control of RNA processing and cellular metabolism.\u003c/p\u003e\u003cp\u003eVB12 is an essential micronutrient critical for human health, particularly in preventing anemia and neurological dysfunction\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Clinically, VB12 supplements address malabsorption disorders such as pernicious anemia and gastrointestinal disorders\u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e. Beyond these classic roles, emerging studies demonstrate that VB12 enhances somatic cell reprogramming and tissue repair in mammalian models\u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eC. elegans\u003c/em\u003e, VB12 deficiency induces severe phenotypes, including cognitive impairment, growth retardation, infertility, and shortened lifespan\u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e,\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e. Supplementation of VB12 mitigates amyloid-beta and dithiothreitol-induced toxicity in \u003cem\u003eC. elegans\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e,\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e, highlighting its essential role in metabolic homeostasis. Our work extends these insights by demonstrating that VB12 supplementation rescues RNP-6 deficiency-associated spliceosomopathy, confirming the critical function of VB12 under both physiological and disease conditions.\u003c/p\u003e\u003cp\u003eBeyond the central role of SAM/SAH balance and phospholipid metabolism, our data also implicate additional metabolic pathways and organelles as contributors to VRJS-like defect manifestations. For example, metabolites related to the tricarboxylic acid (TCA) cycle (e.g., malate, phosphoenolpyruvate) and mitochondrial β-oxidation (propionylcarnitine and butyrylcarnitine) are reduced in \u003cem\u003ernp-6\u003c/em\u003e mutants, implying mitochondrial dysfunction. Supporting this, the mitochondrial stress reporter \u003cem\u003ehsp-6\u003c/em\u003e is strongly induced in \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants and fully suppressed by the K12-type \u003cem\u003eE. coli\u003c/em\u003e strain BW25113 (Figure S9a\u0026ndash;b). This is also consistent with previous reports that link VB12 supplementation to improved mitochondrial function\u003csup\u003e\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e,\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u003c/sup\u003e. Moreover, crucial metabolites in the \u003cem\u003ede novo\u003c/em\u003e NAD\u003csup\u003e+\u003c/sup\u003e synthesis pathway, such as kynurenine and tryptophan, are reduced in \u003cem\u003ernp-6\u003c/em\u003e mutants and restored by BW25113 \u003cem\u003eE. coli\u003c/em\u003e or VB12, hinting at dysregulated NAD\u003csup\u003e+\u003c/sup\u003e metabolism in \u003cem\u003ernp-6(G281D)\u003c/em\u003e pathology. Additionally, metabolites associated with the glucosamine pathway appear dysregulated. A unifying notion is that \u003cem\u003ernp-6\u003c/em\u003e mutation induces a catabolic state of lower methylation capacity, mitochondrial function, and glucose metabolism, while VB12 reverses these features and re-establishes a more anabolic state, consistent with the activation of mTOR signaling.\u003c/p\u003e\u003cp\u003eOf particular interest is the induction of the integrated stress response in \u003cem\u003ernp-6\u003c/em\u003e mutants. The ISR is a conserved pathway for eukaryotic cells that limits protein synthesis and secretion to cope with diverse stresses\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. The impact of the splicing machinery on ISR has been little explored, though a recent investigation shows that aberrant splicing of protein translation genes induces the ISR and inflammation-related gene expression in acute myeloid leukemia patients\u003csup\u003e\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e\u003c/sup\u003e. Our results provide direct evidence that splicing factor dysfunction triggers the ISR in \u003cem\u003eC. elegans\u003c/em\u003e, in this case through phospholipid remodeling. Further research should help elucidate the detailed mechanisms.\u003c/p\u003e\u003cp\u003eOur study demonstrates that VB12 supplementation activates mTORC1 signaling to rescue growth and metabolic defects in the \u003cem\u003ernp-6\u003c/em\u003e mutant, revealing a novel mechanistic link between micronutrient status and spliceosomal dysfunction. Previous work showed that methylcobalamin, a VB12 analog, activates mTOR via Akt to promote neuronal outgrowth, supporting VB12\u0026rsquo;s capacity to stimulate mTOR in a cellular context\u003csup\u003e\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e\u003c/sup\u003e. In contrast, recent studies report VB12 inhibits mTOR phosphorylation to induce autophagy in disease models\u003csup\u003e\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e\u003c/sup\u003e. Our data uniquely position VB12-induced mTORC1 activation as an anabolic rescue mechanism critical for correcting spliceosomal metabolic defects. Although mTOR activation typically limits longevity in \u003cem\u003eC. elegans\u003c/em\u003e, the lack of lifespan limitation by VB12 may reflect tissue-specific effects of mTORC1, which restricts lifespan in neurons but promotes growth in somatic tissues\u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. We speculate that VB12 preferentially activates mTORC1 in non-neuronal tissues to support development without affecting longevity. Dissecting the tissue-specific roles of RNP-6/NHR-114 signaling, SAM/SAH\u0026ndash;PC/PE