Cytoplasmic RBMX coordinates selective mRNA translation to suppress senescence in cancer

Cytoplasmic RBMX coordinates selective mRNA translation to suppress senescence in cancer | 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 Cytoplasmic RBMX coordinates selective mRNA translation to suppress senescence in cancer Ho Jin You, Jeeho Kim, In-Youb Chang, Young Jin Jeon, Jeongsik Yong This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8859405/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Although RBMX has been studied primarily as a nuclear RNA-binding protein involved in splicing and genome maintenance, we identify a previously unrecognized cytoplasmic role for RBMX in regulating senescence entry through selective mRNA translation in cancer cells. We show that a reversible methylation-dependent switch promotes RBMX relocalization to the cytoplasm, where RBMX directly engages the eIF3i/eIF3F initiation module to regulate translation initiation. Cytoplasmic RBMX does not globally enhance protein synthesis; instead, it functions as a translation-specificity factor that preferentially promotes translation of oncogenic mRNAs, including YBX1. Disruption of this translational program reduces YBX1 protein output without affecting mRNA abundance, leading to p53-independent induction of p21 and a robust senescence response. These findings establish selective translational control by cytoplasmic RBMX as a decisive upstream mechanism governing senescence fate in cancer cells. Biological sciences/Cancer/Cancer models Biological sciences/Biochemistry/Proteins/Transcription factors Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION RNA-binding proteins (RBPs) are central regulators of post-transcriptional gene expression, coordinating alternative splicing, mRNA stability, localization, and translation 1 , 2 . Within this broad class, the heterogeneous nuclear ribonucleoprotein (hnRNP) family comprises a major group of RBPs that control nuclear RNA metabolism in development and disease 3 . RBMX (RNA-binding motif protein X-linked, also known as hnRNPG) is one of the most structurally conserved hnRNPs and occupies a key position at the interface of RNA processing and genome maintenance 4 , 5 . Across human cancers, RBMX has emerged as a context-dependent regulator whose function can be either oncogenic or tumor suppressive. RBMX is frequently dysregulated in malignancies, and changes in its expression have been linked to tumor progression, remodeling of the immune microenvironment, therapy resistance, and clinical outcome 4 , 6 . In hepatocellular carcinoma, RBMX reprograms oncogenic metabolic and transcriptional pathways to promote proliferation, invasion, metastasis, and sorafenib resistance; in osteosarcoma, it shapes an immunosuppressive tumor microenvironment by modulating CD8⁺ T-cell infiltration and effector function; and in colorectal cancer, tumor-derived exosomal LINC01615 recruits RBMX to an EZH2 regulatory axis that enhances EZH2 expression and drives M2 polarization of tumor-associated macrophages 6 , 7 , 8 , 9 , 10 . In contrast, in bladder cancer, RBMX restrains tumorigenicity and invasion by regulating hnRNPA1-dependent PKM splicing and cancer metabolism 11 , and in oral squamous cell carcinoma, RBMX acts as a tumor suppressor that limits proliferation and transformation 12 . Together, these observations position RBMX as a lineage- and context-dependent regulatory node whose biological output—pro-tumorigenic or tumor-suppressive—is shaped by tissue identity, genetic background, and upstream signaling 4 , 6 , 7 , 8 , 9 , 10 , 11 , 12 . At the molecular level, RBMX contains a single RNA recognition motif (RRM) and a C-terminal RGG/RG-rich domain, enabling interactions with both structured and intrinsically disordered RNA elements as well as core spliceosomal components 4 , 5 . Consistent with this domain architecture, RBMX predominantly localizes to the nucleus, where it regulates exon definition, splice-site choice, and alternative splicing programs 4 , 5 , 13 . Beyond canonical splicing control, RBMX participates in co-transcriptional RNA processing and transcription–splicing coupling, thereby enhancing the fidelity of nascent mRNA production 4 , 13 , 14 . RBMX also safeguards genome integrity by suppressing harmful R-loops, coordinating ATR-dependent DNA damage responses, mitigating replication stress, and promoting accurate processing of pre-mRNAs containing ultra-long introns and repetitive sequences 13 , 14 . These activities collectively underscore RBMX as a multifunctional nuclear regulator that couples RNA metabolism to genomic stability 4 , 13 , 14 . Protein arginine methyltransferase 5 (PRMT5) is the major type II arginine methyltransferase that catalyzes symmetric dimethylation within RGG/RG motifs of RBPs and other nuclear proteins 15 , 16 , 17 . Through methylation of splicing factors, transcriptional regulators, chromatin-associated proteins, and components of the RNA-processing and translational machinery, PRMT5 integrates signals that control transcription, pre-mRNA splicing, translation, stemness, and DNA damage repair 15 , 16 , 17 . In cancer, PRMT5 rewires RNA-metabolic networks by modifying multiple RBPs—including