Aging-Associated Modulation of UFMylation Impairs Proteostasis in C. elegans

Aging-Associated Modulation of UFMylation Impairs Proteostasis in C. elegans | 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 Aging-Associated Modulation of UFMylation Impairs Proteostasis in C. elegans Ehud Cohen, Reut Bruck-Haimson, Hana Boocholez, Huadong Zhu, Adam Zaretsky, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6064783/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 The attachment of Post-Translational Modifications (PTMs) to proteins plays key roles in the regulation of the activity and stability of various proteins. Here we utilized the nematode Caenorhabditis elegans to test whether UFMylation, a PTM that was found to be essential for key biological functions, is involved in the regulation of aging and protein homeostasis (proteostasis). Our results indicate that lowering UFMylation extends lifespan and mitigates the toxicity of aggregative proteins that underlie the development of neurodegenerative disorders in humans. Mass spectrometric analysis unveiled that UFMylation of aging-regulating proteins, including components of the nucleolar FIB-1-NOL-56 complex and the germline resident proteins CAR-1 and CGH-1, governs proteostasis across tissues. Functional analyses indicate that the proteostasis-regulating transcription factors DAF-16 and SKN-1 are crucial for the counter proteotoxic effect of reduced UFMylation which is mediated by reduced rate of aggregation and enhanced protein degradation. These insights highlight the important roles of PTMs in the regulation of proteostasis and point at research directions for the development of new therapies for neurodegenerative disorders. Biological sciences/Cell biology/Protein folding/Protein aggregation Biological sciences/Cell biology/Proteolysis/Protein quality control Aging UFMylation germline lifespan proteostasis C. elegans Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Protein maturation is a complex, multi-step process that is critical for cellular and organismal functionality and viability 1 . Right after synthesis, the nascent polypeptide has to attain its desired spatial structure, get proper post-translational modifications and often interact with other proteins to form functional complexes. Although these maturation steps are tightly supervised and assisted by a nexus of chaperones and specialized mechanisms 2 , not all nascent polypeptides maturate properly. Early in life, unfolded polypeptides are either refolded or, when terminally misfolded, cleared by highly conserved degradation mechanisms, namely the ubiquitin proteasome system (UPS) 3 and autophagy 4 . Alas, as the organism ages, the competence of the protein homeostasis (proteostasis) network declines, and proteins that bear an inherent propensity to misfold, accumulate in the cell and form hazardous aggregates 5 . This process jeopardizes cellular viability and can underlie the development of late-onset disorders that are collectively known as “proteinopathies” 6 . Neurodegenerative diseases such as Alzheimer’s disease (AD) and Huntington’s disease (HD) consist a prominent class of proteinopathies. Accordingly, aging is the most prominent risk factor for the development of these maladies which emanate from aberrant protein aggregation 7 . Alzheimer’s disease (AD), the most prevalent dementia causing disorder, stems from the cleavage of the trans membranal “Amyloid Precursor Protein” (APP), by two proteolytic entities, the β and ɣ secretases. This dual digestion releases a family of aggregation-prone peptides that were collectively termed the amyloid β (Aβ) peptides. The accumulation and aggregation of Aβ peptides lead to the formation of toxic oligomers 8 that initiate a pathophysiological process which results in neuronal loss and dementia 9 . Similarly, abnormally long stretches of poly glutamine (polyQ) in the sequences of different proteins, render these polypeptides prone to aggregate, and underlie the development of at least nine late-onset human neurodegenerative illnesses 10 . Among these maladies, Huntington’s disease (HD) is most prevalent. The observation that neurodegenerative disorders manifest in late stages of life, raised the question of whether aging and toxic protein aggregation (proteotoxicity) are mechanistically linked. To address this, we and others employed the nematode Caenorhabditis elegans ( C. elegans ), a preferred model organism for the study of aging and proteostasis 11 . The alteration of aging, by either knocking down the expression of the sole insulin/IGF1 receptor daf-2 , or by dietary restriction (DR), both known as aging regulating manipulations 12 mitigate proteotoxicity that emanated from the expression of neurodegeneration-causing aggregation-prone proteins 13 – 15 . These counter-proteotoxic effects were shown to be conserved from worms to mammals 16 , 17 , and to be entirely dependent upon the activities of aging-controlling transcription factors. These include DAF-16/FOXO, SKN-1/NRF (hereafter DAF-16 and SKN-1 respectively), the ParaQuat Methylviologen responsive (PQM-1) factor 18 and the heat shock factor 1 (HSF-1) 19 . While the modulation of gene expression plays key roles in the regulation of aging and proteostasis, post-translational modifications (PTMs) of proteins are also involved in these mechanisms. First, ubiquitination variations were found to be involved in the regulation of aging 20 . In addition, SUMOylation, the attachment of a small ubiquitin-like modifier (SUMO) to specific lysine residues, regulates the activity of the protein CAR-1 which controls signaling that stems from the reproductive system, and affects lifespan and proteostasis 21 . Importantly, SUMOylation also plays key roles in the development of distinct neurodegenerative disorders 22 , and PTM patterns of the protein TAU are correlated with heterogeneity of Alzheimer’s patients 23 . These findings highlight the key roles of PTMs and raise the question of which additional PTMs are involved in the regulation of the mechanisms of aging and proteostasis. Here we asked whether UFMylation, a protein modification mechanism which attaches a polypeptide of 94 amino acids, known as “ubiquitin-fold modifier 1” (UFM-1), to lysine residues of its target proteins 24 , is involved in the regulation of proteostasis in the nematode. In mammals, the UFMylation pathway is initiated by the cleavage of UFM-1’s C-terminus by two UFM-1 specific cysteine proteases UFSP1 and UFSP2, to expose the glycine residue. Thereafter, the UFM-1 conjugation cascade consists of UBA-5 (ubiquitin-like modifier activating enzyme, E1), UFC-1 (UFM-1 conjugating enzyme 1, E2), and UFL-1 (UFM-1-specific ligase 1, E3) 24 . uba-5 mutant worms exhibited increased resistant to stresses such as oxidation and elevated temperatures 25 . In mammals, mutations in the sequence of uba5 cause fatal congenital neuropathy 26 , directly links UFMylation with brain development. Genetic studies in humans revealed that variants in human uba5 , ufc1 , and ufm1 are associated with a number of neurodevelopmental diseases, including infantile-onset encephalopathy 27 . Surprisingly, we found that reducing the rate of UFMylation by RNAi-mediated knockdown of ufm-1 or of uba-5 , mitigates proteotoxicity that emanates from the expression of Aβ or of polyQ35-YFP. It also slightly extended the lifespan of worms that express these aggregative proteins. Functional assays unveiled that the transcription factors DAF-16 and SKN-1, as well as the chaperones DAF-21 and SIP-1 are critically needed for this protection, which is mediated, at least partially by the enhancement of protein degradation. Our results imply that UFMylation impairs proteostasis in the face of chronic proteotoxic stress and suggest that a selective manipulation of this PTM could be useful for the treatment of these proteinopathies. Results Reduced UFMylation mitigates proteotoxicity and extends lifespan We first sought to test whether aging and reducing the activity of the insulin/IGF1 signaling cascade (IIS), modulate global protein UFMylation in adult worms. To avoid possible background from developing embryos we utilized CF512, worms that are feminized when exposed to 25°C during development. Synchronized eggs were placed on plates that were seeded with daf-2 RNAi bacteria or with control bacteria harboring the empty RNAi vector (EV). The plates were incubated at 25°C for 48 hours and transferred to 20°C until harvested at either day 1, 5, 10 or 15 of adulthood. The worms were homogenized and subjected to Western blot analysis using a UFM-1 antibody, to compare protein UFMylation patterns (Fig. 1 a). We observed several proteins that their UFMylation levels were increased with age, as well as in worms that were treated with daf-2 RNAi (black arrows). At least one protein exhibited reduced level of UFMylation upon IIS reduction (arrowhead). These results indicated that the UFMylation pattern of the C. elegans proteome is modulated by aging and by IIS reduction. The aging-associated variations in protein UFMylation has led us to wonder whether these changes affect proteostasis. To address this, we employed worms that express Aβ in their body wall muscle (strain CL2006 28 ), thereby exhibiting a progressive paralysis phenotype within the population 14 . This phenomenon can be used for the measurement of Aβ-mediated proteotoxicity. Aβ worms were grown from hatching on ufm-1 RNAi (Fig. S1 , a and b), and subjected to the paralysis assay. Surprisingly, four independent experiments showed that the knockdown of ufm-1 significantly mitigates Aβ-mediated proteotoxicity, as at day 12 of adulthood the rate of paralysis among ufm-1 RNAi treated Aβ worms was approximately 35% lower than in their untreated counterparts (Fig. 1 b). To further scrutinize this observation, we knocked down the expression of uba-5 by RNAi (Fig. S1 , c and d), and observed an analogous protection from Aβ proteotoxicity (Fig. 2 c). Similar results were obtained when we reduced UFMylation by treating Aβ worms with RNAi toward odr-8 (Fig. S1 , e-g), a gene that encodes a specific protease whose activity is critically needed for the maturation of UFM-1 29 . As certain components of the proteostasis network can differentially respond to distinct proteotoxic challenges 30 we tested whether the counter-proteotoxic effect of reduced UFMylation is Aβ specific. To address this, we employed worms that express a stretch of 35 glutamines fused to the y ellow f luorescent p rotein (polyQ35-YFP), in their body wall muscles (strain AM140). This aggregation-prone protein, impairs motility, a phenotype that can be followed by the thrashing assay 11 . We found that the knockdown of both, ufm-1 and of uba-5 alleviates the proteotoxic effect of polyQ35-YFP. However, while the counter-proteotoxic effect of ufm-1 RNAi was significant at day 4 and 6 old worms, but not in young animals (day 2 of adulthood, Fig. 1 d), the knockdown of uba-5 significantly reduced the polyQ35-YFP-mediated toxicity at all ages (Fig. 1 e). Similar protection was observed when UFMylation was reduced in worms that express polyQ35-YFP in their neurons (AM1126), by ufm-1 RNAi (Fig. 1 f) or uba-5 RNAi (Fig. S1 h). These results indicate that reduced UFMylation levels alleviate proteotoxicity that stems from distinct aggregation-prone proteins. They also show that reduced UFMylation mitigates polyQ35-YFP-mediated toxicity, regardless of whether they are expressed in muscles or neurons. Given in proteostasis enhancement and longevity are not necessarily coupled 31 , we asked whether reducing UFMylation affects lifespan. Wild-type worms (strain N2) were left untreated (EV) or fed from hatching with RNAi bacteria toward either ufm-1 or uba-5 and living animals were scored daily. While the knockdown of ufm-1 slightly, but significantly (p = 0.0057), extended lifespan, no extension was observed in uba-5 treated animals (Fig. 1 g and Supplemental table 1 ). Similarly, the knockdown of ufm-1 , but not of uba-5 , extended the lifespan of CF512 worms (Fig S1 I, p = 0.0024). Next, we tested whether the reduced UFMylation is capable of extending the lifespans of worms that are challenged by proteotoxicity. Treating AM140 worms (Fig. 1 h) and CL2006 (Fig. 1 i) animals with either ufm-1 or uba-5 RNAi, we found that the knockdown of either ufm-1 or of uba-5 significantly extend lifespans of animals of both proteotoxicity models by approximately 13% compared to their untreated counterparts (Supplemental table 1 ). In sum, our results show that reduced rates of UFMylation extend lifespan, however this effect is more prominent when the animals are challenged by proteotoxic proteins. UFMylation of proteostasis modulating proteins affects proteotoxicity To elucidate the mechanism which promotes proteostasis upon UFMylation reduction, we sought to identify the proteins that are differentially UFMylated with age. Using CRISPR cas9 technology we tagged the endogenous ufm-1 gene with a triple HA sequence (Fig. 2 a, strain MQD2801). This tagging enabled us to specifically immuno-precipitate UFMylated proteins, using an HA antibody, and identify them by mass-spectrometry. We cultured synchronized populations of MQD2801 worms on control bacteria (EV) and harvested them at either day 1 or 5 of adulthood. The worms were homogenized, debris were sedimented, UFMylated proteins were immuno-precipitated from the cleared supernatants and identified by mass spectrometry (MS) (Fig. 2 b, complete MS dataset is available at: PXD060906). Total of 155 UFMylated proteins were identified whereas 94 (60.7%) showed no significant difference in UFMylation levels between days 1 and 5, 23 showed increased (14.8%) and 38 exhibited a reduced (24.5%) UFMylation levels (Fig. 2 b and Supplemental tables 2 and 3). Functional clustering of proteins that exhibited modulated UFMylation levels at day 5 of adulthood (Fig. 2 , c and d) indicated that ribosomal proteins are most abundant, suggesting that UFMylation may regulate translation. However, aging-regulating proteins were also identified. These include NOL-56 and FIB-1, two components of a nucleolar complex which we recently found to regulate organismal proteostasis by modulating TGFβ signaling 32 . Both, NOL-56 and FIB-1 exhibit aging-associated increased UFMylation rates, implying that this PTM positively regulates the activity of the FIB-1-NOL-56 complex. Accordingly, aging-associated increase in the rate of UFMylation enhances the activity of the FIB-1-NOL-56 complex thereby promoting proteotoxicity. To functionally test the possible role of FIB-1 in the regulation of proteostasis by UFMylation, we created a dual-silencing RNAi cassette that concurrently knocks down the expression of ufm-1 and fib-1 (the dilution of ufm-1 RNAi lowers the efficiency of its counter-proteotoxic effect (Fig. S2 a)). Aβ worms were either left untreated (EV) or treated with RNAi toward ufm-1, fib-1 or with the ufm-1/fib-1 cassette, and subjected to a paralysis assay. We found that a concomitant knockdown of ufm-1 and fib-1 shows no reduction in the rate of paralysis below the level which is mediated by a sole knockdown of fib-1 (Fig. S2 b). These results culminate to show that the rate of UFMylation of FIB-1 and NOL-56 is increased with age and infer that the reduction of UFMylation promotes proteostasis, at least partially, via the mechanism downstream of the FIB-1-NOL-56 complex. An additional interesting finding is the decreased UFMylation levels of CAR-1 and CGH-1 in day 5 old animals. These two proteins negatively regulate notch signaling during oogenesis 33 , and are modulators of proteostasis across tissues. Interestingly, these functions are governed by SUMOylation which is controlled by the IIS 21 . The age-associated decreased UFMylation levels of these proteins infers that the knockdown of ufm-1 also controls proteostasis by modulating notch signaling, which originates from the germline and regulate lifespan 34 . According to this notion, the counter-proteotoxic effect of ufm-1 RNAi is expected to be dependent on DAF-16, which is needed for the longevity phenotype of worms that express a mutated, hyper-active CAR-1 21 and to be orchestrated by inter-tissue communication. Two chaperones have also shown modulations in UFMylation levels with age. SIP-1, a small heat shock protein that plays key roles in the maintenance of proteostasis 35 exhibited decreased UFMylation at day 5 old worms compared to their day 1 old counterparts. In addition, HSP-1, a HSP-70 family member, showed increased UFMylation in day 5 old animals. Finally, HSP-90 (encoded by daf-21 ), a chaperone which is crucial for the mediation of proteostasis 36 , is also a UFMylated protein (Fig. 2 b). Clustering the differentially UFMylated proteins according to the tissue of expression (Fig. 2 e) showed enrichment for proteins that reside in muscles, the tissue that is challenged by proteotoxicity in these animals as well as in the neuronal and reproductive systems. We further examined the physiological roles of UFMylation in the regulation of proteostasis by creating dual silencing RNAi cassettes that concomitantly knockdown ufm-1 and either sip-1, daf-21 or hsp-1 (Fig. S3 , a-f). CL2006 were grown on bacteria that express either one of these RNAi cassettes, the corresponding chaperone or ufm-1 RNAi, and paralysis assays were conducted. While treatment with RNAi toward sip-1 was initiated from hatching, genes that encode the other chaperones were knocked down from day 1 of adulthood, to allow complete development of the worms. Our results show that a concurrent knockdown of ufm-1 and of each of these chaperone-encoding genes, completely abolishes the counter-proteotoxic effect of reduced UFMylation (Fig. 3 , a-c). These results infer that these three chaperones are crucial for the alleviation of Aβ-mediated proteotoxicity by ufm-1 RNAi. To expand our investigation, we created silencing cassettes which enabled us to test whether seven additional proteins that exhibited differential UFMylation levels among day 1 and 5 old untreated worms (Fig. 