metabolism, and mTOR within this metabolic network will be important to elucidate. While K12-type \u003cem\u003eE. coli\u003c/em\u003e (BW25113 and HT115) and VB12 exert largely overlapping effects in \u003cem\u003ernp-6(G281D)\u003c/em\u003e mutants, the bacterial diet may also regulate physiology through VB12-independent mechanisms. For instance, K12-type \u003cem\u003eE. coli\u003c/em\u003e suppresses \u003cem\u003ernp-6(G281D)\u003c/em\u003e-associated longevity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), whereas VB12 treatment shows little effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg), suggesting bacterial metabolites or lipids beyond VB12 contribute to lifespan regulation\u003csup\u003e\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e\u003c/sup\u003e. Together, our findings highlight the pleiotropic and context-dependent modulation of mTOR by VB12 and advance current paradigms by identifying mTORC1 as a promising therapeutic target in spliceosomal diseases.\u003c/p\u003e\u003cp\u003eOur analysis of splicing changes in the PC9 cell line suggests that RNP-6/PUF60 might play conserved roles in regulating methionine and phospholipid metabolism, as we observed enrichment of aberrantly spliced genes in choline, propanoate, and methionine/cysteine metabolism. Though we did not find (significant) splicing changes in \u003cem\u003eHNF4\u003c/em\u003e (functional homolog of \u003cem\u003enhr-114)\u003c/em\u003e, we did see splicing changes in \u003cem\u003eNR1H3\u003c/em\u003e (Table S13) a related nuclear receptor that also regulates lipid metabolism\u003csup\u003e\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e\u003c/sup\u003e, suggesting that RNP-6 and PUF60 may affect similar and different splicing targets, dependent on cell type. It is also interesting to note that mTOR signaling pathway is significantly enriched in the PUF60-dependent splicing genes, including NPRL2, NPRL3, RPS6KB2, RPS6KA3 and TSC1 (Table S14). The aberrant splicing with mTOR complex core components might contribute to mTORC1 inhibition under PUF60 deficiency conditions\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. By performing metabolic and lipidomic assays in VRJS plasma, we provide evidence that the PUF60 pathogenic mutation might dysregulate methionine/cysteine and phospholipid metabolism. Of note, some metabolites (such as cysteine, tryptophan, PEP, pantothenate, adenine and long chain TGs), were changed in the same direction in \u003cem\u003ernp-6\u003c/em\u003e mutants and VRJS patients, while other metabolites (such as citrate, malate and fumarate) and lipids (such as PE, hexose ceramide, sphingomyelin lipid and PC/PE ratio) were changed in the opposite direction. Despite these discrepancies, these findings suggest that similar pathways are dysregulated, while the directionality could reflect different metabolic/lipidomic profiles of plasma versus tissues\u003csup\u003e\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e,\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e\u003c/sup\u003e. Moreover, as the plasma samples were obtained from subjects of different ages and genders, this may also partially contribute to the observed changes. It will be important to validate these results in plasma and patient-derived cells (such as fibroblasts) with a larger cohort (age and gender matched) and test the effect of VB12 supplementation.\u003c/p\u003e\u003cp\u003eSplicing is a highly coordinated process that involves hundreds of splicing factors\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. RNP-6/PUF60 functionally and physically interacts with many other splicing factors acting at various stages of spliceosome assembly\u003csup\u003e\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e\u003c/sup\u003e. Mutations in several of these splicing factors, including PRP-19/PRPF19\u003csup\u003e65\u003c/sup\u003e, UAF-1/U2AF2\u003csup\u003e65,99\u003c/sup\u003e, PRP-8/PRPF8\u003csup\u003e100\u003c/sup\u003e, RSP-7/SRSF11\u003csup\u003e101\u003c/sup\u003e and RBM-5/RBM10\u003csup\u003e102\u003c/sup\u003e, have been linked to VRJS-like neurodevelopmental defects in humans, suggesting convergent pathomechanisms. Our data on \u003cem\u003eprp-19\u003c/em\u003e and other splicing factors (Figure S7) indicate that dysregulation of the SAM/SAH balance and phospholipid metabolism is not restricted to PUF60/RNP-6-associated VRJS. The metabolic imbalance identified here may represent a shared downstream pathway regulated by distinct spliceosomal components. It will be of great interest to comprehensively test this hypothesis across human spliceosomopathies and evaluate the therapeutic potential of VB12 supplementation. Furthermore, given the neurological manifestations in VRJS and other spliceosomopathies\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, assessing VB12\u0026rsquo;s effects on neuronal functions constitutes a critical direction for future research.\u003c/p\u003e\u003cp\u003eIn summary, our results illuminate a previously unappreciated metabolic bottleneck underlying RNP-6/PUF60 deficiency and warrant the need for further investigation of vitamin B\u003csub\u003e12\u003c/sub\u003e supplementation as a therapeutic strategy for Verheij syndrome.