hnRNPA1, FUS, and TDP-43—thereby promoting tumor-specific RNA processing and stress-adaptation programs 15 , 16 , 18 . RBMX is a bona fide PRMT5 substrate: symmetric dimethylation of its C-terminal RGG/RG motif modulates its interaction landscape, nuclear complex assembly, and functional specificity 19 , 20 , 21 . Studies of Shashi X-linked intellectual disability have shown that deletion of the RBMX RGG/RG region perturbs p53 signaling, neuronal differentiation, and nuclear RBMX behavior, while biochemical and proteomic analyses have identified RBMX among PRMT5-modified substrates within nuclear ribonucleoprotein assemblies 16 , 19 , 20 , 21 . More broadly, arginine methylation of RGG/RG-containing RBPs operates as a molecular switch that regulates protein–RNA affinity, phase separation, and subcellular organization 15 , 18 , 21 , 22 , 23 . For several RBPs, including hnRNPA1 and related hnRNPs, inhibition or loss of arginine methylation promotes nuclear-to-cytoplasmic translocation and reshapes their cytoplasmic activities, linking PRMT5-mediated modification to nucleo-cytoplasmic trafficking and translational control 15 , 18 , 24 . Despite extensive work on the nuclear functions of RBMX, however, it has remained unclear whether PRMT5 regulates RBMX subcellular distribution or licenses a transition toward cytoplasmic, translation-related functions—representing a critical gap in understanding how RBMX integrates post-translational regulation with cancer phenotypes 4 , 15 , 16 . Recent work has established that specific RBPs can relocalize to the cytoplasm and directly engage the translation initiation machinery to drive tumor-selective translational reprogramming 25 , 26 , 27 . The eukaryotic initiation factor 3 (eIF3) complex is a central orchestrator of such control; its subunits are frequently altered in tumors and regulate mRNA selection, proteome remodeling, and stress adaptation 25 , 28 , 29 . Within this complex, eIF3F and eIF3i have emerged as particularly important subunits. eIF3F displays context-dependent activities, acting as a negative regulator of translation and tumor growth in some settings, while promoting migration, invasion, and metastasis in lung adenocarcinoma through interaction with STAT3 in others 28 , 29 , 30 . eIF3i is a proto-oncogenic factor overexpressed in multiple cancers, including colorectal cancer, where it enhances COX-2 and PHGDH translation, supports proliferation and survival, and drives oncogenesis and metastasis 29 , 31 , 32 , 33 . Although interactions between RBPs and eIF3 subunits have been implicated in establishing malignant translational networks 25 , 26 , 27 , 28 , whether RBMX participates in such mechanisms—and how PRMT5-dependent methylation might tune these interactions—has remained unknown 15 , 16 , 19 . Importantly, Dysregulated translational control has emerged as a critical determinant of cell fate decisions, including senescence, during tumor progression 34 , 35 . In this study, we identify a previously unrecognized cytoplasmic role of RBMX as a PRMT5-regulated translational regulator that governs entry into senescence in cancer cells. We show that RBMX directly interacts with eIF3i and eIF3F through its RGG/RG motif, that this interaction is markedly enhanced in a demethylation-mimetic RBMX mutant, and that cytoplasmic RBMX promotes cap-dependent translation initiation in cancer cells. Using cytoplasmic RIP-seq, we further demonstrate that RBMX selectively regulates the translation of cancer-associated mRNAs, including YBX1. RBMX depletion sharply reduces YBX1 protein levels without altering its mRNA, leading to p21 Cip/Waf1 induction and robust, p53-independent cellular senescence. These findings define a cytoplasmic RBMX–YBX1–p21 Cip/Waf1 translational axis that dictates senescence fate and reveal a methylation-sensitive mechanism linking PRMT5-dependent control of RBMX to cell fate regulation in cancer. Collectively, our work expands the functional landscape of RBMX beyond its canonical nuclear roles and uncovers a PRMT5-RBMX-YBX1-p21 pathway through which selective translation shapes senescence-associated cancer phenotypes. RESULTS Cytoplasmic localization of nuclear RBMX is context dependent RBMX has been reported to exert both oncogenic and tumor-suppressive roles, with its functional impact varying across cancer types 4, 6 . To better define its role in tumorigenesis, we first analyzed RBMX expression patterns across diverse human cancers. TCGA datasets and immunohistochemical (IHC) staining of patient-derived tissues consistently showed that RBMX expression is significantly elevated in multiple tumor types—including lung, colorectal, and liver cancers—compared with corresponding normal tissues. RBMX protein levels were also markedly higher across 11 lung cancer (LC) and 11 colorectal cancer (CRC) cell lines relative to normal lung and colon epithelial cell lines (Supplementary Fig. S1A–D). Functionally, RBMX appeared critical for lung cancer progression. In H1299 cells with stable RBMX knockdown, we observed a substantial reduction in proliferation accompanied by decreased Ki67 expression. RBMX depletion also impaired clonogenic potential and significantly suppressed tumor growth and Ki67 staining