2 b), are also needed for the protection from Aβ that is conferred by the knockdown of ufm-1. Utilizing CL2006 worms and the paralysis assay we found that the knockdown of kin-19 , which encodes a kinase that negatively regulates the Wnt signaling pathway 37 , prevents ufm-1 RNAi from alleviating the paralysis phenotype (Fig. S3 g). Interestingly, the knockdown of atp-2 , that codes for a mitochondrial ATP synthase, alleviated the paralysis phenotype regardless of whether ufm-1 has been knocked down or not (Fig. S3 h). In contrast, a concurrent knockdown of ufm-1 and either one of the other five genes ( lfi-1, ant-1.1, cpl-1, trap-1 and top- 1), did not prevent ufm-1 RNAi from mitigating proteotoxicity (Fig. S3 , i-m). Together, these results confirm that daf-21, sip-1, hsp-1 and kin-19 are crucial for the counter proteotoxic effect of ufm-1 RNAi. DAF-16 and SKN-1 are required for mitigation of proteotoxicity by reduced UFMylation The observations that reducing the levels of daf-21 and of kin-19 prevent the knockdown of ufm-1 from alleviating paralysis, imply that UFMylation regulates proteostasis, at least partially, by modulating the activities of signaling pathways. Since aging-regulating transcription factors have been shown to be needed for the promotion of proteostasis by signaling mechanisms 14 , 18 , 38 , we asked whether the transcription factors DAF-16, SKN-1 and/or PQM-1 are needed for the alleviation of proteotoxicity upon the knockdown of ufm-1 (a concurrent knockdown of ufm-1 and of hsf-1 resulted in high rate of early mortality). To address this, we created dual silencing cassettes that simultaneously knockdown the expression of ufm-1 and of either daf-16, skn-1 or pqm-1 , and tested the possible roles of these transcription factors using two proteotoxicity models, CL2006 animals and AM140 worms. The nematodes were treated from hatching with the dual RNAi silencing cassettes or with RNAi toward ufm-1 or the corresponding transcription factor, and subjected to paralysis assays (CL2006) or thrashing assays (AM140). We found that a simultaneous knockdown of ufm-1 and daf-16 abolishes the protection from Aβ that was conferred by a sole knockdown of ufm-1 (Fig. 4 a). Similar results were obtained when AM140 worms were used (Fig. 4 b), indicating that DAF-16 is critically needed for the mitigation of proteotoxicity that stems from both aggregative proteins. Analogous results were obtained when the ufm-1 and skn-1 were concurrently knocked down (Fig. 4 , c and d) implying that SKN-1 is also critically needed for reduced UFMylation to protect from proteotoxicity of both Aβ and polyQ35-YFP. In contrast, no change in the rates of counter proteotoxic effect was seen when pqm-1 was concomitantly knocked down with ufm-1 (Fig. S4 , a and b). These results infer that the reduction of UFMylation modulates gene expression to mitigate proteotoxicity. Reducing UFMylation modulates the transcriptomic landscape of the worm Reducing UFMylation modulates the transcriptomic landscape of the worm In order to characterize possible modulations in gene expression upon the knockdown of ufm-1 , we compared the transcriptomic landscapes of 6 days old CL2006 worms, an age in which proteotoxicity is apparent, that were treated for two generations with RNAi toward ufm-1 and of their untreated counterparts (EV), using RNA sequencing (data are available at GSE289271). 143 genes exhibited modulated expression levels, whereas 89 showed elevated levels (62%) and 54 genes exhibited reduced levels (37%) upon the knockdown of ufm-1 by RNAi (Figs. 5 a and S5a). To characterize the biological processes that are modulated by reducing UFMylation we used the DAVID bioinformatic tool to cluster genes that showed increased or decreased expression levels upon the knockdown of ufm-1 (Fig. 5 b). Interestingly, the knockdown of ufm-1 has led to increased expression of genes that are related with the innate immune response (defense category), a mechanism that was found to be associated with proteostasis 39 . In addition, genes that their products are extracellular or presented on the cell surface, were most abundant among those which exhibited elevated levels. Among the downregulated genes we identified proteins that are involved in reproduction. Some interesting genes were among those which showed modulated expression levels in ufm-1 RNAi treated animals (Fig. 5 c), including fbxb-45 and fbxb-66 , both contain F-box domains, and may be components of the SCF E3 ubiquitin ligase complex. We previously found proteins of this family to regulate proteostasis 40 . Therefore, the increase in the expression of these genes may be related with enhanced proteasome activity. Surprisingly, the knockdown of ufm-1 elevated the levels of cav-1 , a gene that is upregulated by the IIS and whose knockdown by RNAi, mitigates Aβ proteotoxicity 38 . An additional unexpected observation is the increase in the levels of several genes that encode for transthyretin-related genes ( ttr genes). In fact, the clearance of TTR has been shown to alleviate proteotoxicity in C. elegans 41 . An increased level of the cysteine protease inhibitor cpi-1 was also a result of ufm-1 RNAi treatment. Protease activity has been shown to be tightly linked with neurodegeneration in mice 42 and with proteotoxicity in worms 43 . Among the downregulated genes we found klc-1 whose product binds kinesin, an activity that was implicated in Alzheimer’s disease 44 and the cysteine protease inhibitor cpi-1 , whose human orthologue, cystatin C is associated with aging and Alzheimer’s disease 45 . We also found lst-1 , which is known to be a target of notch signaling 46 . This is in line with the modulation of UFMylated of CAR-1 and CGH-1 that we identified in our MS experiment (Fig. 2 b). We further clustered the modulated genes and identified enrichment of genes that their products reside in the neuronal and reproductive systems (Fig. S5 b). Finally, targets of DAF-16 and SKN-1 were relatively abundant among genes that exhibited modulated expression level upon the knockdown of ufm-1 (Fig. S5 c). Reduced UFMylation enhances proteasome activity and reduce polyQ35-YFP aggregation Since small oligomers, rather than large molecular aggregates, have been shown to be the most toxic species in worms 14 and mice 8 , we asked whether the knockdown of ufm-1 mitigates Aβ proteotoxicity by modulating its rate of aggregation. CL2006 worms were treated from hatching with RNAi toward ufm-1 or left untreated (EV) and harvested at either day 1 or 6 of adulthood. The worms were homogenized, spun to separate supernatants from debris, and the different fractions were subjected to Western blot analysis to compare the levels of Aβ. While no significant difference in Aβ levels has been observed at supernatants and debris of day 1 old worms, clear reduction in Aβ levels was observed in supernatants, but not in debris, of 6 days-old worms that were treated with ufm-1 RNAi (Fig. 6 , a and b). These observations suggest that upon reduction of UFMylation, soluble Aβ is more efficiently degraded by protein clearance mechanisms. To investigate how the inhibition of UFMylation affects the rate of polyQ35-YFP aggregation, we first visualized AM140 that were either treated with ufm-1 RNAi or left untreated (EV) using fluorescent microscopy, and counted the number of foci in these animals at days 4 and 6 of adulthood (Fig. S6a). We found that the knockdown of UFMylation leads to an increase in the number of foci at both ages (Fig. S6b). Next, we employed the filter-trap assay to compare the aggregation rates of polyQ35-YFP in 5-days old controls worms (EV) and in their aged-matched ufm-1 RNAi-treated counterparts. Reduced rate of UFMylation lowers polyQ35-YFP aggregation in five independent experiments (Fig. 6 , c and d). Collectively, these results infer that an inhibition of UFMylation affects the aggregation rates of both, Aβ and of polyQ35-YFP and suggest that protein degradation mechanisms may be also affected by ufm-1 RNAi in Aβ expressing worms. To examine this, we cultured synchronized CL2006 worm populations on either control bacteria (EV) or treated them with ufm-1 RNAi. The worms were harvested at day 1 or 6 of adulthood, homogenized and subjected to WB analysis to compare the relative amounts of highly ubiquitinated proteins. While no difference in the levels of ubiquitinated proteins was observed among untreated and ufm-1 RNAi-treated, day 1 old animals (Fig. S6, c and d), 6 days old CL2006 worms exhibited reduced level upon the knockdown of ufm-1 (Fig. 6 , e and f). These results are in line with the observation that reduced UFMylation reduces Aβ levels in supernatants of day 6 but not day 1 old worms (Fig. 6 , a and b) and propose that ufm-1 RNAi treatment enhances proteasome activity. A significant enhancement of proteasome activity was also observed in day 1 old worms that express the short-lived Ub G76V -GFP proteasome sensor (Fig. S6, e and f 47 ). Reduced UFMylation regulates proteotoxicity in cell-autonomous and non-autonomous manners The prominence of inter-tissue communication as a key regulator of proteostasis 1 and the major effect of ufm-1 on the expression levels of genes that are mainly expressed in the neuronal and reproductive systems (Fig. S5 b) have led us to ask whether UFMylation governs proteostasis at the organismal level. To address this, we crossed AM1126 worms with TU3401 animals to obtain worms that express polyQ35-YFP in their neurons and are amenable to RNAi-mediated knockdown solely in neurons (Strain EHC145). Synchronized populations of EHC145 worms were either treated from hatching with ufm-1 RNAi or left untreated and subjected to the thrashing assay at days 2, 4 and 6 of adulthood. While no mitigation of proteotoxicity was observed in day 2 old worms, the knockdown of ufm-1 protected day 4 and 6-old worms from polyQ35-YFP-mediated toxicity (Fig. 7 a). These results show that the knockdown of ufm-1 exclusively in neurons and throughout the nematode (Fig. 1 f), protect the worms from neuronally expressed polyQ35-YFP, and indicate that reduced UFMylation mitigates proteotoxicity cell-autonomously. Next, we sought to test whether reduction of UFMylation also regulates proteostasis cell-non-autonomously. To address this, we used worms that express polyQ35-YFP in their neurons and process RNAi exclusively in the germline (strain EHC212) or in muscles (strain EHC211 32 ). The worms were treated from hatching with ufm-1 RNAi or left untreated, and subjected to proteotoxicity assays. A thrashing assay using EHC212 animals indicated that knocking down ufm-1 solely in the germline alleviates proteotoxicity that stems from the expression of polyQ35-YFP in neurons. This effect was apparent at all tested ages, day 2, 4 and 6 of adulthood (Fig. 7 b). Since worms that express polyQ35-YFP in neurons and process RNAi merely in muscles (EHC211) are relatively motile, we used to paralysis assay to measure proteotoxicity and found that the knockdown of ufm-1 mitigates the paralysis phenotype (Fig. 7 c). Together these results show that UFMylation levels affect polyQ35-YFP proteotoxicity both cell-autonomously and cell-non-autonomously. We next adopted a similar approach to test whether the knockdown of ufm-1 analogously affects Aβ-mediated proteotoxicity. A paralysis assay using worms that express Aβ in muscles and are amenable to RNAi-mediated gene knockdown solely in the same tissue (strain EHC208) showed that the knockdown of ufm-1 alleviates the toxicity of Aβ in a cell-autonomously fashion (Fig. 7 d). Surprisingly, the knockdown of ufm-1 exclusively in neurons (strain EHC207, Fig. 7 e), germline (strain EHC206, Fig. 7 f) or the intestine (strain EHC205, Fig. S7a) did not affect the toxicity of Aβ in muscles. Discussion The attachment of different PTMs to proteins have key roles in the regulation of various biological traits including aging and proteostasis. While SUMOylation and Ubiquitination have been shown to be regulators of these processes 20 , 21 , the possible roles of UFMylayion in the regulation of organismal proteostasis, are largely unknown. Here we used the nematode C. elegans , and found that reducing the rates of UFMylation by ufm-1 RNAi, protects model worms from the toxicity of the aggregation-prone proteins Aβ and polyQ35-YFP, and slightly extends their lifespans. To identify proteins that exhibit differential UFMylation with age, we tagged the endogenous ufm-1 gene with a triple HA tag and compared the rates of protein UFMylation in day 1 and 5 old worms. We found that components of two known aging-regulating pathways exhibit differential rates of UFMylation with age. First, FIB-1 and NOL-56, both are components of a nucleolar complex that regulates proteostasis across tissues by modulating TGFβ signaling 32 , showed increased UFMylation levels at day 5, suggesting that this PTM enhances their activity. Accordingly, the knockdown of either ufm-1 or nol-56 by RNAi leads to similar outcomes of mitigated proteotoxicity. It also proposes that these UFMylation events occur in ASI neurons where the FIB-1-NOL-56 complex functions as a regulator of proteostasis 32 (Fig. 7 g-i). Secondly, CAR-1 and CGH-1, two germline resident proteins, show reduced UFMylation levels with age (Fig. 7 G-ii). Since these proteins negatively regulate Notch signaling (Fig. 7 g-iii), their activation alleviates proteotoxicity in a DAF-16 dependent manner (Fig. 7 g-iv) 21 . The ufm-1 RNAi-mediated protection from proteotoxicity also requires SKN-1 (Fig. 4 , c and d), however, it is unclear whether this transcription factor is regulated in this context, by Notch signaling, by the FIB-1-NOL-56 complex or by another mechanism (Fig. 7 g-v). This counter-proteotoxic effect is also dependent on at least three chaperones, HSP-1, HSP-90 and SIP-1 which show modulated UFMylation levels with age (Fig. 2 b), and associated with enhanced UPS activity (Fig. 7 g-vi). One fundamental question that arises from our results, is whether the aging-regulating mechanisms downstream of the FIB-1-NOL-56 complex and of CAR-1/CGH-1 act in a coordinated manner, and if yes, whether UFMylation orchestrates them. Several observations suggest that these mechanisms act in concert to regulate proteostasis. First, a concurrent knockdown of fib-1 and ufm-1 show no additive protective effect (Fig. S2 b), suggesting that they may be components of the same pathway. In addition, we previously found that the knockdown of nol-56 reduces the expression levels of car-1 and cgh-1 32 suggesting a direct regulation of Notch signaling by the nucleolar FIB-1-NOL-56 complex. We also observed modulated expression levels of lst-1 , a well characterized target of Notch signaling 46 , upon treatment with nol-56 32 , car-1 21 and ufm-1 RNAi (Fig. 5 c). Together, these results imply that UFMylation enhances proteotoxicity, at least partially, by elevating the activity of Notch signaling. Nevertheless, the FIB-1-NOL-56 complex regulates proteostasis by modulating TGFβ signaling, and is not dependent on DAF-16 for this function 32 . Thus, it is conceivable that the two mechanisms regulate proteostasis by partially overlapping mechanism through which they modify the activity of the Notch pathway, however each may control additional unrelated pathways to promote its counter-proteotoxic roles. It would be important to further elucidate the possible links of UFMylation with TGFβ signaling and the IIS. Our tissue specific RNAi analysis (Fig. 7 , a-f) yielded a puzzling observation. While the knockdown of ufm-1 exclusively in neurons or in germline cells alleviates the toxicity of polyQ35-YFP in neurons, the knockdown of ufm-1 in the same tissues is insufficient to protect worms from Aβ-mediated toxicity in muscles. One possible explanation to this conundrum suggests that reduced UFMylation activates distinct mechanisms in the face of dissimilar proteotoxic challenges. According to this theme, the counter-proteotoxic mechanism that mitigates Aβ-mediated toxicity upon the knockdown of ufm-1 is only cell-autonomous while the one which alleviates the toxicity of polyQ-YFP acts cell-autonomously as well as cell-non-autonomously. It is noteworthy that differential responses of the proteostasis network to these two aggregation-prone proteins has been shown previously 30 . Nevertheless, it is also possible that protection from Aβ in the muscle requires the knockdown of ufm-1 in both tissues, germline and muscle whereas the toxicity that stems from nuclear polyQ35-YFP can be alleviated by the reduction of UFMylation in either one of these tissues. This notion may be supported by the observation that germline-specific knockdown of nol-56 mitigates the toxicity of Aβ in muscles 32 . The regulation of cav-1 by RNAi against both nol-56 32 and ufm-1 (Fig. 5 c) points at neurons, the cells that most prominently express cav-1 38 and at caveolae, as possible sites where UFMylation affects proteostasis-orchestrating signaling. While, further experimental work is needed to fully clarify the communication network between germline, neurons and muscles, our results strongly suggest that reduced UFMylation promotes organismal proteostasis. Yet, it is interesting to note that reduced UFMylation appears to have deleterious effects on proteostasis at the cellular level. For instance, mono-UFMylation was reported to confer the jettison of misfolded α-synuclein from cells 48 . These apparently opposing effects of UFMylation raise the prospect that certain biological entities differentially affect proteostasis at the cellular and organismal levels. Our observations show that in the nematode, the effects of lowering UFMylation culminate to alleviate proteotoxicity. It will be also interesting to investigate how different PTMs control the activity of CAR-1. We have previously shown that this protein undergoes SUMOylation which appears to negatively regulates its activity 21 . Our results (Fig. 2 b) show that the rate of CAR-1 UFMylation is reduced with age, raising the questions of how this PTM affects CAR-1 activity and whether SUMOylation and UFMylation compete as regulators of CAR-1 and Notch signaling. This study sheds new light on the roles of UFMylation as a regulator of proteostasis and sets the basis for future research that would elucidate the mechanisms that are governed by this PTM in the context of aging and aging-associated disorders. Declarations Competing interests The authors have no competing interests. Author contribution EC, RBH designed and initiated this study. RBH performed physiological assays including strain crossing, proteotoxicity, lifespan and thrashing assays, Western blots as well as cloning and qPCR procedures. HB conducted paralysis assays, AZ conducted filter-trap assays, HZ crossed proteotoxicity worm models with animals that process RNAi in a single tissue and, IC performed Western blots. RBH prepared samples for RNA-Seq and MS analyses. YHY performed MS analysis. XF and M-Q.D constructed 3xHA-UFM-1 worms using CRISPR-cas9 technology. EC wrote the manuscript. Acknowledgments This study was generously supported by the Israel-China program (EC #3261/20), the Israel Science Foundation (ISF) EC#543/21, the Israeli Ministry of Science and Technology (MOST)(EC#80884), the Henri J. and Erna D. Leir Chair for Research in Neurodegenerative Diseases, as well as by the National Natural Science Foundation of China (NSFC-ISF 32061143020 to M-Q.D). Data availability The NGS raw data is available at GEO: GSE289271 Mass spectrometry data is available at: PRIDE: PXD060906 References Hoppe T, Cohen E (2020) Organismal Protein Homeost Mech Genet 215:889–901 Jayaraj GG, Hipp MS, Hartl FU (2019) Functional Modules of the Proteostasis Network. Cold Spring Harb Perspect Biol Ahammed MS, Wang X (2024) Promoting proteostasis by cAMP/PKA and cGMP/PKG. Trends Mol Med Fleming A et al (2022) The different autophagy degradation pathways and neurodegeneration. Neuron 110:935–966 Hipp MS, Kasturi P, Hartl FU (2019) The proteostasis network and its decline in ageing. 