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eC. elegans\u003c/b\u003e \u003cb\u003estrains and maintenance\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWorms were maintained at 20\u0026deg;C following standard procedures\u003csup\u003e\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e\u003c/sup\u003e. Detailed information for strains used in this study are found in Supplementary Table S17. All mutant strains obtained from CGC and Sunybiotech (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.sunybiotech.com\u003cspan address=\"http://www.sunybiotech.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were outcrossed with our N2 at least twice. Detailed information regarding CRISPR gene editing can be provided upon request. For all experiments, worm synchronization was done by egg laying.\u003c/p\u003e\n\u003ch3\u003eCompound supplementation assay\u003c/h3\u003e\n\u003cp\u003eFor supplementation of methionine (L-methionine, Sigma-Aldrich: M9625) and choline (choline chloride, Thermo Fisher: A15828.22), methionine/choline was added to NGM medium before pouring to a final concentration of 10 mM. OP50 was seeded on these plates two days before worms lay eggs. For vitamin B\u003csub\u003e12\u003c/sub\u003e supplementation, VB12 (Cyanocobalamin, Sigma-Aldrich: V6629) was mixed with OP50 bacteria and seeded on NGM plates. Worms were cultured on these plates from the egg stage onwards, unless noted otherwise. For the OP50-BW25113 mixture assay, OP50 and BW25113 were cultured to log phase, washed with M9 and concentrated to the same OD600 value. Then OP50 and BW25113 was mixed at different ratios and seeded on peptone-free NGM plates to avoid bacterial growth. Worm images were taken at the young adult stage. For the tunicamycin response assay, tunicamycin (Sigma-Aldrich: T7765) stock solution was added on top of seeded NGM plates (final concentration 2 ug/ml) with synchronized young adult stage worms. After overnight treatment (~\u0026thinsp;16 h), worms were anesthetized with sodium azide (50 mM) and imaged.\u003c/p\u003e\n\u003ch3\u003eWorm imaging\u003c/h3\u003e\n\u003cp\u003eAnalysis of worm reporters Y41C4A.32::mNeonGreen and worm size were performed on a Leica stereo microscope (Leica M165 FC, LAS X) with Leica DFC3000G CCD camera. Analysis of mTOR reporter HLH-30::mNeonGreen was performed on a Zeiss Axioplan2 microscope (Axio Vision SE64, Rel.4.9.1) with a Zeiss AxioCam 506 CCD camera. Fiji software (Version 2.0.0/1.52p)\u003csup\u003e104\u003c/sup\u003e was used for quantifying fluorescent intensity and worm area. For HLH-30::mNeonGreen images, the nuclei of hypodermal cells were selected for quantification. To reduce bias, worms were randomly picked under a dissection microscope and imaged. At least 20 worms per genotype were picked for imaging and all the experiments were independently carried out at least three times unless otherwise indicated.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eDevelopmental rate assay\u003c/h2\u003e\u003cp\u003eWorms were synchronized by short-term (1h) egg lay on the indicated plates. After 48 hours of growth, ~\u0026thinsp;15 worms from each condition were randomly singled to 3 cm NGM plates. After 12 hours, each worm was checked every hour until it laid the first egg. Experiments were repeated at least three times.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBrood size assay\u003c/h3\u003e\n\u003cp\u003eWorms were synchronized by short term (1h) egg lay on the indicated plates. After 48 hours of growth, ~\u0026thinsp;15 L4-stage worms of each condition were singled to 3 cm NGM plates. Worms were transferred every day until egg laying stopped. The total number of hatched larval worms was counted. Experiments were repeated at least three times.\u003c/p\u003e\n\u003ch3\u003eCold tolerance assay\u003c/h3\u003e\n\u003cp\u003eWorms were synchronized and grown on the indicated plates. When the worms reached the young adult stage, they were transferred to a 2\u0026deg;C incubator for 24 hours. Worms were recovered at room temperature for 4 hours and the number of alive and dead worms was scored. Cold survival ratio was measured as the ratio of the number of live worms to the number of total worms. At least 60 worms from each genotype were used in the assay for each replicate. Experiments were repeated at least three times.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eLifespan assay\u003c/h2\u003e\u003cp\u003eAll lifespans were performed at 20\u0026deg;C with the indicated diet or treatment condition for the whole life. Worms were allowed to grow to the young adult stage on standard NGM plates. ~150 young adults were transferred to NGM plates supplemented with 10 \u0026micro;M of FUdR. Survival was monitored every other day. Worms that did not respond to gentle touch by a worm pick were scored as dead and were removed from the plates. Animals that crawled off the plate or had ruptured vulva phenotypes were censored. All lifespan experiments were blinded and performed at least three times. Graphpad Prism (9.0.0) was used to plot survival curves. Survival curves were compared, and p-values were calculated using the log-rank (Mantel-Cox) analysis method. Complete lifespan data are found in Supplementary Table S18.