in xenograft models (Supplementary Fig. S1E-J). These results collectively identify RBMX as a key driver of cancer cell proliferation and tumorigenic growth. Although RBMX has been predominantly studied for its nuclear roles 4, 6 , its mechanistic contribution to oncogenesis remains poorly defined. To explore potential functions beyond the nucleus, we first analyzed its predicted protein interaction landscape. Surprisingly, interrogation of the compartmentalized protein–protein interaction database (comPPI), followed by functional enrichment and STRING analyses, revealed that a substantial proportion of predicted RBMX interactors—66.82%—were nucleocytoplasmic shuttling proteins, and an additional 9.18% were exclusively cytoplasmic (Fig. 1A-B and Supplementary Fig. S1K-L). Contrary to expectations based on prior nuclear-focused studies, these findings suggest that RBMX may also localize to and function within the cytoplasm, prompting us to examine its context-dependent distribution in cancer cells. Because cancer cells often display altered signaling states that drive the cytoplasmic translocation of various RBPs 36, 37, 38 , we hypothesized that RBMX may exhibit context-dependent cytoplasmic localization in tumors. To test this, we performed IHC on tissue microarrays from lung and colorectal cancer patients and quantified RBMX staining specifically within the cytoplasm. Cytoplasmic RBMX levels were significantly higher in tumor tissues than in normal tissues and increased progressively with tumor grade (Fig. 1C and D). Elevated cytoplasmic RBMX expression was also associated with poorer patient prognosis (Fig. 1E). Consistent with these patient data, RBMX protein was detectable in the cytoplasmic fraction of seven cancer cell lines, including H1299 and HCT116 (Fig. 1F and G). Because hyperactive growth factor signaling is a hallmark of cancer progression 39, 40 , we examined whether external stimuli promote cytoplasmic RBMX accumulation. Indeed, treatment with EGF or TPA after serum starvation markedly increased cytoplasmic RBMX levels (Fig. 1H-K and Supplementary Fig. S1M-N). Taken together, these findings demonstrate that RBMX, although classically considered a nuclear RBP, can localize to the cytoplasm in cancer cells. This cytoplasmic localization is enhanced by growth factor signaling and correlates with tumor malignancy and poor clinical outcomes, suggesting that RBMX may exert important cytoplasmic functions during cancer progression. Cytoplasmic RBMX promotes cap-dependent translation initiation To define the physiological relevance of RBMX in the cytoplasm, we first profiled RBMX-associated cytoplasmic proteins. Immunoprecipitation followed by mass spectrometry using cytoplasmic extracts from HEK293T cells overexpressing Flag-tagged RBMX identified 22 proteins that selectively interacted with cytoplasmic RBMX (Fig. 2A-B and Supplementary Table S1). STRING network analysis revealed that these interactors were enriched for factors involved in translation and methylation pathways (Fig. 2C and Supplementary Fig. S2A). To validate the proteomic findings, we performed co-immunoprecipitation and Western blot analyses in HEK293T cells expressing Flag-RBMX. RBMX interacted with the identified protein candidates even after RNase A treatment, indicating that these associations are RNA-independent (Fig. 2D). Moreover, upon arsenite or thapsigargin treatment—conditions that induce stress granule (SG) formation by causing translation initiation factors to aggregate—RBMX co-localized with eIF3b within SGs and co-immunoprecipitated with eIF3b in cytoplasmic extracts (Supplementary Fig. S2B and C). To further assess the involvement of RBMX in translation, we examined its distribution across ribosomal fractions. Polysome profiling showed that RBMX localized to the 40S, 60S, and 80S ribosomal subunits as well as light polysomes (Supplementary Fig. S2D). EDTA-mediated polysome disassembly caused RBMX, translation initiation factors, and ribosomal proteins to shift in parallel patterns (Fig. 2E and Supplementary Fig. S2E-H), supporting a functional association of RBMX with translational complexes. In line with these observations, knockdown of RBMX in H1299 and H460 cells significantly reduced de novo protein synthesis (Fig. 2F and Supplementary Fig. S2I). Polysome profiling after RBMX depletion demonstrated a pronounced loss of polysomes accompanied by enhanced 40S and 80S peaks. Several translation-related proteins—including eEF2, eIF4A1, eIF3b, eIF3F, eIF3i, rpS3, rpL26, and rpL36a—shifted toward monosome fractions upon RBMX loss (Fig. 2G-H and Supplementary Fig. S2J-K), underscoring the essential role of RBMX in sustaining active protein synthesis. Translation initiation represents a highly regulated rate-limiting step in protein synthesis 34 . To evaluate the functional relevance of RBMX–initiation factor interactions, we examined recruitment of translation initiation components to the mRNA 7-methylguanosine (m7G) cap. In H1299 cells, m7G-cap pulldown assays revealed that RBMX depletion markedly reduced the association of eIF4A1, eIF3b, and eIF3i with the cap structure. Importantly, RBMX itself was detected within the cap-bound initiation complex (Fig. 