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Worms were grown at 20°C (unless indicated otherwise) on nematode growth media (NGM)-ampicillin plates (100 µg/ml ampicillin) and fed with Escherichia coli HT115 bacteria. Worm strains were provided by the CGC and were also crossed in our lab in previous projects (worm strains are listed at Supplemental Table S4). CF512 worms are heat-sensitive feminized and therefore maintained at 15°C. For avoiding progeny during experiments, CF512 worms were let hatch at 20°C, L1 larvae were transferred to 25°C for 48 h, and back to 20°C thereafter. Worm populations were synchronized using sodium hypochlorite (bleach) and potassium hydroxide. Gene knockdown by RNA interference (RNAi) For RNAi-mediated gene knockdown, E. coli harboring the appropriate RNAi clone were grown in LB medium overnight at 37°C and seeded on NGM-ampicillin plates. Before adding worms to plates, 100 mM isopropyl β-d-1-thiogalactopyranoside (IPTG; final concentration of 4mM) was added to the plates to induce the expression of the dsRNA. Empty vector (EV, pAD12) daf-2 (pAD48) RNAi and daf-16 (pAD43) were a gift from Prof. Andrew Dillin (Berkeley). uba-5, skn‐1 RNAi bacteria were obtained from the C. elegans ORF-RNAi feeding library (the Vidal library). hsf-1 RNAi was from the Ahringer library. All the other RNAi plasmids used in this study were created for this project by amplifying the relevant sequences (see Supplemental Table S5) using PCR and cloning it into the pL4440 plasmid using restriction enzymes. Paralysis and thrashing assays Paralysis assays: CL2006 worms were synchronized by bleach. Eggs were placed on NGM-ampicillin plates seeded with the tested bacteria, and allowed to develop to day 1 of adulthood. For each experiment, at least one hundred and twenty animals were transferred onto small NGM plates seeded with the respective E. coli culture (12 animals / plate). Paralyzed worms were scored daily by tapping the worms' "noses" with a platinum wire. A paralyzed animal was defined as an animal than can move its head but is unable to crawl away. Paralysis assay was terminated at day 12 of adulthood. Trashing assay : synchronized populations of AM140 or AM1126 worms were grown, as described above, on control or RNAi bacteria until day 1 of adulthood. Thrashing rates were determined at the indicated age, by transferring an individual worm into a 10 μl drop of M9 buffer, waiting 30 seconds for adaptation and counting the number of body bends during the next 30 seconds. Thrashing rates of twenty animals were recorded for each treatment at each time point. At least three independent experiments were performed. Lifespan assays Synchronized eggs were placed on NG‐ampicillin plates that were seeded with the indicated RNAi bacteria and supplemented with IPTG. At day 1 of adulthood, 120 animals per treatment were transferred onto small NG‐ampicillin plates (12 worms per plate, total of 10 plates per treatment). Worms that failed to move their tips when tapped twice with a platinum wire were scored as dead. Animals that bagged, disappeared or dried out were censored. SDS-PAGE and Western blot analysis For protein blotting, 6000 worms were treated from hatching with RNAi or left untreated as indicated. At the indicated age the worms were collected and homogenized in M9 supplemented with a protease inhibitor cocktail (Millipore, Billerica, MA, USA; #539134) using a Dounce homogenizer. Worm homogenates were spun for 10 min at 10,000 × g at 4°C to sediment debris. The post debris supernatants were collected and total protein concentrations were measured by a BCA kit (Pierce #23227, Thermofisher, Waltham, MA USA). Then, were supplemented with loading buffer (10% glycerol, 125 mM Tris base, 1% SDS) and boiled for 10 min. 100 μg total protein were loaded into each well. Proteins were separated on a polyacrylamide (PAA) gel, transferred onto a PVDF membrane (for UFM-1 conjugates, GFP conjugates, and ubiquitin conjugates blotting). The membranes were probed with the appropriate antibody: anti-UFM-1 (Abcam, Cat# ab109305), GFP (Cell Signaling, #2037) or FK2 Multiubiquitin chain Monoclonal antibody (Cayman chemical, Cat# CAY-14220). To characterize Aβ quantities and aggregation states both, supernatants and debris were loaded on the gels, transferred onto a nitrocellulose membrane (#66485; Pall Corporation), and probed with an Aβ antibody (Cat#803001; clone 6E10, BioLegend). HRP-conjugated secondary antibody (Jackson ImmunoResearch West Grove, Pennsylvania, USA), chemiluminescence system, and a luminescent image analyzer (Chemidoc XRS+; Biorad) were used to detect proteins. The membrane was stripped in 300 mM NaOH buffer for 5 minutes, re-blocked in 5% BSA and probed with antibody against β-Actin (Cat# A5441, Sigma Aldrich) for normalizing the signals of different proteins. Worm visualization by fluorescent microscopy and foci counting To visualize polyQ35-YFP-containing foci, AM140 worms were anesthetized in 20mM sodium azide and mounted on glass slides. Images were taken using a Nikon AZ100 fluorescent microscopy system. To determine the size distribution of foci, we used the ImageJ software. Automatic threshold was determined, which converted the image into a binary. The particles were filtered by area to include ones between 15 and 1000 pixels. These values ensure excluding of noise and large particles deriving from continuous labeling. Filter trap assay AM140 worms were treated with RNAi bacteria from hatching or left untreated as indicated (four thousand worms per treatment). The worms were washed daily with M9 to discard progeny and collected on day 5 of adulthood. The animals were immediately frozen in liquid N2 and kept in -80°C. The samples were thawed on ice, supplemented with lysis buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X‐100) and with an EDTA‐free protease inhibitor cocktail (Millipore, Billerica, MA, USA; #539134) and homogenized using a Dounce homogenizer. Worm homogenates were spun for 10 min at 10,000 × g at 4°C to sediment debris. The post debris supernatants were collected, protein concentrations were measured by a BCA kit (Pierce #23227, Thermofisher, Waltham, MA USA). 100 μg of each protein extract was supplemented with SDS to the final concentration of 0.5% and loaded onto a cellulose acetate membrane assembled in a slot blot apparatus (Bio‐Rad cat#1703938). The membrane was washed with 0.2% SDS, and the retained polyQ35‐YFP was assessed by immunoblotting with a GFP antibody (Cell Signaling, #2037). RNA isolation and quantitative real-time PCR Total RNA was isolated using a NucleoSpin® RNA isolation kit (MACHEREY-NAGEL; #740955). For each time-point, 10,000 synchronized eggs were placed on NG-ampicillin plates seeded with the indicated RNAi bacteria and supplemented with IPTG. The worms were washed daily with M9 to get rid of progeny and adult nematodes were transferred to new plates. At the indicated age the worms were harvested in M9 and frozen at -80 C. The worms were then thawed and homogenized using Dounce homogenizer. Homogenates were transferred to Eppendorf tubes and centrifuged at 14,000 × g for 5 minutes. The supernatants were transferred to NucleoSpin® Filter (NucleoSpin® RNA kit, Macherey-Nagel, Düren Germany), and total RNA was purified according to the manufacturer’s instructions. The RNA was quantified using a NanoDrop2000c spectrophotometer. For qPCR: cDNA was prepared by reverse transcription of the total RNA samples via random-priming using the iScript TM cDNA Synthesis Kit (#170–8890; Bio-Rad, Hercules, CA, USA) as per the manufacturer’s protocol. Analyzes by qPCR were performed using gene-specific primers (Supplemental Table S6). Analyzes by qPCR were performed with Luna® Universal qPCR Master Mix (Biolabs, Cat#M3003G) and expression levels were normalized to the expression levels of cdc-42 and pmp-3 which served as normalizing genes . qPCR reactions for each gene were performed in triplicates. Next Generation Sequencing (NGS) and computational analysis For NGS,we used RNA ScreenTape kit (catalog #5067-5576; Agilent Technologies, Santa Clara, CA), D1000 ScreenTape kit (catalog #5067-5582; Agilent Technologies) Qubit® RNA HS Assay kit (catalog # Q32852; Invitrogen, Carlsbad, CA) and Qubit® DNA HS Assay kit (catalog #32854; Invitrogen). mRNA libraries were prepared using KAPA Stranded mRNA kit with mRNA Capture Beads (KAPA Biosystems, KK8421). In brief, 1µg was used for the library construction; library was eluted in 20µl of elution buffer. All DNA samples libraries were pooled to 10nM sample. Multiplex samples Pool were loaded on NovaSeq 6000 (Illumina), using NovaSeq 6000 SP Reagent Kit v1.5 100 cycles (cat# 20028401), with 122 cycles of single-end sequencing. Computational analyses of next generation sequencing data Raw reads were processed for quality trimming and adaptors removal using fastq_quality_filter v0.0.14 and cutadapt v1.18 (Marcel M. et al., EMBnet.journal 2011, 17.1:10-12). The processed reads were aligned to the Caenorhabditis elegans transcriptome and genome version WBcel235 with annotations from Ensembl release 106 using TopHat v2.1.1 (Kim D et al., Genome Biology 2013, 14:R36). Counts per gene quantification was done with htseq-count v2.6.1 (Anders S et al., Bioinformatics 2015, 31 (2):166-169). Normalization and differential expression analysis were performed using the DESeq2 package (v1.36.0). Genes with a sum of counts less than 10 over all samples were filtered out, then size factors and dispersion were calculated. Normalized counts were used for several quality control assays, such as counts distributions and principal component analysis, which were calculated and visualized in R. Pair-wise comparisons were tested with default parameters (Wald test), except not using the independent filtering algorithm. Significance threshold was taken as padj<0.01 (default). Finally, results were combined with gene details (such as symbol, known transcripts, etc.), taken from the results of a BioMart query (Ensembl, release 106), to produce the final Excel file. Gene Ontology Classification and Secreted peptide analysis: All significant gene entries were subjected to KEGG and GO classification (http://www.geneontology.org). Significant over-representation of KEGG and GO-classified biological processes was assessed by comparing the number of pertinent genes in a given biological process to the total number of the relevant genes printed on the array for that particular biological process (Fisher exact test) using the publicly accessible software DAVID (http://david.abcc.ncifcrf.gov/summary.jsp). Immuno-precipitation and MS of UFMylated proteins MQD2801 generation : MQD2801 strain ( ufm-1(hq493[ufm-1p::3XHA::ufm-1]) III ) was generated using the CRISPR/Cas9 method. The strain was generated in N2 background and N-terminal 3XHA tag was inserted into the endogenous ufm-1 locus. Then the trangenic worms were crossed for 6 times with N2 worms to clean up the genetic background. For immuno-precipitation , 12,000 MQD2801 worms per sample were grown from hatching on ufm-1 RNAi or left untreated (EV). Half of each sample was collected on day 1 of adulthood, while the remaining worms were washed in M9 daily and transffered to new plates to get rid of progeny. Worms of the second half were collected on day 5 of adulthood. Worms of the two groups were homogenized in 1 ml of lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 1% Triton-X100 (2% for Membrane protein), 10% glycerol, 0.1~2mM PMSF +10 mM NaF +PI (1:1000), using Dounce homogenizer on ice. Worm lysates were transferred into 1.5 ml Eppendorf tubes and centrifuged at 10,000 × g, at 4°C for 30 minutes. Thereafter, The post debris supernatants were collected and transferred to fresh 1.5 ml Eppendorf tubes. Protein concentrations were measured by a BCA kit (Pierce #23227, Thermofisher, Waltham, MA USA) and 1 mg of total protein in a volume of 200 μl lysis buffer was used for immuno-precipitation. 20 μl of Pierce Anti-HA Magnetic Beads were added to worm lysate and samples were rotated for 4 hours at 4°C followed by centrifugation at 1,000 × g, 4°C for 1 minute, and flow throgh was discarded. Then, beads were washed 3 additional times with cold worm lysis buffer (supplemented with protease and phosphatase inhibitors) by rotating 5 min at 4°C and centrifuging at 1,000 × g, 4°C for 1 minute. Finally, beads were washed twice with 1.5 mL/tube 1x cold worm lysis buffer without detergent (tritone) and supernatants were discarded. Tubes were kept in -80°C until processed for MS. Protein identification by MS : Sample preparation for MS analysis : Following immunoprecipitation and washing, the packed beads were resuspended in 100 μl 8M urea, 10 mM DTT, 25 mM Tris-HCl pH 8.0 and incubated for 30 min at 22°C. Next, Iodoacetamide (55 mM) was added and beads were incubated for 30 min (22°C, in the dark), followed by addition of DTT (20 mM). The Urea was diluted by the addition of 7 volumes of 25 mM Tris-HCl pH 8.0. Trypsin was added (0.3 μg/ sample) and the beads were incubated overnight at 37°C with gentle agitation. The beads were spun down and the peptides in the supernatants were desalted on C18 home-made Stage tips. nanoLC-MS/MS analysis: MS analysis was performed using a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA USA) coupled on-line to a nanoflow UHPLC instrument, Ultimate 3000 Dionex (Thermo Fisher Scientific, Waltham, MA USA). Peptides (1.0 μg, as estimated by O.D.280 nm) were separated over a non-linear 90 min gradient (0 - 80% acetonitrile) run at a flow rate of 0.3 μl/min on a reverse phase 25-cm-long C18 column (75 μm ID, 2 μm, 100Å, Thermo PepMapRSLC). The survey scans (380–2,000 m/z, target value 3E6 charges, maximum ion injection times 50 ms) were acquired and followed by higher energy collisional dissociation (HCD) based fragmentation set at 27. A resolution of 70,000 was used for survey scans and up to 15 dynamically chosen most abundant precursor ions, with “peptide preferable” profile was fragmented (isolation window 1.8 m/z). The MS/MS scans were acquired at a resolution of 17,500 (target value 1E5 charges, maximum ion injection times 120 ms). Dynamic exclusion was 60 sec. Data were acquired using Xcalibur software (Thermo Scientific). To avoid a carryover, the column was washed with 80% acetonitrile, 0.1% formic acid for 25 min between samples. MS data analysis: The Mass Spec data was analyzed by searching for matching sequences in the Swissprot (Uniprot) database of the C. elegans proteome containing 26,768 sequences, both annotated and predicted. The search program (Maxquant version 1.5.3.12.) may also include proteins from other species, if they match the program’s database of commonly used species. Peak lists were searched against translated coding sequences of the human proteome obtained from Uniprot. The search included cysteine carbamidomethylation as a fixed modification and oxidation of methionine as variable modifications, allowing up to two miscleavages. The match-between-runs option was used. Peptides with a length of at least seven amino-acids were considered and the required FDR was set to 1% at the peptide and protein level. Protein identification required at least 2 unique or razor peptides per protein. Relative protein quantification in MaxQuant was performed using the label-free quantification (LFQ) algorithm. Protein contaminants and proteins identified by less than 2 peptides were excluded from the analysis. For comparing protein abundances between samples, LFQ intensity (Label Free Quantification) is the accepted parameter. It represents the total protein intensity based on the combined intensities of all the identified peptides of the protein. LFQ intensities are normalized across all the samples, enabling inter-sample comparison of relative protein abundance. LFQ = 0, means the level of protein are either truly 0 or may just be below the threshold needed for calculating LFQ. The analysis was done only on proteins which have a valid LFQ value in at least 4 of the samples in at least one group. Of those proteins left in the analysis, all missing LFQ values were replaced by random low numbers