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eRNA interference\u003c/h2\u003e\u003cp\u003eRNAi experiments were performed as previously described\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eE. coli\u003c/em\u003e HT115 (high VB12) and \u003cem\u003eE. coli OP50(xu363)\u003c/em\u003e (low VB12) bacterial strains were used in this study. HT115 bacteria came from the Vidal or Ahringer library. \u003cem\u003eOP50(xu363)\u003c/em\u003e competent bacteria were transformed with dsRNA expression plasmids, which were extracted from the respective HT115 bacterial strains. The RNAi bacteria were grown in LB medium supplemented with 100 \u0026micro;g/mL ampicillin at 37 ̊C to reach log phase, washed with fresh medium, and concentrated 5-fold. Bacteria was then spread on RNAi plates, which are NGM plates containing 100 \u0026micro;g/mL ampicillin and 0.4 mM isopropyl \u0026szlig;-D-1-thiogalactopyranoside (IPTG). dsRNA-expressing bacteria were grown on plates at room temperature for two days. RNAi was initiated by letting the animals feed on the desired RNAi bacteria. Luciferase (L4440::luc, i.e., luci) RNAi vector was used as a non-targeting control in all experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eKeio library screen\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003ernp-6(dh1127);Y41C4A.32(syb2725)\u003c/em\u003e reporter strain was used for screening. The \u003cem\u003eE. coli\u003c/em\u003e Keio knockout collection comprised two independent deletion strains for each of the 3985 nonessential genes, yielding a total of 7970 strains. All strains were tested as follows: briefly, Keio library bacteria were inoculated in 96-well plates overnight in LB (+\u0026thinsp;kanamycin) medium and seeded on 3 cm NGM plates. After two days of growth, ~\u0026thinsp;30 synchronized worm eggs (generated by bleaching) were seeded on the plates. The worms were scored after three days. For the primary screen, the plates were checked manually under a Leica stereo microscope (Leica M165 FC, LAS X). Mutant bacterial strains that resulted in more than 3 worms showing a bright mNeonGreen signal were marked and selected for further validation. In the confirmation screen, OP50 and the parental BW25113 strain were used as positive and negative control, respectively. Images of worms were captured for fluorescence intensity quantification. Bacterial hits were confirmed by Sanger sequencing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eEMS mutagenesis screen\u003c/h2\u003e\u003cp\u003eEMS mutagenesis was performed as described previously\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Briefly, ~\u0026thinsp;1,000 synchronized L4 larvae worms of \u003cem\u003ernp-6(dh1127);Y41C4A.32(syb2725)\u003c/em\u003e reporter strain were exposed to 0.15% ethyl methanesulfonate in M9 buffer for 4h at room temperature, and then washed and transferred to normal NGM plates for recovery. After overnight growth, P0 adult animals were transferred to new plates seeded with OP50 for egg laying. After 3 days of growing, adult F1 worms were bleached, and eggs were seeded onto NGM plates seeded with BW25113 bacteria. After 3- and 4-day growth, the plates were scored, and F2 worms that showed a bright fluorescence signal were singled to individual NGM plates. To exclude the false positive hits, the reporter fluorescence intensity of mutants growing on OP50 or BW25113 was measured, and the rescue efficiency (RE) of BW25113 diet was calculated. The mutants with RE value less than 0.5 were selected for further characterizations. The \u003cem\u003ernp-6(dh1127);Y41C4A.32(syb2725)\u003c/em\u003e animals were used as negative control in all the assays. To map causative mutations, Hawaiian-SNP mapping and whole genome sequencing were used as previously described\u003csup\u003e\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e\u003c/sup\u003e. In brief, EMS mutants were crossed with Hawaiian strain (CB4856) males. The non-fluorescent F1 worms were picked, raised until adulthood, and allowed to lay eggs on NGM plates seeded with BW25113. Fluorescent F2 adult worms were singled. After 5-days of growth, worms were then pooled together, and their genomic DNA purified using Gentra Puregene Kit (Qiagen). The pooled DNA was sequenced on an Illumina HiSeq platform (paired-end 150 nucleotide). MiModD pipeline (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.celegans.de/en/mimodd\u003c/span\u003e\u003cspan address=\"http://www.celegans.de/en/mimodd\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to narrow down the causative mutations. The WBcel235/ce11 \u003cem\u003eC. elegans\u003c/em\u003e assembly was used as a reference genome. Causative mutations were confirmed by multiple outcrosses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eAID degradation experiments\u003c/h2\u003e\u003cp\u003eThe AID inducible-degradation system was designed following previously described protocols \u003csup\u003e\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e\u003c/sup\u003e. Degron and GFP were inserted at the N-terminus of endogenous RNP-6. The transgenic line was crossed with TIR1-expression strain to generate the inducible-degradation strain AA5498 (\u003cem\u003ernp-6\u003c/em\u003e(\u003cem\u003esyb6010\u003c/em\u003e [degron::3XFLAG::GFP::RNP-6]);Si57 [Peft-3::TIR1::mRuby::unc-54 3'UTR\u0026thinsp;+\u0026thinsp;Cbr-unc-119(+)]). To induce RNP-6 degradation, strain AA5498 was grown on plates supplemented with different concentrations of Potassium Naphthaleneacetic Acid (K-NAA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eRNA extraction and cDNA synthesis\u003c/h2\u003e\u003cp\u003e\u003cem\u003eC. elegans\u003c/em\u003e were lysed with QIAzol Lysis Reagent. RNA was extracted using chloroform extraction. Samples were then purified using RNeasy Mini Kit (Qiagen). Purity and concentration of the RNA samples were assessed using a NanoDrop 2000c (peqLab). cDNA synthesis was performed using iScript cDNA synthesis kit (Bio-Rad). Standard manufacturer protocols were followed for all mentioned commercial kits.