2I). Conversely, treatment with EGF—a potent stimulator of global protein synthesis—significantly enhanced the recruitment of RBMX, eIF4G1, eIF3b, and eIF4A1 to the m7G cap (Fig. 2J). Consistent with increased translational activity, EGF treatment elevated polysome abundance and shifted RBMX, eIF3b, eIF3F, eIF3i, rpS6, rpS3, and rpL26 into heavy polysome fractions. These effects were entirely absent in RBMX-depleted cells (Fig. 2K and L). m7G-cap pulldown assays further demonstrated that EGF-induced recruitment of eIF3b, eIF3i, eIF3F, and eIF4A1 to the initiation complex required RBMX, as this enhancement was abolished upon RBMX knockdown (Fig. 2M). Collectively, these results demonstrate that cytoplasmic RBMX facilitates protein synthesis by engaging translation initiation factors and promoting assembly of the cap-dependent translation initiation complex. Cytoplasmic RBMX is a specificity factor for oncogenic mRNA translation Several RNA-binding proteins (RBPs) that relocalize to the cytoplasm can selectively engage specific transcripts and modulate their stability or translation 15, 18, 24 . Although global mapping of RBP-bound RNAs has provided important insights into such mechanisms, previous studies examining the RBMX interactome relied on whole-cell extracts without distinguishing between nuclear and cytoplasmic RNA pools 41 . Given that RBMX is predominantly nuclear, its cytoplasmic RNA targets were likely obscured in these earlier analyses. To define the cytoplasmic RNA interactome of RBMX, we expressed Flag-RBMX in HEK293T cells, isolated cytoplasmic fractions, and performed RNA immunoprecipitation followed by RNA sequencing. This approach identified a distinct set of RBMX-bound RNAs compared with controls (Fig. 3A-C and Supplementary Fig. S3A-B). Most cytoplasmic RBMX-associated transcripts were mRNAs (74.48%), with additional binding to lncRNAs (19.27%) and pseudogenes (5.04%) (Fig. 3D and Supplementary Table S2). Functional annotation of significantly enriched mRNAs (RIP/Input ≥ 1.5-fold, p < 0.05) highlighted genes involved in transcription (18.08%), membrane protein trafficking (15.88%), and metabolic processes (15.72%). KEGG pathway analysis further revealed enrichment of cancer-associated pathways, including ribosome biogenesis and MAPK, Notch, and mTOR signaling (Fig. 3E-F and Supplementary Fig. S3C and Supplementary Tables S3 and S4). Comparison with previously published PAR-CLIP datasets generated from whole-cell extracts identified 539 overlapping transcripts. Among these, YBX1 mRNA ranked among the most enriched shared targets (Fig. 3G and Supplementary Table S5). To validate these interactions, we performed qRT-PCR and RT-PCR analyses, confirming that RBMX specifically bound YBX1, PPARγ, PARG, MAN1A2, FKBP3, PPDPF, EphA8, and BACH1 mRNAs, but not YTHDF1, ARFGAP1, β-actin, or GMEB2 (Fig. 3H and Supplementary Fig. S3D). We next examined the functional consequences of RBMX binding on gene expression. In H1299, HCT116, and SW480 cells, RBMX depletion markedly reduced the protein levels—but not the mRNA abundance—of YBX1, PARG, BACH1, and FKBP3 (Fig. 3I-J and Supplementary Fig. S3E-F). These observations suggest that RBMX enhances the translation of its bound mRNAs. Supporting this conclusion, polysome profiling in RBMX-depleted H1299 cells showed that YBX1, PARG, FKBP3, and BACH1 transcripts shifted from heavy to light polysomes, consistent with reduced translational efficiency. In contrast, non-bound transcripts such as β-actin, ARFGAP1, and YTHDF1 displayed unchanged polysome distributions (Fig. 3K and L). Among the cytoplasmic RBMX targets, YBX1 was of particular interest due to its strong enrichment and established oncogenic function. Analysis of TCGA lung cancer RNA-seq datasets revealed a significant positive correlation between RBMX expression and the expression of YBX1 target genes (Fig. 3M and Supplementary Fig. S3G), whereas no such correlation was observed for non-target genes (Supplementary Fig. S3H). Consistent with these findings, knockdown of either RBMX or YBX1 in H1299, HCT116, and SW480 cells reduced the expression of canonical YBX1 targets, including SRSF7, hnRNPL, CDK2, eIF5, eEF1A1, and C11orf58 (Fig. 3N and Supplementary Fig. S3I-J). Importantly, overexpression of Myc-tagged YBX1 restored the expression of these targets in RBMX-depleted cells (Supplementary Fig. S3K-L), demonstrating that impaired YBX1 translation mediates the downstream effects of RBMX loss. Finally, clinical tissue analyses revealed that cytoplasmic RBMX expression positively correlated with YBX1 protein levels in lung cancer patient samples (Fig. 3O-Q). Similar correlations were observed for PARG, FKBP3, and BACH1 (Supplementary Fig. S3M-O), extending the relevance of RBMX-mediated translational regulation to human tumors. Collectively, these findings establish cytoplasmic RBMX as a specificity factor that selectively enhances the translation of oncogenic mRNAs, thereby contributing to cancer progression through targeted modulation of the translational landscape. eIF3i and eIF3F mediate cytoplasmic RBMX function in translational regulation Translation initiation is the rate-limiting step of protein synthesis and a key regulatory node in translational control 