out of a normal distribution to allow for statistical analysis. Statistical analysis and software Statistical analyses were performed using Graph Pad Prism 9 (GraphPad Software, Inc., La Jolla, USA). The statistical tests used, statistical significance, error bars, and sample sizes can be found in the corresponding figure legends. “Statistically significant” was defined as a minimum of p-value < 0.05. Additional Declarations There is NO Competing Interest. Supplementary Files SupplementalTable2incresedUFMylatedMSproteins.xlsx Supplemental table 2 SupplementalTable3decreasedUFMylatedMSproteins.xlsx Supplemental table 3 SupplementalTables46.docx Supplemental tables 4-6 BruckHaimsonetalUFMylationSupplementalfiguresandtext.pdf Supplemental figures and legends Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6064783","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":430224611,"identity":"075e821d-a94c-4f0d-a908-869c0589151c","order_by":0,"name":"Ehud Cohen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYDACCRCqYDBgYEhgY2DgkQALHnhAUMsZdC0JhLQwtsG0wAA+LfzSPYY3fs6zMdZtT2B7zCNjwcDffoARry2Sc84YW/ZuSzMzO/OA3ZgH6DCJMwn4HWZwI8dMmnHbYRuzGwls0iAtDDcI+MUerGXOf4QWeUJaDCRAWhoOmMG1GBDSInEjrdiy51iysdmZh22Sc3gkeAzPJDbg1cI/I3njjR81dobbjicfk3jbUycnd/zw4Q8f8GhBAowNDIw9DDxgBgngBymKR8EoGAWjYKQAAElDSA8aUcVzAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-5552-7086","institution":"The Hebrew University of Jerusalem","correspondingAuthor":true,"prefix":"","firstName":"Ehud","middleName":"","lastName":"Cohen","suffix":""},{"id":430224612,"identity":"5fd6025b-4ebd-412c-aa94-5b2ef53522c6","order_by":1,"name":"Reut Bruck-Haimson","email":"","orcid":"","institution":"The Hebrew University of Jerusalem","correspondingAuthor":false,"prefix":"","firstName":"Reut","middleName":"","lastName":"Bruck-Haimson","suffix":""},{"id":430224613,"identity":"3bec8ae0-7837-4c9a-8b95-b2d5fbafed54","order_by":2,"name":"Hana Boocholez","email":"","orcid":"","institution":"The Hebrew University of Jerusalem","correspondingAuthor":false,"prefix":"","firstName":"Hana","middleName":"","lastName":"Boocholez","suffix":""},{"id":430224614,"identity":"6934cb2f-ab4a-48ed-a735-868e4dc2af63","order_by":3,"name":"Huadong Zhu","email":"","orcid":"","institution":"The Hebrew University of Jerusalem","correspondingAuthor":false,"prefix":"","firstName":"Huadong","middleName":"","lastName":"Zhu","suffix":""},{"id":430224615,"identity":"58f7e668-5629-4c32-a0b9-76fae07655bf","order_by":4,"name":"Adam Zaretsky","email":"","orcid":"","institution":"The Hebrew University of Jerusalem","correspondingAuthor":false,"prefix":"","firstName":"Adam","middleName":"","lastName":"Zaretsky","suffix":""},{"id":430224616,"identity":"0dfc0050-33b3-4a7f-8cf7-64fb7d335d4b","order_by":5,"name":"Irit Cohen","email":"","orcid":"","institution":"The Hebrew University of Jerusalem","correspondingAuthor":false,"prefix":"","firstName":"Irit","middleName":"","lastName":"Cohen","suffix":""},{"id":430224617,"identity":"d6220d54-c8e9-4f94-b498-db27563b98b1","order_by":6,"name":"Xiaofeng Feng","email":"","orcid":"","institution":"National Institute of Biological Sciences (NIBS)","correspondingAuthor":false,"prefix":"","firstName":"Xiaofeng","middleName":"","lastName":"Feng","suffix":""},{"id":430224618,"identity":"0bfa3a92-9e41-4b81-a7e6-412104de3536","order_by":7,"name":"Yonghong Yan","email":"","orcid":"","institution":"National Institute of Biological Sciences (NIBS)","correspondingAuthor":false,"prefix":"","firstName":"Yonghong","middleName":"","lastName":"Yan","suffix":""},{"id":430224619,"identity":"f849ee02-86ed-452b-83a6-e137bd289188","order_by":8,"name":"Meng-Qiu Dong","email":"","orcid":"https://orcid.org/0000-0002-6094-1182","institution":"National Institute of Biological Sciences","correspondingAuthor":false,"prefix":"","firstName":"Meng-Qiu","middleName":"","lastName":"Dong","suffix":""}],"badges":[],"createdAt":"2025-02-19 14:00:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6064783/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6064783/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79073266,"identity":"dfafa2dc-d443-489d-9333-5d727c4b84f5","added_by":"auto","created_at":"2025-03-24 06:37:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":771294,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUFMylation reduction protects against proteotoxicity and extends lifespan.\u003c/strong\u003e\u003cbr\u003e\n(a) Western blot analysis of global protein UFMylation patterns in homogenates of 1, 5, 10 and 15 days old CF512 worms that were treated with \u003cem\u003edaf-2\u003c/em\u003e RNAi or left untreated. Arrows indicate proteins which exhibit increased UFMylation levels, while the arrowhead marks a protein that shows reduced level of UFMylation upon IIS reduction. (b-c) Paralysis assays of CL2006 worms expressing Aβ in body wall muscles, treated with \u003cem\u003eufm-1\u003c/em\u003e RNAi (b) or \u003cem\u003euba-\u003c/em\u003e5 (c) compared control bacteria (EV), showing reduced paralysis rates upon \u003cem\u003eufm-1\u003c/em\u003eor \u003cem\u003euba-\u003c/em\u003e5 knockdown. N=4 independent experiments, bars represent average daily rates of paralysis in the population± SEM. Statistical test used: Two-way ANOVA followed by Holm-Sidak correction for multiple comparisons, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. (d-e) Thrashing assays of AM140 worms expressing polyQ35-YFP in muscles that were treated with \u003cem\u003eufm-1\u003c/em\u003eRNAi (d) or \u003cem\u003euba-5\u003c/em\u003e RNAi (e). Proteotoxicity is significantly mitigated at days 4 and 6, but only \u003cem\u003euba-5\u003c/em\u003e RNAi also alleviates proteotoxicity at day 2 of adulthood. N=4 (d) and N=3 (e) independent experiments. Bars represent average thrashing rate within the population ± SEM. Statistical test used: Two-way ANOVA followed by Holm-Sidak correction for multiple comparisons **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. (f) Similar protection was observed in AM1126 worms expressing polyQ35-YFP in neurons, upon \u003cem\u003eufm-1\u003c/em\u003e RNAi. N=3 independent experiments. Bars represent average thrashing rate within the population ± SEM. Statistical test used: Two-way ANOVA followed by Holm-Sidak correction for multiple comparisons **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. (g) Lifespan assays using N2 worms treated with \u003cem\u003eufm-1\u003c/em\u003e or \u003cem\u003euba-5\u003c/em\u003eRNAi, showing modest but significant lifespan extension upon \u003cem\u003eufm-1\u003c/em\u003eknockdown. (h, i) Lifespan assays of AM140 (h) and CL2006 (i) worms that were treated with either \u003cem\u003eufm-1\u003c/em\u003e or \u003cem\u003euba-5\u003c/em\u003e RNAi, demonstrating that reduced UFMylation extends lifespan in worms expressing proteotoxic proteins.\u003c/p\u003e","description":"","filename":"BruckHaimsonetalUFMylationFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6064783/v1/4db51cb5780571f2a731bc2b.png"},{"id":79072823,"identity":"3cc8f57e-0054-4219-b209-054057b6465a","added_by":"auto","created_at":"2025-03-24 06:29:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":355619,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtein UFMylation is modulated with age. \u003c/strong\u003e(a) An illustration of the experimental workflow for the isolation of differentially UFMylated proteins. We created transgenic worms, expressing 3xHA-tagged endogenous UFM-1 (strain MQD2801). Synchronized MQD2801 worms were grown on control bacteria, harvested at either day 1 or 5 of adulthood, homogenized and UFMylated proteins were pulled down by an HA kit. Sedimented proteins were identified by mass spectrometry. (b) A volcano plot of the immuno-precipitated, differentially UFMylated proteins in day 1 and 5 adult worms, revealing 155 UFMylated proteins, 38 downregulated and 23 upregulated with age. (c, d) Functional clustering of differentially UFMylated proteins, highlighting ribosomal proteins as well as lifespan regulating proteins. (e) Tissue enrichment analysis of UFMylated proteins, showing significant representation in muscles, neurons, and germline.\u003c/p\u003e","description":"","filename":"BruckHaimsonetalUFMylationFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6064783/v1/57092b845d494e078cb61073.png"},{"id":79073927,"identity":"a304c3bb-4cb6-4d7c-ba88-52ebbcc1901c","added_by":"auto","created_at":"2025-03-24 06:46:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":327067,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentially UFMylated chaperones are crucial for the counter proteotoxic effect of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eufm-1 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eRNAi. \u003c/strong\u003e(a) Paralysis assay of CL2006 worms grown on EV\u003cem\u003e, ufm-1\u003c/em\u003e RNAi, \u003cem\u003esip-1\u003c/em\u003e RNAi or \u003cem\u003eufm-1/sip-1\u003c/em\u003e dual silencing cassette, showing that a concurrent knockdown of \u003cem\u003esip-1\u003c/em\u003e and \u003cem\u003eufm-1 \u003c/em\u003eabolishes the protective effect of \u003cem\u003eufm-1\u003c/em\u003e RNAi. N=240 worms per treatment in two independent experiments. (b) Paralysis assay of CL2006 worms grown on EV\u003cem\u003e, ufm-1\u003c/em\u003e RNAi, \u003cem\u003edaf-21\u003c/em\u003e RNAi or \u003cem\u003eufm-1/daf-21\u003c/em\u003e dual silencing cassette, showing that the concurrent KD knockdown of \u003cem\u003edaf-21\u003c/em\u003e and \u003cem\u003eufm-1 \u003c/em\u003eabolishes the protective effect of \u003cem\u003eufm-1\u003c/em\u003e RNAi. N=360 worms per treatment in two independent experiments, Bars represent average daily rates of paralysis within the population± SEM. (c) Paralysis assay of CL2006 worms grown on EV\u003cem\u003e, ufm-1\u003c/em\u003e RNAi, \u003cem\u003ehsp-1\u003c/em\u003e RNAi or \u003cem\u003eufm-1/hsp-1\u003c/em\u003e dual silencing cassette, showing that the concurrent KD of \u003cem\u003ehsp-1\u003c/em\u003e and \u003cem\u003eufm-1 \u003c/em\u003eabolishes the protective effect of \u003cem\u003eufm-1\u003c/em\u003e RNAi. N=360 worms per treatment in three independent experiments. For all paralysis assays displayed in this figure, bars represent average daily rates of paralysis of the population± SEM. Statistical test used: One way ANOVA followed by Tukey-Kramer correction for multiple comparisons, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"BruckHaimsonetalUFMylationFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6064783/v1/a0311847f8e40740f345abeb.png"},{"id":79073264,"identity":"990f2add-445c-4190-a9b6-bd0c35d1fd11","added_by":"auto","created_at":"2025-03-24 06:37:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":472748,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe transcription factors DAF-16 and SKN-1 are required for the counter-proteotoxic effect of UFMylation reduction. \u003c/strong\u003e(a-d) Paralysis assay using CL2006 worms (a, c) and thrashing assay utilizing AM140 animals (b, d). The worms were treated from hatching with the indicated dual RNAi silencing cassettes or with RNAi toward \u003cem\u003eufm-1 \u003c/em\u003eor the corresponding transcription factor. (a-b) The concurrent knockdown of \u003cem\u003eufm-1\u003c/em\u003e and \u003cem\u003edaf-16\u003c/em\u003eabolished the protection from Aβ (a) and polyQ35-YFP (b), that was observed when \u003cem\u003eufm-1 \u003c/em\u003ewas exclusively knocked down. (c-d) Similarly, a concurrent knockdown of \u003cem\u003eufm-1\u003c/em\u003e and \u003cem\u003eskn-1\u003c/em\u003e by RNAi abolished the UFMylation reduction-mediated protection from proteotoxicity both in CL2006 (c) and in AM140 (d) worms. (a, c) N=3 independent experiments, 590 CL2006 worms per treatment. Bars represent average daily rates of paralysis within the population± SEM. Statistical test used: two-way ANOVA followed by Holm-Sidak correction for multiple comparisons, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. (b, d) N=3 independent experiments, 60 AM140 worms per treatment. Bars represent average thrashing rate within the population ± SEM. Statistical test used: two-way ANOVA followed by Holm-Sidak correction for multiple comparisons **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"BruckHaimsonetalUFMylationFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6064783/v1/4f8d78b845579890363f4761.png"},{"id":79072837,"identity":"df16eff5-fc6a-478c-9cf1-5a8d820d1b63","added_by":"auto","created_at":"2025-03-24 06:29:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":267737,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReduced UFMylation modulates Transcriptomic landscape. \u003c/strong\u003e(a) RNA sequencing analysis to compare gene expression profiles in untreated CL2006 worms and their counterparts that were treated with \u003cem\u003eufm-1\u003c/em\u003e RNAi for two generations. 143 genes exhibited differential expression levels, whereas 89 genes showed upregulation and 54 genes showed downregulation upon the knockdown of \u003cem\u003eufm-1\u003c/em\u003e. (b) Gene ontology clustering of the upregulated genes showed an enrichment in immune response and extracellular signaling. Downregulated genes showed enrichment of for reproduction-associated functions. (c) Heatmap of a selected set of genes that were identified in the RNA-sequencing experiment, including proteostasis regulators (\u003cem\u003efbxb-45, fbxb-66, cav-1\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"BruckHaimsonetalUFMylationFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6064783/v1/e293860bc79c4b166c57234e.png"},{"id":79072830,"identity":"64d59a64-8269-4c13-8f29-4fdc52a11c02","added_by":"auto","created_at":"2025-03-24 06:29:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":648008,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUFMylation reduction decreases protein aggregation and enhances proteasome activity. \u003c/strong\u003e\u003cbr\u003e\n(a, b) Western blot analysis of Aβ in homogenates of day 1 and 6 old, CL2006 worms that were treated with \u003cem\u003eufm-1\u003c/em\u003e RNAi or left untreated. Aβ levels were reduced in the soluble phase of day 6 old worms but not at day 1, nor in insoluble phases at both ages. Bars represent average signal intensity of 4 replicates ± SEM. Statistical test used: Unpaired, two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. Confidence level 95% *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. (c, d) Filter-trap assay using day 5 old AM140 worms, shows decreased levels of SDS resistant polyQ35-YFP aggregates in homogenates of \u003cem\u003eufm-1\u003c/em\u003e RNAi treated worms. Bars represent average signal intensity of 5 replicates ± SEM. Statistical test used: Unpaired, two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. (e, f) Western blot analysis of ubiquitinated proteins in day 6 old CL2006 worms, revealing reduced level of ubiquitin conjugates in homogenates of day 6 old \u003cem\u003eufm-1\u003c/em\u003eRNAi-treated animals compared to their untreated counterparts, indicative of enhanced proteasome activity. Bars represent average signal intensity of 4 replicates ± SEM. Statistical test used: Unpaired, two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"BruckHaimsonetalUFMylationFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6064783/v1/9fc627ff5923ed90c9340587.png"},{"id":79074593,"identity":"261fec82-61d4-4381-a7be-5fc7c8ad82af","added_by":"auto","created_at":"2025-03-24 06:54:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":569102,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUFMylation reduction regulates proteostasis both cell-autonomously and non-autonomously. \u003c/strong\u003e(a) Neuronal-specific knockdown of \u003cem\u003eufm-1\u003c/em\u003eprotects the worms from neuronal polyQ35-YFP as measured by thrashing assay. (b) The knockdown of \u003cem\u003eufm-1\u003c/em\u003e solely in the germline protects worms from neuronal polyQ35-YFP, as measured by thrashing assay. For A and B panels: N=3 independent experiments, bars represent average thrashing rate within the population ± SEM. Statistical test used: Two-way ANOVA followed by Holm-Sidak correction for multiple comparisons ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. (c) Muscle-specific knockdown of \u003cem\u003eufm-1\u003c/em\u003emitigates the toxicity of neuronal polyQ35-YFP as measured by paralysis assay, indicating a non-autonomous proteostasis regulation. (d) Muscle-specific \u003cem\u003eufm-1\u003c/em\u003eRNAi alleviates the toxicity of Aβ in a cell-autonomously fashion. For c and d: N=3 independent experiments, bars represent average daily rates of paralysis of the population± SEM. Statistical test used: Two-way ANOVA followed by Holm-Sidak correction for multiple comparisons, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. (e-f) Neuron-specific knockdown of \u003cem\u003eufm-1\u003c/em\u003e (e) or germling-specific knockdown of \u003cem\u003eufm-1\u003c/em\u003e (f) did not affect the toxicity of Aβ in muscles. Statistical test used within 10 plates/treatment of the experiment: Two-way ANOVA followed by Holm-Sidak correction for multiple comparisons. (g) Model depicting the regulation of UFMylation on proteostasis. UFMylation affects at least two aging-regulating protein complexes, the neuronal FIB-1/NOL-56 complex (i) and the germline resident CAR-1/CGH-1 mechanism (ii). Accordingly, the knockdown of \u003cem\u003eufm-1\u003c/em\u003e reduces the age-associated increase the activity of FIB-1/NOL-56 complex and elevates the activity of CAR-1/CGH-1 thereby inhibiting GLP-1 activity (iii). These modulations enhance the activity of DAF-16 (iv) and possibly of SKN-1. The increased activities of these transcription factors elevate the expression of certain chaperones and enhance UPS activity (v). These activities promote proteostasis in muscles and neurons (vi).