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eRNA-Seq and bioinformatic analysis\u003c/h2\u003e\u003cp\u003e1 \u0026micro;g of total RNA was used per sample for library preparation. The protocol of Illumina Tru-Seq stranded RiboZero was used for RNA preparation. After purification and validation (2200 TapeStation; Agilent Technologies), libraries were pooled for quantification using the KAPA Library Quantification kit (Peqlab) and the 7900HT Sequence Detection System (Applied Biosystems). The libraries were then sequenced with Illumina HiSeq4000 sequencing system using the paired-end 2\u0026times;100 bp sequencing protocol. For data analysis, Wormbase genome (WBcel235_89) was used for alignment of the reads. Kallisto was used to map raw reads to reference transcriptome and quantify transcript abundance\u003csup\u003e\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e\u003c/sup\u003e. DESeq2 was used for each pairwise comparison\u003csup\u003e\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e\u003c/sup\u003e. For the splicing analysis, SAJR\u003csup\u003e\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e\u003c/sup\u003e and IRFinder\u003csup\u003e110\u003c/sup\u003e pipelines were used. The significant events from different pipelines were combined, and the unique events were kept for further analysis. Row Z-score heatmaps were generated by using the iHeatmap function from FLASKI (Version 2.0.0) (DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.5281/zenodo.5254193\u003c/span\u003e\u003cspan address=\"10.5281/zenodo.5254193\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Adjusted p-value or q-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 is considered to be significant for gene expression and splicing. Gene enrichment visualization was performed with WormCat 2.0\u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eAlternative splicing PCR assay (RT-PCR)\u003c/h2\u003e\u003cp\u003eA new batch of samples (different from the samples used for RNAseq) was used for RNA preparation and cDNA synthesis. Phusion Polymerase (Thermo Fisher) was used to amplify the \u003cem\u003etos-1, tcer-1 and nhr-114\u003c/em\u003e segments. PCR reactions were cycled 30 times with an annealing temperature of 53\u0026deg;C. RT-PCR products were visualized with ChemiDoc Imager (BioRad, ChemiDoc MP, Image Lab 6.1) after staining with Roti-GelStain (Carl Roth). Sequences of the primers used in the RT\u0026ndash;PCR assays are found in Supplementary Table S17.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eQuantitative reverse transcription PCR (RT\u0026ndash;qPCR)\u003c/h2\u003e\u003cp\u003ePower SYBR Green master mix (Applied Biosystems) was used for RT-qPCR experiments. A JANUS automated workstation (PerkinElmer) was used for pipetting the reagents and cDNA samples into a 384 well plate. Thermal cycling was performed using a ViiA7 384 Real-Time PCR System machine (Applied Biosystems). \u003cem\u003eact-1\u003c/em\u003e and \u003cem\u003ecdc-42\u003c/em\u003e were used for internal normalization. Relative expression levels were calculated using the comparative CT method. Sequences of the primers used in the RT\u0026ndash;qPCR assays are provided in Supplementary Table S17.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eWestern blot\u003c/h2\u003e\u003cp\u003eFor \u003cem\u003eC. elegans\u003c/em\u003e samples, animals were first washed with M9 buffer. Worm pellets were resuspended in 4% SDS buffer (4% SDS in 0.1 M Tris/HCl pH 8 with 1 mM EDTA) supplemented with cOmplete Protease Inhibitor (Roche) and PhosSTOP (Roche) and snap frozen in liquid nitrogen. Thawed samples were lysed using Bioruptor Sonication System (Diagenode), centrifuged (20,000g, 10 min) and protein concentrations were measured with Pierce BCA kit. Protein samples were then heated to 95 ̊C for 10 min in Laemmli buffer with 0.8% 2-mercaptoethanol in order to denature proteins. 10 ug protein samples were loaded on 4\u0026ndash;15% Mini PROTEAN TGXTM Precast Protein Gels (Bio-Rad), and electrophoresis was performed at a constant voltage of 200V for around 40 min. After separation, the proteins were transferred to PVDF membranes using Trans-Blot TurboTM Transfer System (BioRad). 5% bovine serum albumin (BSA) or 5% milk in Tris-buffered Saline and Tween20 (TBST) were used for blocking of the membranes. After antibody incubations (anti-HA 1:1000, anti-Phospho-AMPKα (Thr172) 1:2000, anti-beta Actin 1:5000, Anti-Mouse HRP 1:5000, Anti-Rabbit HRP 1:5000 and Anti-Rat HRP 1:5000) and washing with TBST buffer, imaging of the membranes was performed with ChemiDoc Imager (BioRad, ChemiDoc MP, Image Lab 6.1). Western Lightning Plus Enhanced Chemiluminescence Substrate (PerkinElmer) was used as the chemiluminescence reagent. A list of antibodies is provided in Supplementary Table S17.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eMetabolomics and lipidomics sample preparation\u003c/h2\u003e\u003cp\u003eWorms were synchronized by egg laying and collected when they reached the young adult stage. For each sample, ~\u0026thinsp;2000 worms were washed three times with ddH₂O, snap-frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C before use. For the metabolite/lipid extraction, 500 \u0026micro;L extraction buffer (MTBE:MeOH:H2O, 50:30:20) supplemented with internal standards was added to each sample. Worm pellets were homogenized with ~\u0026thinsp;100 \u0026micro;L 1 mm zirconia beads using a TissueLyser (Qiagen) at 50 Hz and 4\u0026deg;C for 20 min. After initial homogenization, 500 \u0026micro;L extraction buffer was added to each sample and homogenization resumed for 5 min. Worm lysates were centrifuged at 21000 x g and 4\u0026deg;C for 10 min. Supernatant was transferred to new tubes. Residual buffer was removed before drying protein pellets under a fume hood overnight. Protein concentration was determined using a BCA kit (ThermoFisher). The cleared supernatant was mixed with 200 \u0026micro;L MTBE (Sigma) and 150 \u0026micro;L H2O, and incubated at 15\u0026deg;C for 10 min. Samples were centrifuged at 15\u0026deg;C and 16000 x g for 10 min to obtain phase separation (top lipid phase, bottom polar metabolites phase). 