34 . To elucidate how RBMX promotes translation, we first examined its interactions with major components of the translation initiation machinery. HEK293T cells were transfected with a panel of Flag-tagged initiation factors—including eIF2α, eIF3F, eIF3i, eIF3h, eIF3g, eIF4A1, eIF5A, eIF4G1, and eEF1α1—followed by anti-Flag immunoprecipitation. Immunoblot analysis revealed that RBMX associates with eIF3F, eIF3i, eIF3g, eIF4A1, and eIF4G1 (Fig. 4A). To determine whether these interactions are direct, we generated recombinant full-length RBMX using a cell-free protein synthesis (CFPS) system and purified His x6 -tagged recombinant initiation factors (eIF3e, eIF3F, eIF3g, eIF3h, eIF3i, eIF3m, eIF2α, and eIF4A1). Direct binding assays demonstrated that only eIF3i and eIF3F directly interact with RBMX (Fig. 4B). We next mapped the RBMX domains required for these interactions. Using GST-fused deletion mutants of RBMX in direct pull-down assays with His x6 -tagged eIF3i or eIF3F, we found that only the RGG domain of RBMX was capable of binding both proteins (Fig. 4C and Supplementary Fig. S4A). These findings indicate that the RGG domain serves as the primary interface through which RBMX engages eIF3i and eIF3F. To explore the clinical relevance of these interactions, we performed proximity ligation assays (PLA) on tissue microarrays containing normal, primary, and various tumor-grade samples from lung cancer and colorectal cancer patients. PLA signals corresponding to RBMX–eIF3i and RBMX–eIF3F interactions were markedly elevated in tumor tissues relative to normal tissues and increased progressively with tumor grade (Fig. 4D-E and Supplementary Fig. S4B–C). Importantly, patients with higher RBMX–eIF3i/eIF3F interaction levels exhibited significantly poorer prognosis in both lung cancer and colorectal cancer cohorts (Fig. 4F and Supplementary Fig. S4D), underscoring the clinical significance of these interactions. Because the RGG domain of RBMX is known to be methylated by PRMT5 (Fig.4G) 19, 42 , and because PRMT5–MEP50–pICln methylosome components were identified among RBMX-interacting proteins in cytoplasmic extracts (Fig. 2A-C), we next examined the methylation status of RBMX. Fractionation analyses revealed that RBMX in the cytoplasm predominantly existed in a demethylated form, whereas methylated RBMX was enriched in the nucleus (Fig. 4H-I). We then asked how PRMT5-mediated methylation influences RBMX localization and function. Pharmacological inhibition of PRMT5 promoted cytoplasmic translocation of RBMX, as observed by confocal microscopy and immunoblotting (Fig. 4J and Supplementary Fig. S4E). Consistent with this shift, treatment of Flag-RBMX–expressing HEK293T cells with a PRMT5 inhibitor substantially enhanced the interaction between RBMX and multiple translation initiation factors, including eIF4G, eIF3b, eIF3i, and eIF3F (Fig. 4K-L). Collectively, these findings demonstrate that eIF3i and eIF3F directly engage the RGG domain of RBMX to facilitate translation initiation, and that PRMT5-mediated methylation modulates RBMX cytoplasmic localization and interaction with the initiation machinery. These results highlight the mechanistic and clinical significance of RBMX–eIF3 interactions in translational regulation. Demethylation of the RBMX RGG motif drives its cytoplasmic localization and control of translation Because PRMT5 methylates a broad spectrum of substrates 15, 16, 17 , the effects of PRMT5 inhibition on RBMX function could include indirect contributions. To directly assess how RBMX demethylation influences its activity, we generated a methylation-deficient mutant by substituting four arginine residues within the RGG domain (R369K, R373K, R377K, and R384K) with lysines (RBMX_4RK). Consistent with our hypothesis, the demethylation-mimetic RBMX_4RK mutant exhibited markedly enhanced interactions with translation initiation factors compared with wild-type RBMX (Fig. 5A-B and Supplementary Fig. S5A), along with a more pronounced cytoplasmic localization (Fig. 5C-D). These findings align with prior reports showing that demethylation of RGG-containing RBPs promotes their cytoplasmic translocation 18, 24, 43 . We next examined whether demethylated RBMX more actively stimulates translation. Overexpression of V5-tagged RBMX_WT or RBMX_4RK in HEK293T cells followed by polysome profiling revealed that RBMX_WT increased polysome abundance and shifted eIF3b, eIF3i, eIF3F, and ribosomal protein L26 toward heavy polysome fractions. Notably, these effects were significantly amplified in RBMX_4RK-expressing cells (Fig. 5E-F). Consistently, m7G-cap pulldown analyses showed that RBMX_4RK enhanced the recruitment of multiple initiation factors to the cap structure more effectively than RBMX_WT (Fig. 5G). Functionally, RBMX_4RK promoted cell migration and anchorage-independent growth in H1299 cells to a considerably greater extent than wild-type RBMX (Supplementary Fig. S5B and C). Together, these findings demonstrate that demethylation of the RBMX RGG domain strengthens its interactions with translation initiation factors, enhances translational output, and contributes to tumorigenic phenotypes. Cytoplasmic RBMX–YBX1– p21 cip/waf1 axis prevents cellular senescence Loss