\u003c/p\u003e","description":"","filename":"BruckHaimsonetalUFMylationFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6064783/v1/9ba5e146ca6ff47bcc596646.png"},{"id":79074778,"identity":"d11c259b-e5b3-4b88-a576-39c06af8fdfe","added_by":"auto","created_at":"2025-03-24 07:02:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4864465,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6064783/v1/81033ab6-2268-44e0-9bbc-b8eb96c637be.pdf"},{"id":79073924,"identity":"6ed5dbcb-701c-450f-a6d8-12c8d5095b76","added_by":"auto","created_at":"2025-03-24 06:45:51","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11550,"visible":true,"origin":"","legend":"Supplemental table 2","description":"","filename":"SupplementalTable2incresedUFMylatedMSproteins.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6064783/v1/0eace903ef23e7d00fa0adba.xlsx"},{"id":79072818,"identity":"a0d82ccd-3b54-4c57-97e5-5226477e9eba","added_by":"auto","created_at":"2025-03-24 06:29:51","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12733,"visible":true,"origin":"","legend":"Supplemental table 3","description":"","filename":"SupplementalTable3decreasedUFMylatedMSproteins.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6064783/v1/f0c5f31b3e24c340daae3ba1.xlsx"},{"id":79072819,"identity":"1d01cc93-0332-4d8f-a0df-0902cc83969f","added_by":"auto","created_at":"2025-03-24 06:29:51","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":20247,"visible":true,"origin":"","legend":"Supplemental tables 4-6","description":"","filename":"SupplementalTables46.docx","url":"https://assets-eu.researchsquare.com/files/rs-6064783/v1/b98b0834298e4ef42df78743.docx"},{"id":79074581,"identity":"ac7a243c-b777-4303-b4c7-07f95c923746","added_by":"auto","created_at":"2025-03-24 06:53:56","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":750044,"visible":true,"origin":"","legend":"Supplemental figures and legends","description":"","filename":"BruckHaimsonetalUFMylationSupplementalfiguresandtext.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6064783/v1/9176d3cdd5cf063f9442214f.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Aging-Associated Modulation of UFMylation Impairs Proteostasis in C. elegans","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProtein maturation is a complex, multi-step process that is critical for cellular and organismal functionality and viability \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Right after synthesis, the nascent polypeptide has to attain its desired spatial structure, get proper post-translational modifications and often interact with other proteins to form functional complexes. Although these maturation steps are tightly supervised and assisted by a nexus of chaperones and specialized mechanisms \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, not all nascent polypeptides maturate properly. Early in life, unfolded polypeptides are either refolded or, when terminally misfolded, cleared by highly conserved degradation mechanisms, namely the ubiquitin proteasome system (UPS) \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e and autophagy \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Alas, as the organism ages, the competence of the protein homeostasis (proteostasis) network declines, and proteins that bear an inherent propensity to misfold, accumulate in the cell and form hazardous aggregates \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. This process jeopardizes cellular viability and can underlie the development of late-onset disorders that are collectively known as \u0026ldquo;proteinopathies\u0026rdquo; \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Neurodegenerative diseases such as Alzheimer\u0026rsquo;s disease (AD) and Huntington\u0026rsquo;s disease (HD) consist a prominent class of proteinopathies. Accordingly, aging is the most prominent risk factor for the development of these maladies which emanate from aberrant protein aggregation \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlzheimer\u0026rsquo;s disease (AD), the most prevalent dementia causing disorder, stems from the cleavage of the trans membranal \u0026ldquo;Amyloid Precursor Protein\u0026rdquo; (APP), by two proteolytic entities, the β and ɣ secretases. This dual digestion releases a family of aggregation-prone peptides that were collectively termed the amyloid β (Aβ) peptides. The accumulation and aggregation of Aβ peptides lead to the formation of toxic oligomers \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e that initiate a pathophysiological process which results in neuronal loss and dementia \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Similarly, abnormally long stretches of poly glutamine (polyQ) in the sequences of different proteins, render these polypeptides prone to aggregate, and underlie the development of at least nine late-onset human neurodegenerative illnesses \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Among these maladies, Huntington\u0026rsquo;s disease (HD) is most prevalent.\u003c/p\u003e \u003cp\u003eThe observation that neurodegenerative disorders manifest in late stages of life, raised the question of whether aging and toxic protein aggregation (proteotoxicity) are mechanistically linked. To address this, we and others employed the nematode \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e (\u003cem\u003eC. elegans\u003c/em\u003e), a preferred model organism for the study of aging and proteostasis \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The alteration of aging, by either knocking down the expression of the sole insulin/IGF1 receptor \u003cem\u003edaf-2\u003c/em\u003e, or by dietary restriction (DR), both known as aging regulating manipulations \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e mitigate proteotoxicity that emanated from the expression of neurodegeneration-causing aggregation-prone proteins \u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. These counter-proteotoxic effects were shown to be conserved from worms to mammals \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, and to be entirely dependent upon the activities of aging-controlling transcription factors. These include DAF-16/FOXO, SKN-1/NRF (hereafter DAF-16 and SKN-1 respectively), the ParaQuat Methylviologen responsive (PQM-1) factor \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and the heat shock factor 1 (HSF-1) \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhile the modulation of gene expression plays key roles in the regulation of aging and proteostasis, post-translational modifications (PTMs) of proteins are also involved in these mechanisms. First, ubiquitination variations were found to be involved in the regulation of aging \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In addition, SUMOylation, the attachment of a small ubiquitin-like modifier (SUMO) to specific lysine residues, regulates the activity of the protein CAR-1 which controls signaling that stems from the reproductive system, and affects lifespan and proteostasis \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Importantly, SUMOylation also plays key roles in the development of distinct neurodegenerative disorders \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, and PTM patterns of the protein TAU are correlated with heterogeneity of Alzheimer\u0026rsquo;s patients \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. These findings highlight the key roles of PTMs and raise the question of which additional PTMs are involved in the regulation of the mechanisms of aging and proteostasis.\u003c/p\u003e \u003cp\u003eHere we asked whether UFMylation, a protein modification mechanism which attaches a polypeptide of 94 amino acids, known as \u0026ldquo;ubiquitin-fold modifier 1\u0026rdquo; (UFM-1), to lysine residues of its target proteins \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, is involved in the regulation of proteostasis in the nematode. In mammals, the UFMylation pathway is initiated by the cleavage of UFM-1\u0026rsquo;s C-terminus by two UFM-1 specific cysteine proteases UFSP1 and UFSP2, to expose the glycine residue. Thereafter, the UFM-1 conjugation cascade consists of UBA-5 (ubiquitin-like modifier activating enzyme, E1), UFC-1 (UFM-1 conjugating enzyme 1, E2), and UFL-1 (UFM-1-specific ligase 1, E3) \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003euba-5\u003c/em\u003e mutant worms exhibited increased resistant to stresses such as oxidation and elevated temperatures \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In mammals, mutations in the sequence of \u003cem\u003euba5\u003c/em\u003e cause fatal congenital neuropathy \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, directly links UFMylation with brain development. Genetic studies in humans revealed that variants in human \u003cem\u003euba5\u003c/em\u003e, \u003cem\u003eufc1\u003c/em\u003e, and \u003cem\u003eufm1\u003c/em\u003e are associated with a number of neurodevelopmental diseases, including infantile-onset encephalopathy \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Surprisingly, we found that reducing the rate of UFMylation by RNAi-mediated knockdown of \u003cem\u003eufm-1\u003c/em\u003e or of \u003cem\u003euba-5\u003c/em\u003e, mitigates proteotoxicity that emanates from the expression of Aβ or of polyQ35-YFP. It also slightly extended the lifespan of worms that express these aggregative proteins. Functional assays unveiled that the transcription factors DAF-16 and SKN-1, as well as the chaperones DAF-21 and SIP-1 are critically needed for this protection, which is mediated, at least partially by the enhancement of protein degradation. Our results imply that UFMylation impairs proteostasis in the face of chronic proteotoxic stress and suggest that a selective manipulation of this PTM could be useful for the treatment of these proteinopathies.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReduced UFMylation mitigates proteotoxicity and extends lifespan\u003c/h2\u003e \u003cp\u003eWe first sought to test whether aging and reducing the activity of the insulin/IGF1 signaling cascade (IIS), modulate global protein UFMylation in adult worms. To avoid possible background from developing embryos we utilized CF512, worms that are feminized when exposed to 25\u0026deg;C during development. Synchronized eggs were placed on plates that were seeded with \u003cem\u003edaf-2\u003c/em\u003e RNAi bacteria or with control bacteria harboring the empty RNAi vector (EV). The plates were incubated at 25\u0026deg;C for 48 hours and transferred to 20\u0026deg;C until harvested at either day 1, 5, 10 or 15 of adulthood. The worms were homogenized and subjected to Western blot analysis using a UFM-1 antibody, to compare protein UFMylation patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). We observed several proteins that their UFMylation levels were increased with age, as well as in worms that were treated with \u003cem\u003edaf-2\u003c/em\u003e RNAi (black arrows). At least one protein exhibited reduced level of UFMylation upon IIS reduction (arrowhead). These results indicated that the UFMylation pattern of the \u003cem\u003eC. elegans\u003c/em\u003e proteome is modulated by aging and by IIS reduction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe aging-associated variations in protein UFMylation has led us to wonder whether these changes affect proteostasis. To address this, we employed worms that express Aβ in their body wall muscle (strain CL2006 \u003csup\u003e28\u003c/sup\u003e), thereby exhibiting a progressive paralysis phenotype within the population \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. This phenomenon can be used for the measurement of Aβ-mediated proteotoxicity. Aβ worms were grown from hatching on \u003cem\u003eufm-1\u003c/em\u003e RNAi (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, a and b), and subjected to the paralysis assay. Surprisingly, four independent experiments showed that the knockdown of \u003cem\u003eufm-1\u003c/em\u003e significantly mitigates Aβ-mediated proteotoxicity, as at day 12 of adulthood the rate of paralysis among \u003cem\u003eufm-1\u003c/em\u003e RNAi treated Aβ worms was approximately 35% lower than in their untreated counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). To further scrutinize this observation, we knocked down the expression of \u003cem\u003euba-5\u003c/em\u003e by RNAi (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, c and d), and observed an analogous protection from Aβ proteotoxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Similar results were obtained when we reduced UFMylation by treating Aβ worms with RNAi toward \u003cem\u003eodr-8\u003c/em\u003e (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, e-g), a gene that encodes a specific protease whose activity is critically needed for the maturation of UFM-1 \u003csup\u003e29\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs certain components of the proteostasis network can differentially respond to distinct proteotoxic challenges \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e we tested whether the counter-proteotoxic effect of reduced UFMylation is Aβ specific. To address this, we employed worms that express a stretch of 35 glutamines fused to the \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ey\u003c/span\u003eellow \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ef\u003c/span\u003eluorescent \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ep\u003c/span\u003erotein (polyQ35-YFP), in their body wall muscles (strain AM140). This aggregation-prone protein, impairs motility, a phenotype that can be followed by the thrashing assay \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. We found that the knockdown of both, \u003cem\u003eufm-1\u003c/em\u003e and of \u003cem\u003euba-5\u003c/em\u003e alleviates the proteotoxic effect of polyQ35-YFP. However, while the counter-proteotoxic effect of \u003cem\u003eufm-1\u003c/em\u003e RNAi was significant at day 4 and 6 old worms, but not in young animals (day 2 of adulthood, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), the knockdown of \u003cem\u003euba-5\u003c/em\u003e significantly reduced the polyQ35-YFP-mediated toxicity at all ages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eSimilar protection was observed when UFMylation was reduced in worms that express polyQ35-YFP in their neurons (AM1126), by \u003cem\u003eufm-1\u003c/em\u003e RNAi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef) or \u003cem\u003euba-5\u003c/em\u003e RNAi (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003eThese results indicate that reduced UFMylation levels alleviate proteotoxicity that stems from distinct aggregation-prone proteins. They also show that reduced UFMylation mitigates polyQ35-YFP-mediated toxicity, regardless of whether they are expressed in muscles or neurons.\u003c/p\u003e \u003cp\u003eGiven in proteostasis enhancement and longevity are not necessarily coupled \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, we asked whether reducing UFMylation affects lifespan. Wild-type worms (strain N2) were left untreated (EV) or fed from hatching with RNAi bacteria toward either \u003cem\u003eufm-1\u003c/em\u003e or \u003cem\u003euba-5\u003c/em\u003e and living animals were scored daily. While the knockdown of \u003cem\u003eufm-1\u003c/em\u003e slightly, but significantly (p\u0026thinsp;=\u0026thinsp;0.0057), extended lifespan, no extension was observed in \u003cem\u003euba-5\u003c/em\u003e treated animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg and Supplemental table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Similarly, the knockdown of \u003cem\u003eufm-1\u003c/em\u003e, but not of \u003cem\u003euba-5\u003c/em\u003e, extended the lifespan of CF512 worms (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eI, p\u0026thinsp;=\u0026thinsp;0.0024). Next, we tested whether the reduced UFMylation is capable of extending the lifespans of worms that are challenged by proteotoxicity. Treating AM140 worms (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh) and CL2006 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei) animals with either \u003cem\u003eufm-1\u003c/em\u003e or \u003cem\u003euba-5\u003c/em\u003e RNAi, we found that the knockdown of either \u003cem\u003eufm-1\u003c/em\u003e or of \u003cem\u003euba-5\u003c/em\u003e significantly extend lifespans of animals of both proteotoxicity models by approximately 13% compared to their untreated counterparts (Supplemental table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn sum, our results show that reduced rates of UFMylation extend lifespan, however this effect is more prominent when the animals are challenged by proteotoxic proteins.