650 \u0026micro;L of the lipid phase was transferred to new tubes and dried in a SpeedVac concentrator at 20\u0026deg;C and 1000 rpm for ~\u0026thinsp;2 h. 600 \u0026micro;L polar phase solution was transferred to new tubes and dried in a Speed Vac concentrator at 20\u0026deg;C and 1000 rpm for ~\u0026thinsp;6 h. Samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further processing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eSemi-targeted liquid chromatography-high-resolution mass spectrometry-based (LC-HRS-MS) analysis of amine-containing metabolites\u003c/h2\u003e\u003cp\u003eAmine-containing compounds were analyzed using a Q-Exactive Plus high-resolution mass spectrometer coupled to a Vanquish UHPLC chromatography system (Thermo Fisher Scientific). As previously described, dried extracts were reconstituted in 150 \u0026micro;L LC-MS-grade water at 4\u0026deg;C and 1500 rpm shaking for 10 min. After centrifugation, 50 \u0026micro;L supernatant was mixed with 25 \u0026micro;L 100 mM sodium carbonate, followed by 25 \u0026micro;L 2% (v/v) benzoyl chloride in acetonitrile (UPLC/MS-grade, Biosolve) \u003csup\u003e111\u003c/sup\u003e. Samples were mixed and stored at 20\u0026deg;C until analysis. 1 \u0026micro;L of derivatized sample was injected onto a 100 \u0026times; 2.1 mm HSS T3 UPLC column (Waters) at 40\u0026deg;C, 400 \u0026micro;L/min, using a binary buffer system: buffer A (10 mM ammonium formate, 0.15% formic acid in water) and buffer B (acetonitrile). LC gradient: 0% B (0 min); 0\u0026ndash;15% B (0\u0026ndash;4.1 min); 15\u0026ndash;17% B (4.1\u0026ndash;4.5 min); 17\u0026ndash;55% B (4.5\u0026ndash;11 min); 55\u0026ndash;70% B (11\u0026ndash;11.5 min); 70\u0026ndash;100% B (11.5\u0026ndash;13 min); 100% B (13\u0026ndash;14 min); 100\u0026ndash;0% B (14\u0026ndash;14.1 min); 0% B (14.1\u0026ndash;19 min). MS acquisition was performed in positive ionization mode (m/z 100\u0026ndash;1000), with the following source settings: 3.5 kV spray voltage, capillary temperature 300\u0026deg;C, sheath gas 60 AU, aux gas 20 AU at 330\u0026deg;C, sweep gas 2 AU, RF lens 60. Raw mass spectra files were converted to mzML using MSConvert (v3.0.22060) \u003csup\u003e112\u003c/sup\u003e, and analyzed in El Maven (v0.12.0) \u003csup\u003e113\u003c/sup\u003e. Area of the protonated [M\u0026thinsp;+\u0026thinsp;nBz\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e mass peaks of every required compound was extracted and integrated (mass accuracy of \u0026lt;\u0026thinsp;5 ppm, retention time tolerance of \u0026lt;\u0026thinsp;0.05 min compared to reference compounds). Data were normalized to internal standards and protein content.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eAnion-exchange chromatography mass spectrometry (AEX-MS) for the analysis of anionic metabolites\u003c/h2\u003e\u003cp\u003eExtracted metabolites were reconstituted in 150 \u0026micro;L UPLC/MS grade water (Biosolve) of which 100 \u0026micro;L was transferred to polypropylene autosampler vials (Chromatography Accessories Trott). Analysis was performed as previously described, using a Dionex Integrion ion chromatography system (Thermo Fisher Scientific)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. 5 \u0026micro;L of extract was injected (push-full mode, overfill factor 1) onto a Dionex IonPac AS11-HC column (2 \u0026times; 250 mm, 4 \u0026micro;m particle size) equipped with a AG11-HC guard column (2 \u0026times; 50 mm, 4 \u0026micro;m) at 30\u0026deg;C and autosampler at 6\u0026deg;C. Metabolites were separated at 380 \u0026micro;L/min using a KOH cartridge (Eluent Generator, Thermo Scientific) with the following gradient: 0\u0026ndash;3 min, 10 mM; 3\u0026ndash;12 min, 10\u0026ndash;50 mM; 12\u0026ndash;19 min, 50\u0026ndash;100 mM; 19\u0026ndash;22 min, 100 mM; 22\u0026ndash;23 min, 100\u0026ndash;10 mM; re-equilibration at 10 mM (3 min). Eluting metabolites were detected in negative ion mode (m/z 77\u0026ndash;770) on a Q-Exactive HF MS. Source settings: 3.2 kV spray voltage, capillary 300\u0026deg;C, sheath gas 50 AU, aux gas 14 AU at 380\u0026deg;C, sweep gas 3 AU, S-lens 40. Raw mass spectra files were converted to mzML using MSConvert (v3.0.22060)\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, and analyzed in El Maven (v0.12.0)\u003csup\u003e2\u003c/sup\u003e. Area of the deprotonated [M-H\u003csup\u003e+\u003c/sup\u003e]\u003csup\u003e\u0026minus;1\u003c/sup\u003e or doubly deprotonated [M-2H]\u003csup\u003e\u0026minus;2\u003c/sup\u003e isotopologues mass peaks of every required compound was extracted and integrated (mass accuracy of \u0026lt;\u0026thinsp;5 ppm, retention time tolerance of \u0026lt;\u0026thinsp;0.05 min compared to reference compounds). Data were normalized to internal standards and protein content.