of RBMX markedly impaired proliferative capacity and tumor growth in vitro and in vivo (Supplementary Fig. S1E-I). During these experiments, we observed that RBMX knockdown in H1299 and H460 cells induced pronounced increases in cell size and multinucleation (Fig. 6A), morphological features strongly associated with cellular senescence 35, 44 . As shown in Figure 3, RBMX-bound cytoplasmic transcripts were significantly enriched for genes regulating transcription. To characterize downstream changes in gene expression following RBMX loss, we performed ClueGO-based KEGG pathway enrichment using genes altered more than twofold (p < 0.05) after RBMX knockdown. The most prominently affected pathways were related to the cell cycle and cellular senescence (Fig. 6B and C). Among these genes, CDKN1A (p21 cip/waf1 ) showed the strongest induction within both categories (Fig. 6D). To validate these transcriptomic findings, we used a cell stress protein array, which confirmed upregulation of multiple senescence-associated proteins—including p21 cip/waf1 , HSP70, SOD2, Thioredoxin1, and SIRT2—with p21 cip/waf1 showing the most robust increase (Fig. 6E). Consistent with this, depletion of RBMX or YBX1 in H1299 cells greatly increased SA-β-galactosidase staining (Supplementary Fig. S6A) and induced strong p21 expression at both mRNA and protein levels (Fig. 6F and Supplementary Fig. S6B-D). Because YBX1 is a direct target of RBMX-mediated translation and is known to repress p21 expression 45, 46, 47 , we examined whether RBMX loss induces senescence via YBX1 downregulation. Indeed, RBMX knockdown decreased YBX1 levels and increased p21 cip/waf1 expression (Fig. 6F and Supplementary Fig. S6C), consistent with a model in which impaired YBX1 translation drives p21 upregulation. Given that p21 cip/waf1 is a canonical transcriptional target of p53 (Fig. 6G) 48, 49 , we tested whether RBMX regulates p21 in a p53-dependent manner. In p53-null H1299 cells, HCT116 p53⁻/⁻ cells, and HCT116 cells treated with p53 siRNA, RBMX depletion continued to induce p21 expression (Fig. 6F and Supplementary Fig. S6E-H), demonstrating that this effect occurs independently of p53. To further test whether YBX1 mediates RBMX-dependent p21 regulation, we performed rescue experiments. In HCT116 cells, RBMX knockdown increased p21 expression and senescence-associated SA-β-gal positivity; co-expression of Myc-tagged YBX1 abolished both effects (Fig. 6H-J). Importantly, RBMX-mediated regulation of YBX1 and p21 was substantially more pronounced in cells expressing the demethylation-mimetic RBMX_4RK mutant than in those expressing wild-type RBMX (Fig. 6K), linking RBMX post-translational modification to its control of senescence pathways. Collectively, these findings demonstrate that cytoplasmic RBMX prevents cellular senescence by sustaining YBX1 protein expression and repressing p21 Cip/Waf1 expression, thereby promoting tumor cell fitness and malignancy (Fig. 6L). DISCUSSION This study uncovers a regulatory mechanism in which post-translational modification and growth factor signaling cooperate to convert RBMX from a nuclear splicing factor into a cytoplasmic, translation-selective effector of cancer progression. Although RBMX has been associated primarily with genome maintenance and co-transcriptional RNA processing 4 , 13 , 14 , our data show that its oncogenic activity arises through a mechanistically distinct program controlled by PRMT5-dependent arginine methylation of the C-terminal RGG/RG motif 15 , 16 , 19 , 21 . This methylation event functions as a molecular gate: methylated RBMX remains nuclear, whereas demethylated RBMX—promoted by growth factor signaling—translocates to the cytoplasm, where it engages the translation initiation machinery. Mechanistically, demethylation promotes an interaction-competent state of RBMX that enables its association with eIF3i and eIF3F, two subunits critical for mRNA recruitment and start-codon scanning. Our proteomic, biochemical, and ribosomal-engagement analyses demonstrate that cytoplasmic RBMX facilitates productive assembly of the cap-dependent initiation complex, thereby enabling translation of specific mRNAs. Importantly, RBMX does not act as a global translational enhancer. Instead, demethylation unmasks a role for RBMX as a translation-specificity factor, preferentially promoting the translation of a restricted gene set enriched for oncogenic regulators. This property distinguishes RBMX from shuttling RBPs such as hnRNPA1, FUS, HUR, and TDP-43 whose cytoplasmic mislocalization drives broader perturbations of RNA metabolism 18 , 24 , 43 , 50 . Our findings introduce the concept that the translational landscape of cancer cells includes PTM-tuned, transcript-selective RBPs that act as regulatory nodes downstream of growth and stress signals. Within this selective network, YBX1 emerges as the principal functional target. YBX1 controls oncogenic transcriptional and translational programs and represses p21-mediated senescence 47 , 49 , 51 . We show that RBMX is required for YBX1 protein synthesis without altering its mRNA abundance, defining a translational dependency. RBMX depletion sharply reduces YBX1 protein levels and triggers p53-independent induction