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eUFMylation of proteostasis modulating proteins affects proteotoxicity\u003c/h3\u003e\n\u003cp\u003eTo elucidate the mechanism which promotes proteostasis upon UFMylation reduction, we sought to identify the proteins that are differentially UFMylated with age. Using CRISPR cas9 technology we tagged the endogenous \u003cem\u003eufm-1\u003c/em\u003e gene with a triple HA sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, strain MQD2801). This tagging enabled us to specifically immuno-precipitate UFMylated proteins, using an HA antibody, and identify them by mass-spectrometry. We cultured synchronized populations of MQD2801 worms on control bacteria (EV) and harvested them at either day 1 or 5 of adulthood. The worms were homogenized, debris were sedimented, UFMylated proteins were immuno-precipitated from the cleared supernatants and identified by mass spectrometry (MS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, complete MS dataset is available at: PXD060906). Total of 155 UFMylated proteins were identified whereas 94 (60.7%) showed no significant difference in UFMylation levels between days 1 and 5, 23 showed increased (14.8%) and 38 exhibited a reduced (24.5%) UFMylation levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and Supplemental tables 2 and 3). Functional clustering of proteins that exhibited modulated UFMylation levels at day 5 of adulthood (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, c and d) indicated that ribosomal proteins are most abundant, suggesting that UFMylation may regulate translation. However, aging-regulating proteins were also identified. These include NOL-56 and FIB-1, two components of a nucleolar complex which we recently found to regulate organismal proteostasis by modulating TGFβ signaling \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Both, NOL-56 and FIB-1 exhibit aging-associated increased UFMylation rates, implying that this PTM positively regulates the activity of the FIB-1-NOL-56 complex. Accordingly, aging-associated increase in the rate of UFMylation enhances the activity of the FIB-1-NOL-56 complex thereby promoting proteotoxicity. To functionally test the possible role of FIB-1 in the regulation of proteostasis by UFMylation, we created a dual-silencing RNAi cassette that concurrently knocks down the expression of \u003cem\u003eufm-1\u003c/em\u003e and \u003cem\u003efib-1\u003c/em\u003e (the dilution of \u003cem\u003eufm-1\u003c/em\u003e RNAi lowers the efficiency of its counter-proteotoxic effect (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ea)). Aβ worms were either left untreated (EV) or treated with RNAi toward \u003cem\u003eufm-1, fib-1\u003c/em\u003e or with the \u003cem\u003eufm-1/fib-1\u003c/em\u003e cassette, and subjected to a paralysis assay. We found that a concomitant knockdown of \u003cem\u003eufm-1\u003c/em\u003e and \u003cem\u003efib-1\u003c/em\u003e shows no reduction in the rate of paralysis below the level which is mediated by a sole knockdown of \u003cem\u003efib-1\u003c/em\u003e (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eb). These results culminate to show that the rate of UFMylation of FIB-1 and NOL-56 is increased with age and infer that the reduction of UFMylation promotes proteostasis, at least partially, via the mechanism downstream of the FIB-1-NOL-56 complex.\u003c/p\u003e \u003cp\u003eAn additional interesting finding is the decreased UFMylation levels of CAR-1 and CGH-1 in day 5 old animals. These two proteins negatively regulate notch signaling during oogenesis \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, and are modulators of proteostasis across tissues. Interestingly, these functions are governed by SUMOylation which is controlled by the IIS \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The age-associated decreased UFMylation levels of these proteins infers that the knockdown of \u003cem\u003eufm-1\u003c/em\u003e also controls proteostasis by modulating notch signaling, which originates from the germline and regulate lifespan \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. According to this notion, the counter-proteotoxic effect of \u003cem\u003eufm-1\u003c/em\u003e RNAi is expected to be dependent on DAF-16, which is needed for the longevity phenotype of worms that express a mutated, hyper-active CAR-1 \u003csup\u003e21\u003c/sup\u003e and to be orchestrated by inter-tissue communication.\u003c/p\u003e \u003cp\u003eTwo chaperones have also shown modulations in UFMylation levels with age. SIP-1, a small heat shock protein that plays key roles in the maintenance of proteostasis \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e exhibited decreased UFMylation at day 5 old worms compared to their day 1 old counterparts. In addition, HSP-1, a HSP-70 family member, showed increased UFMylation in day 5 old animals.\u003c/p\u003e \u003cp\u003eFinally, HSP-90 (encoded by \u003cem\u003edaf-21\u003c/em\u003e), a chaperone which is crucial for the mediation of proteostasis \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, is also a UFMylated protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eClustering the differentially UFMylated proteins according to the tissue of expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee) showed enrichment for proteins that reside in muscles, the tissue that is challenged by proteotoxicity in these animals as well as in the neuronal and reproductive systems.\u003c/p\u003e \u003cp\u003eWe further examined the physiological roles of UFMylation in the regulation of proteostasis by creating dual silencing RNAi cassettes that concomitantly knockdown \u003cem\u003eufm-1\u003c/em\u003e and either \u003cem\u003esip-1, daf-21\u003c/em\u003e or \u003cem\u003ehsp-1\u003c/em\u003e (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e, a-f). CL2006 were grown on bacteria that express either one of these RNAi cassettes, the corresponding chaperone or \u003cem\u003eufm-1\u003c/em\u003e RNAi, and paralysis assays were conducted. While treatment with RNAi toward \u003cem\u003esip-1\u003c/em\u003e was initiated from hatching, genes that encode the other chaperones were knocked down from day 1 of adulthood, to allow complete development of the worms. Our results show that a concurrent knockdown of \u003cem\u003eufm-1\u003c/em\u003e and of each of these chaperone-encoding genes, completely abolishes the counter-proteotoxic effect of reduced UFMylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, a-c). These results infer that these three chaperones are crucial for the alleviation of Aβ-mediated proteotoxicity by \u003cem\u003eufm-1\u003c/em\u003e RNAi. To expand our investigation, we created silencing cassettes which enabled us to test whether seven additional proteins that exhibited differential UFMylation levels among day 1 and 5 old untreated worms (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), are also needed for the protection from Aβ that is conferred by the knockdown of \u003cem\u003eufm-1.\u003c/em\u003e Utilizing CL2006 worms and the paralysis assay we found that the knockdown of \u003cem\u003ekin-19\u003c/em\u003e, which encodes a kinase that negatively regulates the Wnt signaling pathway \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, prevents \u003cem\u003eufm-1\u003c/em\u003e RNAi from alleviating the paralysis phenotype (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eg). Interestingly, the knockdown of \u003cem\u003eatp-2\u003c/em\u003e, that codes for a mitochondrial ATP synthase, alleviated the paralysis phenotype regardless of whether \u003cem\u003eufm-1\u003c/em\u003e has been knocked down or not (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eh). In contrast, a concurrent knockdown of \u003cem\u003eufm-1\u003c/em\u003e and either one of the other five genes (\u003cem\u003elfi-1, ant-1.1, cpl-1, trap-1\u003c/em\u003e and \u003cem\u003etop-\u003c/em\u003e1), did not prevent \u003cem\u003eufm-1\u003c/em\u003e RNAi from mitigating proteotoxicity (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e, i-m). Together, these results confirm that \u003cem\u003edaf-21, sip-1, hsp-1\u003c/em\u003e and \u003cem\u003ekin-19\u003c/em\u003e are crucial for the counter proteotoxic effect of \u003cem\u003eufm-1\u003c/em\u003e RNAi.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eDAF-16 and SKN-1 are required for mitigation of proteotoxicity by reduced UFMylation\u003c/h3\u003e\n\u003cp\u003eThe observations that reducing the levels of \u003cem\u003edaf-21\u003c/em\u003e and of \u003cem\u003ekin-19\u003c/em\u003e prevent the knockdown of \u003cem\u003eufm-1\u003c/em\u003e from alleviating paralysis, imply that UFMylation regulates proteostasis, at least partially, by modulating the activities of signaling pathways. Since aging-regulating transcription factors have been shown to be needed for the promotion of proteostasis by signaling mechanisms \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, we asked whether the transcription factors DAF-16, SKN-1 and/or PQM-1 are needed for the alleviation of proteotoxicity upon the knockdown of \u003cem\u003eufm-1\u003c/em\u003e (a concurrent knockdown of \u003cem\u003eufm-1\u003c/em\u003e and of \u003cem\u003ehsf-1\u003c/em\u003e resulted in high rate of early mortality). To address this, we created dual silencing cassettes that simultaneously knockdown the expression of \u003cem\u003eufm-1\u003c/em\u003e and of either \u003cem\u003edaf-16, skn-1\u003c/em\u003e or \u003cem\u003epqm-1\u003c/em\u003e, and tested the possible roles of these transcription factors using two proteotoxicity models, CL2006 animals and AM140 worms. The nematodes were treated from hatching with the dual RNAi silencing cassettes or with RNAi toward \u003cem\u003eufm-1\u003c/em\u003e or the corresponding transcription factor, and subjected to paralysis assays (CL2006) or thrashing assays (AM140). We found that a simultaneous knockdown of \u003cem\u003eufm-1\u003c/em\u003e and \u003cem\u003edaf-16\u003c/em\u003e abolishes the protection from Aβ that was conferred by a sole knockdown of \u003cem\u003eufm-1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Similar results were obtained when AM140 worms were used (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), indicating that DAF-16 is critically needed for the mitigation of proteotoxicity that stems from both aggregative proteins.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalogous results were obtained when the \u003cem\u003eufm-1\u003c/em\u003e and \u003cem\u003eskn-1\u003c/em\u003e were concurrently knocked down (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, c and d) implying that SKN-1 is also critically needed for reduced UFMylation to protect from proteotoxicity of both Aβ and polyQ35-YFP. In contrast, no change in the rates of counter proteotoxic effect was seen when \u003cem\u003epqm-1\u003c/em\u003e was concomitantly knocked down with \u003cem\u003eufm-1\u003c/em\u003e (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e, a and b). These results infer that the reduction of UFMylation modulates gene expression to mitigate proteotoxicity.\u003c/p\u003e\n\u003ch3\u003eReducing UFMylation modulates the transcriptomic landscape of the worm\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eReducing UFMylation modulates the transcriptomic landscape of the worm\u003c/div\u003e \u003cp\u003eIn order to characterize possible modulations in gene expression upon the knockdown of \u003cem\u003eufm-1\u003c/em\u003e, we compared the transcriptomic landscapes of 6 days old CL2006 worms, an age in which proteotoxicity is apparent, that were treated for two generations with RNAi toward \u003cem\u003eufm-1\u003c/em\u003e and of their untreated counterparts (EV), using RNA sequencing (data are available at GSE289271). 143 genes exhibited modulated expression levels, whereas 89 showed elevated levels (62%) and 54 genes exhibited reduced levels (37%) upon the knockdown of \u003cem\u003eufm-1\u003c/em\u003e by RNAi (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and S5a).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo characterize the biological processes that are modulated by reducing UFMylation we used the DAVID bioinformatic tool to cluster genes that showed increased or decreased expression levels upon the knockdown of \u003cem\u003eufm-1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Interestingly, the knockdown of \u003cem\u003eufm-1\u003c/em\u003e has led to increased expression of genes that are related with the innate immune response (defense category), a mechanism that was found to be associated with proteostasis \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In addition, genes that their products are extracellular or presented on the cell surface, were most abundant among those which exhibited elevated levels. Among the downregulated genes we identified proteins that are involved in reproduction.\u003c/p\u003e \u003cp\u003eSome interesting genes were among those which showed modulated expression levels in \u003cem\u003eufm-1\u003c/em\u003e RNAi treated animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), including \u003cem\u003efbxb-45\u003c/em\u003e and \u003cem\u003efbxb-66\u003c/em\u003e, both contain F-box domains, and may be components of the SCF E3 ubiquitin ligase complex. We previously found proteins of this family to regulate proteostasis \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Therefore, the increase in the expression of these genes may be related with enhanced proteasome activity. Surprisingly, the knockdown of \u003cem\u003eufm-1\u003c/em\u003e elevated the levels of \u003cem\u003ecav-1\u003c/em\u003e, a gene that is upregulated by the IIS and whose knockdown by RNAi, mitigates Aβ proteotoxicity \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. An additional unexpected observation is the increase in the levels of several genes that encode for transthyretin-related genes (\u003cem\u003ettr\u003c/em\u003e genes). In fact, the clearance of TTR has been shown to alleviate proteotoxicity in \u003cem\u003eC. elegans\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. An increased level of the cysteine protease inhibitor \u003cem\u003ecpi-1\u003c/em\u003e was also a result of \u003cem\u003eufm-1\u003c/em\u003e RNAi treatment. Protease activity has been shown to be tightly linked with neurodegeneration in mice \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e and with proteotoxicity in worms \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Among the downregulated genes we found \u003cem\u003eklc-1\u003c/em\u003e whose product binds kinesin, an activity that was implicated in Alzheimer\u0026rsquo;s disease \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e and the cysteine protease inhibitor \u003cem\u003ecpi-1\u003c/em\u003e, whose human orthologue, cystatin C is associated with aging and Alzheimer\u0026rsquo;s disease \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. We also found \u003cem\u003elst-1\u003c/em\u003e, which is known to be a target of notch signaling \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. This is in line with the modulation of UFMylated of CAR-1 and CGH-1 that we identified in our MS experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eWe further clustered the modulated genes and identified enrichment of genes that their products reside in the neuronal and reproductive systems (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eb). Finally, targets of DAF-16 and SKN-1 were relatively abundant among genes that exhibited modulated expression level upon the knockdown of \u003cem\u003eufm-1\u003c/em\u003e (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003ec).\u003c/p\u003e\n\u003ch3\u003eReduced UFMylation enhances proteasome activity and reduce polyQ35-YFP aggregation\u003c/h3\u003e\n\u003cp\u003eSince small oligomers, rather than large molecular aggregates, have been shown to be the most toxic species in worms \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e and mice \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, we asked whether the knockdown of \u003cem\u003eufm-1\u003c/em\u003e mitigates Aβ proteotoxicity by modulating its rate of aggregation. CL2006 worms were treated from hatching with RNAi toward \u003cem\u003eufm-1\u003c/em\u003e or left untreated (EV) and harvested at either day 1 or 6 of adulthood. The worms were homogenized, spun to separate supernatants from debris, and the different fractions were subjected to Western blot analysis to compare the levels of Aβ. While no significant difference in Aβ levels has been observed at supernatants and debris of day 1 old worms, clear reduction in Aβ levels was observed in supernatants, but not in debris, of 6 days-old worms that were treated with \u003cem\u003eufm-1\u003c/em\u003e RNAi (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, a and b). These observations suggest that upon reduction of UFMylation, soluble Aβ is more efficiently degraded by protein clearance mechanisms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate how the inhibition of UFMylation affects the rate of polyQ35-YFP aggregation, we first visualized AM140 that were either treated with \u003cem\u003eufm-1\u003c/em\u003e RNAi or left untreated (EV) using fluorescent microscopy, and counted the number of foci in these animals at days 4 and 6 of adulthood (Fig. S6a). We found that the knockdown of UFMylation leads to an increase in the number of foci at both ages (Fig. S6b). Next, we employed the filter-trap assay to compare the aggregation rates of polyQ35-YFP in 5-days old controls worms (EV) and in their aged-matched \u003cem\u003eufm-1\u003c/em\u003e RNAi-treated counterparts. Reduced rate of UFMylation lowers polyQ35-YFP aggregation in five independent experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, c and d).