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eLiquid Chromatography-High Resolution Mass Spectrometry-based (LC-HRMS) analysis of lipids\u003c/h2\u003e\u003cp\u003eStored (-80\u0026deg;C) lipid extracts were reconstituted in 150 \u0026micro;L UPLC-grade acetonitrile:isopropanol (70:30, v/v). After vortexing, samples were incubated for 10 min at 4\u0026deg;C with continuous shaking. Samples were clarified by centrifugation at 16000 x g, 4\u0026deg;C, for 5 min. Supernatants were transferred to 2 mL glass vials equipped with 300 \u0026micro;L glass inserts (Chromatography Zubeh\u0026ouml;r Trott). Aliquots of 20 \u0026micro;L from each sample were pooled to generate quality control (QC) samples, which were injected after every 10th sample or after each replicate group. Samples and QCs were maintained at 6\u0026deg;C in a Vanquish UHPLC system (Thermo Fisher Scientific) fitted with a quaternary pump and coupled to a TimsTOF Pro 2 HRMS with a heated ESI (VIP-HESI) source (Bruker Daltonics). For each run, 1 \u0026micro;L sample was injected onto a 100 x 2.1 mm CSH C18 UPLC column (1.7 \u0026micro;m, Waters). The chromatographic gradient was performed at 400 \u0026micro;L/min using buffer A (10 mM ammonium formate, 0.1% formic acid in acetonitrile:water, 60:40, v/v) and buffer B (10 mM ammonium formate, 0.1% formic acid in isopropanol:acetonitrile, 90:10, v/v) as follows: 0\u0026ndash;0.5 min, 45\u0026ndash;48% B; 0.5\u0026ndash;1 min, 48\u0026ndash;55% B; 1\u0026ndash;1.8 min, 55\u0026ndash;60% B; 1.8\u0026ndash;10 min, 60\u0026ndash;85% B; 10\u0026ndash;11 min, 85\u0026ndash;99% B; 11\u0026ndash;11.5 min, 99% B; 11.7\u0026ndash;15 min, re-equilibration at 45% B (total run time: 15 min/sample). Prior to each batch, mass and mobility calibrations were performed using a 1:1 mixture of 10 mM sodium formate and ESI-L Low Concentration Tuning Mix (Agilent). Data were acquired in data-dependent PASEF mode, primarily in positive ionization mode (source settings: 4.5 kV capillary, 500 V end plate offset, nebulizer 2 bar, dry gas 8 L/min at 230\u0026deg;C, sheath gas 4 L/min at 400\u0026deg;C). For MS/MS, isolation width was set to 2 mD and collision energy to 30 eV. Pooled QC samples were additionally injected in negative mode (collision energy 40 eV, -3.5 kV capillary) for further fatty acid annotation. Samples were analyzed in randomized order. QC samples were analyzed after every 10th injection in both positive and negative ionization modes to ensure data quality and stability. Raw spectra were processed in MetaboScape (v2024) to extract features and annotate lipid species, using pooled QC samples for validation. Lipids were only included in downstream analysis if their abundance in QC samples was at least threefold higher than in extraction blanks.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eHuman sample handling and preparation\u003c/h2\u003e\u003cp\u003eThe peripheral blood samples of VRJS patients were collected in the Center for Rare Diseases, University Hospital of Cologne, Germany. All patients and/or custodians gave informed consent according to local institutional review board approval (20-1711, Medical Faculty at University of Cologne). The plasma was isolated with standard protocol and processed for metabolomic and lipidomic analysis in the Metabolomic Core Facility of the Max Planck Institute for Biology of Ageing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eStatistics \u0026amp; Reproducibility\u003c/h2\u003e\u003cp\u003eIn all figures, the numbers of independent replicates and the total number of animals analyzed are indicated in each panel. All statistical analyses were performed in GraphPad Prism (Version 9.0.0 (86)). Asterisks denote corresponding statistical significance *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ****P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. Data distribution was assumed to be normal but this was not formally tested. No statistical method was used to predetermine sample size but our sample sizes are similar to those reported in previous publications\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e,114,115\u003c/sup\u003e. Missing or outlier data points were excluded from the analyses. At least three independent experiments for each assay were performed to verify the reproducibility of the findings, unless indicated otherwise. For worm experiments, samples preparation and data collection were randomized. For lifespan experiments, all the genotypes were blinded before the assays. For cold tolerance, developmental rate, body size, brood size, Western blot, and imaging experiments, the genotypes were not blinded before assay, as mutant worms have obvious phenotypes that revealed the sample identity (body size and developmental rate). However, worms were randomly picked and assigned to the different treatment conditions in a random order. For RNA-seq experiments, the genotypes were not blinded before collecting samples. Once the RNA samples were ready, they were processed at the Cologne Center for Genomics (CCG) in a blinded manner.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA-seq datasets generated in this study are available in the GEO datasets with the accession number GSE307065 as of the date of publication. The \u003cem\u003eprp-19\u003c/em\u003e RNAseq data was obtained under accession number GSE191294\u003csup\u003e66\u003c/sup\u003e. The list of \u003cem\u003enhr-114\u003c/em\u003e target genes was as defined\u003csup\u003e60\u003c/sup\u003e, available under the accession number GSE211747. The siPUF60-treated RNAseq data was obtained under accession number OEP004324\u003csup\u003e79\u003c/sup\u003e in NODE (The National Omics Data Encyclopedia) database.