of p21, resulting in a robust senescence response. The demethylation-mimetic RBMX mutant (4RK) strongly amplifies YBX1 translation and suppresses senescence, demonstrating that RGG demethylation is the switch that determines whether RBMX exerts nuclear or cytoplasmic functions. This establishes a mechanistic RBMX–YBX1–p21 axis, linking extracellular cues and PTM control to cell-cycle arrest and proliferative escape. The mechanistic insights uncovered here hold major translational implications. Cytoplasmic RBMX abundance, RBMX–eIF3 engagement, and YBX1 expression correlate strongly across patient tumors and align with poor survival outcomes in lung and colorectal cancers. These findings suggest that tumors with high RBMX cytoplasmic activity may represent a distinct subclass driven by PTM-controlled translational plasticity. Our data also carry substantial relevance for cancer therapies targeting PRMT5. While PRMT5 is widely regarded as oncogenic 52 , 53 , our results reveal a paradox: PRMT5-dependent methylation restrains RBMX cytoplasmic activity, and PRMT5 inhibition inadvertently enhances RBMX-driven selective translation, thereby potentiating tumor aggressiveness. This mechanistic duality mirrors observations in other methylation-sensitive RBPs, where loss of arginine methylation promotes cytoplasmic accumulation and pathological translation 18 , 22 , 23 , 43 . These insights raise the possibility that PRMT5 inhibitors—currently advancing in clinical trials—may carry unintended risks in tumors with high RBMX expression or dependency. Collectively, this study positions RBMX as a methylation- and signal-tuned regulator of selective translation whose cytoplasmic activation promotes YBX1 synthesis, suppresses p21-driven senescence, and accelerates tumor progression. By defining demethylation as a key activating PTM and revealing RBMX’s function as a translation-specificity factor, we provide a mechanistic framework that connects extracellular signaling, PTM remodeling, translational selectivity, and cancer cell fate. These findings suggest that therapeutic strategies targeting RBMX’s methylation state, cytoplasmic trafficking, or translational interactions may offer new opportunities to disrupt oncogenic translational circuits—particularly in RBMX-high tumors. Future work will be required to delineate the complete RBMX-dependent translational portfolio, to resolve how RBMX integrates with other RBPs and stress-response networks, and to evaluate whether dual modulation of RBMX and PRMT5 can yield tumor-selective vulnerabilities. MATERIALS AND METHODS Cell culture and transfection H1299 and H460 cells were cultured in RPMI-1640 medium (Invitrogen), and HEK293T, SW480, U2OS, HCT116, and HCT116 p53⁻/⁻ cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen). All cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, and cells were maintained at 37 °C in a humidified incubator with 5% CO₂. Plasmid transfections were performed using TurboFect (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Sodium arsenite, EGF, and TPA were purchased from Sigma-Aldrich (St. Louis, MO, USA), and the PRMT5 inhibitor GSK3235025 was obtained from Selleckchem (Houston, TX, USA). Plasmid constructs and cloning Human RBMX cDNA was amplified by RT-PCR and cloned into pCI-neo-Flag or V5 expression vectors (Promega). Serial deletion constructs of RBMX were generated by PCR amplification of individual fragments using pCI-neo-Flag-RBMX as a template, followed by insertion into XhoI and NotI sites of the pCI-neo-Flag vector. For in vitro GST pulldown assays, RBMX fragments were subcloned into pGEX4T-1 (GE Healthcare). The pCI-neo-Flag or V5-RBMX_4RK constructs (R369K, R373K, R377K, and R384K) were generated by site-directed mutagenesis using the QuikChange II Site-Directed Mutagenesis kit (Agilent Technologies). His x6 -tagged eIF3e, eIF3F, eIF3g, eIF3h, eIF3i, eIF3m, eIF2α, and eIF4A1 constructs used for recombinant protein isolation have been described previously 54 . The pDEST-myc-YBX1 plasmid (#19878) was obtained from Addgene (Watertown, MA, USA). All PCR primer sequences are listed in Supplementary Table S6. Quantitative real-time PCR (RT-qPCR) Total RNA was extracted using TRIzol reagent (Invitrogen), and 2 µg of RNA was reverse transcribed with oligo(dT) primers and M-MuLV Reverse Transcriptase (Invitrogen). RT-qPCR was performed using gene-specific primers and the SYBR Premix Ex Taq™ kit (TaKaRa Bio, Shiga, Japan) on a CFX96 Real-Time PCR Detection System (Bio-Rad, CA, USA). Target transcripts included RBMX, YBX1, PPARγ, MAN1A2, FKBP3, PPDPF, EphA8, BACH1, YTHDF1, ARFGAP1, GMEB2, SRSF7, hnRNPL, CDK2, eIF5, ZNF207, UBXN4, C11orf58, eIF1α1, SSRP1, PRPF40, IPO5, and β-actin. Each sample was analyzed in triplicate. Ct values for each gene were normalized to β-actin, and relative expression was calculated using the comparative Ct method (ΔCt = Ct(β-actin) – Ct(target)). Fold changes in expression relative to control were calculated as 2^−ΔΔCt. Primer sequences used for RT-qPCR are listed in Supplementary Table S6. RNA interference For transient knockdown, cells were transfected with siRNAs (40 nM) using Lipofectamine RNAiMAX (Invitrogen). After 36 h, cells were trypsinized, re-plated, and