\u003c/p\u003e \u003cp\u003eCollectively, these results infer that an inhibition of UFMylation affects the aggregation rates of both, Aβ and of polyQ35-YFP and suggest that protein degradation mechanisms may be also affected by \u003cem\u003eufm-1\u003c/em\u003e RNAi in Aβ expressing worms. To examine this, we cultured synchronized CL2006 worm populations on either control bacteria (EV) or treated them with \u003cem\u003eufm-1\u003c/em\u003e RNAi. The worms were harvested at day 1 or 6 of adulthood, homogenized and subjected to WB analysis to compare the relative amounts of highly ubiquitinated proteins. While no difference in the levels of ubiquitinated proteins was observed among untreated and \u003cem\u003eufm-1\u003c/em\u003e RNAi-treated, day 1 old animals (Fig. S6, c and d), 6 days old CL2006 worms exhibited reduced level upon the knockdown of \u003cem\u003eufm-1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, e and f). These results are in line with the observation that reduced UFMylation reduces Aβ levels in supernatants of day 6 but not day 1 old worms (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, a and b) and propose that \u003cem\u003eufm-1\u003c/em\u003e RNAi treatment enhances proteasome activity. A significant enhancement of proteasome activity was also observed in day 1 old worms that express the short-lived Ub\u003csup\u003eG76V\u003c/sup\u003e-GFP proteasome sensor (Fig. S6, e and f \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eReduced UFMylation regulates proteotoxicity in cell-autonomous and non-autonomous manners\u003c/h2\u003e \u003cp\u003eThe prominence of inter-tissue communication as a key regulator of proteostasis \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and the major effect of \u003cem\u003eufm-1\u003c/em\u003e on the expression levels of genes that are mainly expressed in the neuronal and reproductive systems (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eb) have led us to ask whether UFMylation governs proteostasis at the organismal level. To address this, we crossed AM1126 worms with TU3401 animals to obtain worms that express polyQ35-YFP in their neurons and are amenable to RNAi-mediated knockdown solely in neurons (Strain EHC145). Synchronized populations of EHC145 worms were either treated from hatching with \u003cem\u003eufm-1\u003c/em\u003e RNAi or left untreated and subjected to the thrashing assay at days 2, 4 and 6 of adulthood. While no mitigation of proteotoxicity was observed in day 2 old worms, the knockdown of \u003cem\u003eufm-1\u003c/em\u003e protected day 4 and 6-old worms from polyQ35-YFP-mediated toxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese results show that the knockdown of \u003cem\u003eufm-1\u003c/em\u003e exclusively in neurons and throughout the nematode (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef), protect the worms from neuronally expressed polyQ35-YFP, and indicate that reduced UFMylation mitigates proteotoxicity cell-autonomously. Next, we sought to test whether reduction of UFMylation also regulates proteostasis cell-non-autonomously. To address this, we used worms that express polyQ35-YFP in their neurons and process RNAi exclusively in the germline (strain EHC212) or in muscles (strain EHC211 \u003csup\u003e32\u003c/sup\u003e). The worms were treated from hatching with \u003cem\u003eufm-1\u003c/em\u003e RNAi or left untreated, and subjected to proteotoxicity assays. A thrashing assay using EHC212 animals indicated that knocking down \u003cem\u003eufm-1\u003c/em\u003e solely in the germline alleviates proteotoxicity that stems from the expression of polyQ35-YFP in neurons. This effect was apparent at all tested ages, day 2, 4 and 6 of adulthood (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). Since worms that express polyQ35-YFP in neurons and process RNAi merely in muscles (EHC211) are relatively motile, we used to paralysis assay to measure proteotoxicity and found that the knockdown of \u003cem\u003eufm-1\u003c/em\u003e mitigates the paralysis phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). Together these results show that UFMylation levels affect polyQ35-YFP proteotoxicity both cell-autonomously and cell-non-autonomously.\u003c/p\u003e \u003cp\u003eWe next adopted a similar approach to test whether the knockdown of \u003cem\u003eufm-1\u003c/em\u003e analogously affects Aβ-mediated proteotoxicity. A paralysis assay using worms that express Aβ in muscles and are amenable to RNAi-mediated gene knockdown solely in the same tissue (strain EHC208) showed that the knockdown of \u003cem\u003eufm-1\u003c/em\u003e alleviates the toxicity of Aβ in a cell-autonomously fashion (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). Surprisingly, the knockdown of \u003cem\u003eufm-1\u003c/em\u003e exclusively in neurons (strain EHC207, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee), germline (strain EHC206, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef) or the intestine (strain EHC205, Fig. S7a) did not affect the toxicity of Aβ in muscles.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe attachment of different PTMs to proteins have key roles in the regulation of various biological traits including aging and proteostasis. While SUMOylation and Ubiquitination have been shown to be regulators of these processes \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, the possible roles of UFMylayion in the regulation of organismal proteostasis, are largely unknown. Here we used the nematode \u003cem\u003eC. elegans\u003c/em\u003e, and found that reducing the rates of UFMylation by \u003cem\u003eufm-1\u003c/em\u003e RNAi, protects model worms from the toxicity of the aggregation-prone proteins Aβ and polyQ35-YFP, and slightly extends their lifespans. To identify proteins that exhibit differential UFMylation with age, we tagged the endogenous \u003cem\u003eufm-1\u003c/em\u003e gene with a triple HA tag and compared the rates of protein UFMylation in day 1 and 5 old worms. We found that components of two known aging-regulating pathways exhibit differential rates of UFMylation with age. First, FIB-1 and NOL-56, both are components of a nucleolar complex that regulates proteostasis across tissues by modulating TGFβ signaling \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, showed increased UFMylation levels at day 5, suggesting that this PTM enhances their activity. Accordingly, the knockdown of either \u003cem\u003eufm-1\u003c/em\u003e or \u003cem\u003enol-56\u003c/em\u003e by RNAi leads to similar outcomes of mitigated proteotoxicity. It also proposes that these UFMylation events occur in ASI neurons where the FIB-1-NOL-56 complex functions as a regulator of proteostasis \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg-i). Secondly, CAR-1 and CGH-1, two germline resident proteins, show reduced UFMylation levels with age (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG-ii). Since these proteins negatively regulate Notch signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg-iii), their activation alleviates proteotoxicity in a DAF-16 dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg-iv) \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003eufm-1\u003c/em\u003e RNAi-mediated protection from proteotoxicity also requires SKN-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, c and d), however, it is unclear whether this transcription factor is regulated in this context, by Notch signaling, by the FIB-1-NOL-56 complex or by another mechanism (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg-v). This counter-proteotoxic effect is also dependent on at least three chaperones, HSP-1, HSP-90 and SIP-1 which show modulated UFMylation levels with age (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), and associated with enhanced UPS activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg-vi).\u003c/p\u003e \u003cp\u003eOne fundamental question that arises from our results, is whether the aging-regulating mechanisms downstream of the FIB-1-NOL-56 complex and of CAR-1/CGH-1 act in a coordinated manner, and if yes, whether UFMylation orchestrates them. Several observations suggest that these mechanisms act in concert to regulate proteostasis. First, a concurrent knockdown of \u003cem\u003efib-1\u003c/em\u003e and \u003cem\u003eufm-1\u003c/em\u003e show no additive protective effect (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eb), suggesting that they may be components of the same pathway. In addition, we previously found that the knockdown of \u003cem\u003enol-56\u003c/em\u003e reduces the expression levels of \u003cem\u003ecar-1\u003c/em\u003e and \u003cem\u003ecgh-1\u003c/em\u003e \u003csup\u003e\u003cem\u003e32\u003c/em\u003e\u003c/sup\u003e suggesting a direct regulation of Notch signaling by the nucleolar FIB-1-NOL-56 complex. We also observed modulated expression levels of \u003cem\u003elst-1\u003c/em\u003e, a well characterized target of Notch signaling \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, upon treatment with \u003cem\u003enol-56\u003c/em\u003e \u003csup\u003e32\u003c/sup\u003e, \u003cem\u003ecar-1\u003c/em\u003e \u003csup\u003e21\u003c/sup\u003e and \u003cem\u003eufm-1\u003c/em\u003e RNAi (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Together, these results imply that UFMylation enhances proteotoxicity, at least partially, by elevating the activity of Notch signaling. Nevertheless, the FIB-1-NOL-56 complex regulates proteostasis by modulating TGFβ signaling, and is not dependent on DAF-16 for this function \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Thus, it is conceivable that the two mechanisms regulate proteostasis by partially overlapping mechanism through which they modify the activity of the Notch pathway, however each may control additional unrelated pathways to promote its counter-proteotoxic roles. It would be important to further elucidate the possible links of UFMylation with TGFβ signaling and the IIS.\u003c/p\u003e \u003cp\u003eOur tissue specific RNAi analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, a-f) yielded a puzzling observation. While the knockdown of \u003cem\u003eufm-1\u003c/em\u003e exclusively in neurons or in germline cells alleviates the toxicity of polyQ35-YFP in neurons, the knockdown of \u003cem\u003eufm-1\u003c/em\u003e in the same tissues is insufficient to protect worms from Aβ-mediated toxicity in muscles. One possible explanation to this conundrum suggests that reduced UFMylation activates distinct mechanisms in the face of dissimilar proteotoxic challenges. According to this theme, the counter-proteotoxic mechanism that mitigates Aβ-mediated toxicity upon the knockdown of \u003cem\u003eufm-1\u003c/em\u003e is only cell-autonomous while the one which alleviates the toxicity of polyQ-YFP acts cell-autonomously as well as cell-non-autonomously. It is noteworthy that differential responses of the proteostasis network to these two aggregation-prone proteins has been shown previously \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Nevertheless, it is also possible that protection from Aβ in the muscle requires the knockdown of \u003cem\u003eufm-1\u003c/em\u003e in both tissues, germline and muscle whereas the toxicity that stems from nuclear polyQ35-YFP can be alleviated by the reduction of UFMylation in either one of these tissues. This notion may be supported by the observation that germline-specific knockdown of \u003cem\u003enol-56\u003c/em\u003e mitigates the toxicity of Aβ in muscles \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe regulation of \u003cem\u003ecav-1\u003c/em\u003e by RNAi against both \u003cem\u003enol-56\u003c/em\u003e \u003csup\u003e32\u003c/sup\u003e and \u003cem\u003eufm-1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) points at neurons, the cells that most prominently express \u003cem\u003ecav-1\u003c/em\u003e \u003csup\u003e38\u003c/sup\u003e and at caveolae, as possible sites where UFMylation affects proteostasis-orchestrating signaling. While, further experimental work is needed to fully clarify the communication network between germline, neurons and muscles, our results strongly suggest that reduced UFMylation promotes organismal proteostasis. Yet, it is interesting to note that reduced UFMylation appears to have deleterious effects on proteostasis at the cellular level. For instance, mono-UFMylation was reported to confer the jettison of misfolded α-synuclein from cells \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. These apparently opposing effects of UFMylation raise the prospect that certain biological entities differentially affect proteostasis at the cellular and organismal levels. Our observations show that in the nematode, the effects of lowering UFMylation culminate to alleviate proteotoxicity.\u003c/p\u003e \u003cp\u003eIt will be also interesting to investigate how different PTMs control the activity of CAR-1. We have previously shown that this protein undergoes SUMOylation which appears to negatively regulates its activity \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Our results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) show that the rate of CAR-1 UFMylation is reduced with age, raising the questions of how this PTM affects CAR-1 activity and whether SUMOylation and UFMylation compete as regulators of CAR-1 and Notch signaling.\u003c/p\u003e \u003cp\u003eThis study sheds new light on the roles of UFMylation as a regulator of proteostasis and sets the basis for future research that would elucidate the mechanisms that are governed by this PTM in the context of aging and aging-associated disorders.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors have no competing interests.\u003c/p\u003e\n\u003ch2\u003eAuthor contribution\u003c/h2\u003e\n\u003cp\u003eEC, RBH designed and initiated this study. RBH performed physiological assays including strain crossing, proteotoxicity, lifespan and thrashing assays, Western blots as well as cloning and qPCR procedures. HB conducted paralysis assays, AZ conducted filter-trap assays, HZ crossed proteotoxicity worm models with animals that process RNAi in a single tissue and, IC performed Western blots. RBH prepared samples for RNA-Seq and MS analyses. YHY performed MS analysis. XF and M-Q.D constructed 3xHA-UFM-1 worms using CRISPR-cas9 technology. EC wrote the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eThis study was generously supported by the Israel-China program (EC #3261/20), the Israel Science Foundation (ISF) EC#543/21, the Israeli Ministry of Science and Technology (MOST)(EC#80884), the Henri J. and Erna D. Leir Chair for Research in Neurodegenerative Diseases, as well as by the National Natural Science Foundation of China (NSFC-ISF 32061143020 to M-Q.D).\u003c/p\u003e\n\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eThe NGS raw data is available at GEO: GSE289271\u003c/p\u003e\n\u003cp\u003eMass spectrometry data is available at: PRIDE: PXD060906\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHoppe T, Cohen E (2020) Organismal Protein Homeost Mech Genet 215:889\u0026ndash;901\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJayaraj GG, Hipp MS, Hartl FU (2019) Functional Modules of the Proteostasis Network. Cold Spring Harb Perspect Biol\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhammed MS, Wang X (2024) Promoting proteostasis by cAMP/PKA and cGMP/PKG. 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Sci Adv 10:eadk2542\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCaenorhabditis elegans, maintenance and growth conditions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStandard \u003cem\u003eC.\u0026nbsp;elegans\u003c/em\u003e techniques were used to maintain and manipulate the worm strains. Worms were grown at 20\u0026deg;C (unless indicated otherwise) on nematode growth media (NGM)-ampicillin plates (100 \u0026micro;g/ml ampicillin) and fed with Escherichia coli HT115 bacteria. Worm strains were provided by the CGC and were also crossed in our lab in previous projects (worm strains are listed at Supplemental Table S4). CF512 worms are heat-sensitive feminized and therefore maintained at 15\u0026deg;C. For avoiding progeny during experiments, CF512 worms were let hatch at 20\u0026deg;C, L1 larvae were transferred to 25\u0026deg;C for 48 h, and back to 20\u0026deg;C thereafter. Worm populations were synchronized using sodium hypochlorite (bleach) and potassium hydroxide.\u003c/p\u003e\n\u003ch4\u003eGene knockdown by RNA interference (RNAi)\u003c/h4\u003e\n\u003cp\u003e\u003cem\u003eFor\u003c/em\u003eRNAi-mediated gene knockdown,\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e harboring the appropriate RNAi clone were grown in LB medium overnight at 37\u0026deg;C and seeded on NGM-ampicillin plates. Before adding worms to plates, 100 mM isopropyl \u0026beta;-d-1-thiogalactopyranoside (IPTG; final concentration of 4mM) was added to the plates to induce the expression of the dsRNA. Empty vector (EV, pAD12) \u003cem\u003edaf-2\u003c/em\u003e (pAD48) RNAi and \u003cem\u003edaf-16\u0026nbsp;\u003c/em\u003e(pAD43) were a gift from Prof. Andrew Dillin (Berkeley). \u003cem\u003euba-5, skn‐1 RNAi\u003c/em\u003e bacteria were obtained from the \u003cem\u003eC. elegans\u003c/em\u003e ORF-RNAi feeding library (the Vidal library). \u003cem\u003ehsf-1\u0026nbsp;\u003c/em\u003eRNAi was from the Ahringer library. All the other RNAi plasmids used in this study were created for this project by amplifying the relevant sequences (see Supplemental Table S5) using PCR and cloning it into the pL4440 plasmid using restriction enzymes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eParalysis and thrashing assays\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eParalysis assays:\u003c/u\u003e CL2006 worms were synchronized by bleach. Eggs were placed on NGM-ampicillin plates seeded with the tested bacteria, and allowed to develop to day 1 of adulthood. For each experiment, at least one hundred and twenty animals were transferred onto small NGM plates seeded with the respective \u003cem\u003eE.\u0026nbsp;coli\u003c/em\u003e culture (12 animals / plate). Paralyzed worms were scored daily by tapping the worms\u0026apos; \u0026quot;noses\u0026quot; with a platinum wire. A paralyzed animal was defined as an animal than can move its head but is unable to crawl away. Paralysis assay was terminated at day 12 of adulthood.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eTrashing assay\u003c/u\u003e: synchronized populations of AM140 or AM1126 worms were grown, as described above, on control or RNAi bacteria until day 1 of adulthood. Thrashing rates were determined at the indicated age, by transferring an individual worm into a 10 \u0026mu;l drop of M9 buffer, waiting 30\u0026nbsp;seconds for adaptation and counting the number of body bends during the next 30 seconds. Thrashing rates of twenty animals were recorded for each treatment at each time point. At least three independent experiments were performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLifespan assays\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSynchronized eggs were placed on NG‐ampicillin plates that were seeded with the indicated RNAi bacteria and supplemented with IPTG. At day 1 of adulthood, 120 animals per treatment were transferred onto small NG‐ampicillin plates (12 worms per plate, total of 10 plates per treatment). Worms that failed to move their tips when tapped twice with a platinum wire were scored as dead. Animals that bagged, disappeared or dried out were censored.