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Caenorhabditis Genetics Center (CGC). Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We thank Lianfeng Wu (Westlake University) and William Mair (Harvard T.H. Chan School of Public Health) for kindly providing some \u003cem\u003eC. elegans\u003c/em\u003e strains. We thank the Bioinformatics, Imaging, Metabolomics, and Proteomics core facilities of the Max Planck Institute for Biology of Ageing for their technical support. We would also like to thank the members of the Antebi lab, especially Dr. Kreuz, Dr. Tabrez and Dr. Kawamura for valuable comments on the manuscript. We also thank Dr. Filipe Cabreiro for his valuable feedback on the manuscript. We thank the \u003cem\u003ePostDoc\u003c/em\u003e Seed funding 2023 (W.H.) from CECAD Cluster for Excellence, University of Cologne (Gef\u0026ouml;rdert durch die Deutsche Forschungsgemeinschaft (DFG) im Rahmen der Exzellenzstrategie des Bundes und der L\u0026auml;nder - EXC 2030 \u0026ndash; 390661388),\u0026nbsp;Cologne Graduate School for Ageing Research (J.K.) and the Max Planck Society, Germany (A.A.) for funding this project.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eW.H., J.K. and A.A. conceived and designed the study. W.H., J.K. and A.L. performed the investigation in \u003cem\u003eC. elegans\u003c/em\u003e. K.C. performed the western blot experiment. W.H. and J.K. analyzed the data. D.H. and E.B. collected, processed, and analyzed patient samples. W.H. wrote the original draft of the manuscript; W.H., J.K. and A.A. reviewed and edited the final version. A.A. acquired funding for the project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWill, C. L. \u0026amp; Luhrmann, R. Spliceosome structure and function. \u003cem\u003eCold Spring Harb Perspect Biol\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e (2011). https://doi.org:10.1101/cshperspect.a003707\u003c/li\u003e\n\u003cli\u003eWang, E. \u0026amp; Aifantis, I. RNA Splicing and Cancer. \u003cem\u003eTrends Cancer\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 631-644 (2020). https://doi.org:10.1016/j.trecan.2020.04.011\u003c/li\u003e\n\u003cli\u003eBhadra, M., Howell, P., Dutta, S., Heintz, C. \u0026amp; Mair, W. B. 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G.\u003cem\u003e et al.\u003c/em\u003e NFYB-1 regulates mitochondrial function and longevity via lysosomal prosaposin. \u003cem\u003eNat Metab\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 387-396 (2020). https://doi.org:10.1038/s42255-020-0200-2\u003c/li\u003e\n\u003cli\u003eNakamura, S.\u003cem\u003e et al.\u003c/em\u003e Suppression of autophagic activity by Rubicon is a signature of aging. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 847 (2019). https://doi.org:10.1038/s41467-019-08729-6\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8077579/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8077579/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSpliceosomal dysfunction profoundly impacts cellular metabolism, yet mechanistic links between RNA splicing defects and metabolic rewiring remain limited. Here, we investigate Verheij syndrome (VRJS), a rare disease caused by mutations in the core splicing factor \u003cem\u003ePUF60\u003c/em\u003e. Using a \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e model, human cell lines, and patient-derived samples, we demonstrate that RNP-6/PUF60 deficiency disrupts splicing of genes governing one-carbon metabolism and phospholipid remodeling, culminating in impaired S-adenosylmethionine (SAM)/S-adenosylhomocysteine (SAH) cycling and phosphatidylcholine synthesis. These perturbations trigger the integrated stress response and compromise mTORC1 signaling, causing developmental and growth defects. Vitamin B12 (VB12) supplementation restores metabolic balance by reactivating SAM-dependent phospholipid remodelling and mTORC1 activity, effectively rescuing VRJS-like phenotypes. Similar metabolic responses arise from perturbations in other spliceosomal factors such as PRPF19/PRP-19, indicating a conserved mechanism across spliceosomopathies. Interestingly, we identify intron retention of the \u003cem\u003enhr-114/HNF4\u003c/em\u003e transcription factor as a primary driver of growth defects, and restoring its splicing robustly suppresses these phenotypes. Our findings establish a mechanistic connection between RNA splicing and lipid metabolism, implicating VB12-dependent one-carbon metabolism as a metabolic modulator with broad implications for spliceosome-related diseases, and suggesting VB12 as a potential strategy to mitigate VRJS-related anomalies.\u003c/p\u003e","manuscriptTitle":"Vitamin B12 alleviates spliceosomopathy via phospholipid remodeling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-17 08:08:38","doi":"10.21203/rs.3.rs-8077579/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e9cd6fa7-1f9a-4473-9c5b-4a54e2c973fb","owner":[],"postedDate":"November 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58090050,"name":"Biological sciences/Genetics/RNA splicing"},{"id":58090051,"name":"Biological sciences/Cell biology/Mechanisms of disease"}],"tags":[],"updatedAt":"2026-03-19T18:05:46+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-17 08:08:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8077579","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8077579","identity":"rs-8077579","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}