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSDS-PAGE and Western blot analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor protein blotting, 6000 worms were treated from hatching with RNAi or left untreated as indicated. At the indicated age the worms were collected and homogenized in M9 supplemented with a protease inhibitor cocktail (Millipore, Billerica, MA, USA; #539134) using a Dounce homogenizer. Worm homogenates were spun for 10 min at 10,000 \u0026times; g at 4\u0026deg;C to sediment debris. The post debris supernatants were collected and total protein concentrations were measured by a BCA kit (Pierce #23227, Thermofisher, Waltham, MA USA). Then, were supplemented with loading buffer (10% glycerol, 125 mM Tris base, 1% SDS) and boiled for 10 min. 100 \u0026mu;g total protein were loaded into each well. Proteins were separated on a polyacrylamide (PAA) gel, transferred onto a PVDF membrane (for UFM-1 conjugates, GFP conjugates, and ubiquitin conjugates blotting). The membranes were probed with the appropriate antibody: anti-UFM-1 (Abcam, Cat# ab109305), GFP (Cell Signaling, #2037) or FK2 Multiubiquitin chain Monoclonal antibody (Cayman chemical, Cat# CAY-14220). To characterize A\u0026beta; quantities and aggregation states both, supernatants and debris were loaded on the gels, transferred onto a nitrocellulose membrane (#66485; Pall Corporation), and probed with an A\u0026beta; antibody (Cat#803001; clone 6E10, BioLegend). HRP-conjugated secondary antibody (Jackson ImmunoResearch West Grove, Pennsylvania, USA), chemiluminescence system, and a luminescent image analyzer (Chemidoc XRS+; Biorad) were used to detect proteins. The membrane was stripped in 300 mM NaOH buffer for 5 minutes, re-blocked in 5% BSA and probed with antibody against \u0026beta;-Actin (Cat# A5441, Sigma Aldrich) for normalizing the signals of different proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eWorm visualization by fluorescent microscopy and foci counting\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo visualize polyQ35-YFP-containing foci, AM140 worms were anesthetized in 20mM sodium azide and mounted on glass slides. Images were taken using a Nikon AZ100 fluorescent microscopy system.\u0026nbsp;To determine the size distribution of foci, we used the ImageJ software. Automatic threshold was determined, which converted the image into a binary. The particles were filtered by area to include ones between 15 and 1000 pixels. These values ensure excluding of noise and large particles deriving from continuous labeling.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFilter trap assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAM140 worms were treated with RNAi bacteria from hatching or left untreated as indicated (four thousand worms per treatment). The worms were washed daily with M9 to discard progeny and collected on day 5 of adulthood. The animals were immediately frozen in liquid N2 and kept in -80\u0026deg;C. The samples were thawed on ice, supplemented with lysis buffer (50\u0026thinsp;mM Hepes pH\u0026thinsp;7.4, 150\u0026thinsp;mM NaCl, 1\u0026thinsp;mM EDTA, 1% Triton X‐100) and with an EDTA‐free protease inhibitor cocktail (Millipore, Billerica, MA, USA; #539134) and homogenized using a Dounce homogenizer. Worm homogenates were spun for 10 min at 10,000 \u0026times; g at 4\u0026deg;C to sediment debris. The post debris supernatants were collected, protein concentrations were measured by a BCA kit (Pierce #23227, Thermofisher, Waltham, MA USA). 100\u0026thinsp;\u0026mu;g of each protein extract was supplemented with SDS to the final concentration of 0.5% and loaded onto a cellulose acetate membrane assembled in a slot blot apparatus (Bio‐Rad cat#1703938). The membrane was washed with 0.2% SDS, and the retained polyQ35‐YFP was assessed by immunoblotting with a GFP antibody (Cell Signaling, #2037).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRNA isolation and quantitative real-time PCR\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated using a NucleoSpin\u0026reg; RNA isolation kit (MACHEREY-NAGEL; #740955). For each time-point, 10,000 synchronized eggs were placed on NG-ampicillin plates seeded with the indicated RNAi bacteria and supplemented with IPTG. The worms were washed daily with M9 to get rid of progeny and adult nematodes were transferred to new plates. At the indicated age the worms were harvested in M9 and frozen at -80 C. The worms were then thawed and homogenized using Dounce homogenizer. Homogenates were transferred to Eppendorf tubes and centrifuged at 14,000 \u0026times;\u0026nbsp;\u003cem\u003eg\u003c/em\u003e for 5 minutes. The supernatants were transferred to NucleoSpin\u0026reg; Filter (NucleoSpin\u0026reg; RNA kit, Macherey-Nagel, D\u0026uuml;ren Germany), and total RNA was purified according to the manufacturer\u0026rsquo;s instructions. The RNA was quantified using a NanoDrop2000c spectrophotometer.\u003cbr\u003eFor qPCR: cDNA was prepared by reverse transcription of the total RNA samples via random-priming using the iScript\u003csup\u003eTM\u003c/sup\u003e cDNA Synthesis Kit (#170\u0026ndash;8890; Bio-Rad, Hercules, CA, USA) as per the manufacturer\u0026rsquo;s protocol. Analyzes by qPCR were performed using gene-specific primers (Supplemental Table S6). Analyzes by qPCR were performed with Luna\u0026reg; Universal qPCR Master Mix (Biolabs, Cat#M3003G) and expression levels were normalized to the expression levels of \u003cem\u003ecdc-42\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;pmp-3\u0026nbsp;\u003c/em\u003ewhich served as normalizing genes\u003cem\u003e.\u0026nbsp;\u003c/em\u003eqPCR reactions for each gene were performed in triplicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eNext Generation Sequencing (NGS) and computational analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor NGS,we used RNA\u0026nbsp;ScreenTape kit (catalog #5067-5576; Agilent Technologies, Santa Clara, CA), D1000 ScreenTape kit (catalog #5067-5582; Agilent Technologies) Qubit\u0026reg;\u0026nbsp;RNA\u0026nbsp;HS Assay kit (catalog # Q32852; Invitrogen, Carlsbad, CA) and Qubit\u0026reg; DNA HS Assay kit (catalog #32854; Invitrogen). mRNA libraries were\u0026nbsp;prepared\u0026nbsp;using KAPA Stranded mRNA kit with\u0026nbsp;mRNA\u0026nbsp;Capture Beads (KAPA Biosystems, KK8421).\u0026nbsp;In brief, 1\u0026micro;g was used for the\u0026nbsp;library\u0026nbsp;construction;\u0026nbsp;library\u0026nbsp;was eluted in 20\u0026micro;l of elution buffer. All DNA samples\u0026nbsp;libraries\u0026nbsp;were pooled to\u0026nbsp;10nM sample.\u0026nbsp;Multiplex samples Pool were loaded on\u0026nbsp;NovaSeq 6000 (Illumina), using\u0026nbsp;NovaSeq 6000 SP Reagent Kit v1.5 100 cycles (cat# 20028401), with 122 cycles of single-end sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eComputational analyses of next generation sequencing data\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw reads were processed for quality trimming and adaptors removal using fastq_quality_filter v0.0.14 and cutadapt v1.18 (Marcel M. et al., EMBnet.journal 2011, 17.1:10-12). The processed reads were aligned to the \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e transcriptome and genome version WBcel235 with annotations from Ensembl release 106 using TopHat v2.1.1 (Kim D et al., Genome Biology 2013, 14:R36). Counts per gene quantification was done with htseq-count v2.6.1 (Anders S et al., Bioinformatics 2015, 31 (2):166-169). Normalization and differential expression analysis were performed using the DESeq2 package (v1.36.0). Genes with a sum of counts less than 10 over all samples were filtered out, then size factors and dispersion were calculated. Normalized counts were used for several quality control assays, such as counts distributions and principal component analysis, which were calculated and visualized in R. Pair-wise comparisons were tested with default parameters (Wald test), except not using the independent filtering algorithm. Significance threshold was taken as padj\u0026lt;0.01 (default). Finally, results were combined with gene details (such as symbol, known transcripts, etc.), taken from the results of a BioMart query (Ensembl, release 106), to produce the final Excel file.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGene Ontology Classification and Secreted peptide analysis:\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll significant gene entries were subjected to KEGG and GO classification (http://www.geneontology.org). Significant over-representation of KEGG and GO-classified biological processes was assessed by comparing the number of pertinent genes in a given biological process to the total number of the relevant genes printed on the array for that particular biological process (Fisher exact test) using the publicly accessible software DAVID (http://david.abcc.ncifcrf.gov/summary.jsp).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmuno-precipitation and MS of UFMylated proteins\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eMQD2801 generation\u003c/u\u003e: MQD2801 strain (\u003cu\u003eufm-1(hq493[ufm-1p::3XHA::ufm-1]) III )\u003c/u\u003e was generated using the CRISPR/Cas9 method. The strain was generated in N2 background and N-terminal 3XHA tag was inserted into the endogenous ufm-1 locus. Then the trangenic worms were crossed for 6 times with N2 worms to clean up the genetic background.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eFor immuno-precipitation\u003c/u\u003e, 12,000 MQD2801 worms per sample were grown from hatching on \u003cem\u003eufm-1\u003c/em\u003e RNAi or left untreated (EV). Half of each sample was collected on day 1 of adulthood, while the remaining worms were washed in M9 daily and transffered to new plates to get rid of progeny. Worms of the second half were collected on day 5 of adulthood. Worms of the two groups were homogenized in 1 ml of lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 1% Triton-X100 (2% for Membrane protein), 10% glycerol, 0.1~2mM PMSF +10 mM NaF +PI (1:1000), using Dounce homogenizer on ice. Worm lysates were transferred into 1.5 ml Eppendorf tubes and centrifuged at 10,000 \u0026times; g, at 4\u0026deg;C for 30 minutes. Thereafter, The post debris supernatants were collected and transferred to fresh 1.5 ml Eppendorf tubes. Protein concentrations were measured by a BCA kit (Pierce #23227, Thermofisher, Waltham, MA USA) and 1 mg of total protein in a volume of 200 \u0026mu;l lysis buffer was used for immuno-precipitation.\u0026nbsp;20 \u0026mu;l of Pierce Anti-HA Magnetic Beads\u0026nbsp;were added to worm lysate and samples were rotated for 4 hours at 4\u0026deg;C followed by centrifugation at 1,000 \u0026times; g, 4\u0026deg;C for 1 minute, and flow throgh was discarded. Then, beads were washed 3 additional times with cold worm lysis buffer (supplemented with protease and phosphatase inhibitors) by rotating 5 min at 4\u0026deg;C \u0026nbsp;and centrifuging at 1,000 \u0026times; g, 4\u0026deg;C for 1 minute. Finally, beads were washed twice with 1.5 mL/tube 1x cold worm lysis buffer without detergent (tritone) and supernatants were discarded. Tubes were kept in -80\u0026deg;C until processed for MS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eProtein identification by MS\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eSample preparation for MS analysis\u003c/u\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing immunoprecipitation and washing, the packed beads were resuspended in 100 \u0026mu;l 8M urea, 10 mM DTT, 25 mM Tris-HCl pH 8.0 and incubated for 30 min at 22\u0026deg;C. Next, Iodoacetamide (55 mM) was added and beads were incubated for 30 min (22\u0026deg;C, in the dark), followed by addition of DTT (20 mM). The Urea was diluted by the addition of 7 volumes of 25 mM Tris-HCl pH 8.0. Trypsin was added (0.3 \u0026mu;g/ sample) and the beads were incubated overnight at 37\u0026deg;C with gentle agitation. The beads were spun down and the peptides in the supernatants were desalted on C18 home-made Stage tips.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003enanoLC-MS/MS analysis:\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eMS analysis was performed using a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA USA) coupled on-line to a nanoflow UHPLC instrument, Ultimate 3000 Dionex (Thermo Fisher Scientific, Waltham, MA USA). Peptides (1.0 \u0026mu;g, as estimated by O.D.280 nm) were separated over a non-linear 90 min gradient (0 - 80% acetonitrile) run at a flow rate of 0.3 \u0026mu;l/min on a reverse phase 25-cm-long C18 column (75 \u0026mu;m ID, 2 \u0026mu;m, 100\u0026Aring;, Thermo PepMapRSLC). The survey scans (380\u0026ndash;2,000 m/z, target value 3E6 charges, maximum ion injection times 50 ms) were acquired and followed by higher energy collisional dissociation (HCD) based fragmentation set at 27. A resolution of 70,000 was used for survey scans and up to 15 dynamically chosen most abundant precursor ions, with \u0026ldquo;peptide preferable\u0026rdquo; profile was fragmented (isolation window 1.8 m/z). The MS/MS scans were acquired at a resolution of 17,500 (target value 1E5 charges, maximum ion injection times 120 ms). Dynamic exclusion was 60 sec. Data were acquired using Xcalibur software (Thermo Scientific). To avoid a carryover, the column was washed with 80% acetonitrile, 0.1% formic acid for 25 min between samples.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eMS data analysis:\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe Mass Spec data was analyzed by searching for matching sequences in the Swissprot (Uniprot) database of the \u003cem\u003eC. elegans\u003c/em\u003e proteome containing 26,768 sequences, both annotated and predicted.\u0026nbsp;The search program (Maxquant\u0026nbsp;version 1.5.3.12.) may also include proteins from other species, if they match the program\u0026rsquo;s database of commonly used species. Peak lists were searched against translated coding sequences of the human proteome obtained from Uniprot. The search included cysteine carbamidomethylation as a fixed modification and oxidation of methionine as variable modifications, allowing up to two miscleavages. The match-between-runs option was used. Peptides with a length of at least seven amino-acids were considered and the required FDR was set to 1% at the peptide and protein level. Protein identification required at least 2 unique or razor peptides per protein. Relative protein quantification in MaxQuant was performed using the label-free quantification (LFQ) algorithm. Protein contaminants and proteins identified by less than 2 peptides were excluded from the analysis.\u0026nbsp;For comparing protein abundances between samples, LFQ intensity (Label Free Quantification) is the accepted parameter. It represents the total protein intensity based on the combined intensities of all the identified peptides of the protein. LFQ intensities are normalized across all the samples, enabling inter-sample comparison of relative protein abundance. LFQ = 0, means the level of protein are either truly 0 or may just be below the threshold needed for calculating LFQ. The analysis was done only on proteins which have a valid LFQ value in at least\u0026nbsp;4 of the samples in at least one group. Of those proteins left in the analysis, all missing LFQ values were replaced by random low numbers out of a normal distribution to allow for statistical analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical analysis and software\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using Graph Pad Prism 9 (GraphPad Software, Inc., La Jolla, USA). The statistical tests used, statistical significance, error bars, and sample sizes can be found in the corresponding figure legends. \u0026ldquo;Statistically significant\u0026rdquo; was defined as a minimum of p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"Aging, UFMylation, germline, lifespan, proteostasis, C. elegans","lastPublishedDoi":"10.21203/rs.3.rs-6064783/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6064783/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe attachment of Post-Translational Modifications (PTMs) to proteins plays key roles in the regulation of the activity and stability of various proteins. Here we utilized the nematode \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e to test whether UFMylation, a PTM that was found to be essential for key biological functions, is involved in the regulation of aging and protein homeostasis (proteostasis). Our results indicate that lowering UFMylation extends lifespan and mitigates the toxicity of aggregative proteins that underlie the development of neurodegenerative disorders in humans. Mass spectrometric analysis unveiled that UFMylation of aging-regulating proteins, including components of the nucleolar FIB-1-NOL-56 complex and the germline resident proteins CAR-1 and CGH-1, governs proteostasis across tissues. Functional analyses indicate that the proteostasis-regulating transcription factors DAF-16 and SKN-1 are crucial for the counter proteotoxic effect of reduced UFMylation which is mediated by reduced rate of aggregation and enhanced protein degradation. These insights highlight the important roles of PTMs in the regulation of proteostasis and point at research directions for the development of new therapies for neurodegenerative disorders.\u003c/p\u003e","manuscriptTitle":"Aging-Associated Modulation of UFMylation Impairs Proteostasis in C. elegans","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-24 06:29:46","doi":"10.21203/rs.3.rs-6064783/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":"8e67c23b-1201-421b-9dbc-fa574d1704b4","owner":[],"postedDate":"March 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":45820202,"name":"Biological sciences/Cell biology/Protein folding/Protein aggregation"},{"id":45820203,"name":"Biological sciences/Cell biology/Proteolysis/Protein quality control"}],"tags":[],"updatedAt":"2026-04-17T02:15:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-24 06:29:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6064783","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6064783","identity":"rs-6064783","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}