transfected a second time for an additional 36 h. Knockdown efficiency was confirmed by Western blotting. For stable knockdown of RBMX, cells were transfected with pSilencer2.1-U6-hygro control shRNA or pSilencer2.1-U6-hygro RBMX shRNA using TurboFect and selected in medium containing 500 µg/ml hygromycin for 4–5 weeks. siRNA and shRNA sequences are listed in Supplementary Table S7. Immunoblot and immunoprecipitation analysis Cells were lysed in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitors (1 mM Na₂VO₄, 10 mM NaF, 2 mM PMSF, 5 µg/ml leupeptin, 10 µg/ml aprotinin, 1 µg/ml pepstatin A; Roche). Equal amounts of protein were resolved by SDS-PAGE and transferred to PVDF membranes (Pall Life Sciences, USA). Membranes were incubated with primary antibodies overnight at 4 °C, followed by HRP-conjugated secondary antibodies for 1 h at room temperature. Signals were detected using ECL chemiluminescent reagents (iNtRON Biotechnology). For immunoprecipitation, lysates were pre-cleared with protein G–Sepharose beads (GE Healthcare) and then incubated with the appropriate antibodies. Immune complexes were captured with protein G–Sepharose, washed, and analyzed by immunoblotting. Antibodies used in this study are listed in Supplementary Table S8. Immunohistochemistry (IHC) IHC was performed on tissue microarrays (TMAs) containing lung and colorectal cancer samples of various grades and adjacent normal tissues (Super Bio Chips; CDN4, CD4, CDA3, CCN5, CC5, CCA4; Seoul, South Korea). Heat-induced antigen retrieval was carried out in 1× antigen retrieval buffer (pH 9.0; Abcam) at 95 °C for 15 min. After quenching endogenous peroxidase activity and blocking in 3% H₂O₂, sections were incubated with primary antibodies overnight at 4 °C, followed by HRP-conjugated secondary antibodies for 1 h at room temperature. DAB (3,3′-diaminobenzidine) was used as the chromogen (2 min), and slides were counterstained with Harris’s hematoxylin. Staining intensity was scored from 0 to 4, and the extent of staining from 0% to 100%; final scores were obtained by multiplying intensity and extent. Slides were evaluated independently by two pathologists. For RBMX, cytoplasmic staining intensity was quantified using ImageJ (Fiji) after excluding nuclear regions. Antibodies are listed in Supplementary Table S8. Total RNA sequencing analysis Total RNA was isolated from cells using TRIzol reagent (1 ml per 60-mm dish) and treated with DNase I (Invitrogen). RNA-seq libraries were prepared and sequenced on an Illumina NovaSeq 6000 platform (DNA Link™, Seoul, Korea). Reads were mapped to the human reference genome (GRCh37/hg19) using TopHat v2.0.13 (http://ccb.jhu.edu/software/tophat/). Expression levels and differentially expressed genes (DEGs) were determined using Cuffdiff v2.2.1 (http://cole-trapnell-lab.github.io/cufflinks/papers/), generating FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values. Geometric and pooled normalization methods were applied for library normalization and dispersion estimation. Heatmaps were generated in R 3.4.1 using the heatmap.2 function (gplots package). DEGs between control and RBMX-deficient cells were subjected to KEGG pathway enrichment using ClueGO (Cytoscape v3.10.1). Bubble plots summarizing pathway significance and gene-level parameters were generated using Python (Matplotlib v3.9.0, Pandas v2.2.2). RNA-seq data have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession number GSE310912. RNA immunoprecipitation (RIP), sequencing, and data analysis RIP was performed as described previously 55 with modifications. HEK293T cells were transfected with either empty vector or Flag-RBMX expression plasmid and harvested 48 h later. Cytoplasmic fractions were prepared by removing nuclear components. Cytoplasmic lysates were incubated with anti-Flag M2 antibody (Sigma-Aldrich) coupled to protein G magnetic beads. RNA–protein complexes were extracted in 1 ml TRIzol (Invitrogen), and co-precipitated RNA was purified according to the manufacturer’s protocol and treated with DNase I (Invitrogen). RIP RNA libraries were sequenced on an Illumina NovaSeq 6000 platform (DNA Link). Peak calling and read density visualization were performed using the UCSC Genome Browser. Functional enrichment of RBMX-associated transcripts was carried out using ClueGO (Cytoscape v3.10.1) based on KEGG annotations. Bubble plots were generated in Python (Matplotlib v3.9.0, Pandas v2.2.2), with the x-axis representing the Flag-RBMX RIP/Input ratio, bubble size indicating the percentage of associated genes, and color reflecting –log₁₀(p value) using a viridis_r color scale. RIP-seq data have been deposited in GEO under accession number GSE311136. Human Cell Stress Array Cell lysates from cells treated with siControl or siRBMX for 72 h were analyzed using the Human Cell Stress Array (ARY018; R&D Systems). Two hundred micrograms of protein were incubated with the array membrane according to the manufacturer’s instructions. Bound proteins were detected with a cocktail of biotinylated detection antibodies, streptavidin-HRP (1:2000), and chemiluminescent substrate. Membranes were scanned, and dot intensities were quantified using ImageJ. Polysome profiling analysis HEK293T, U2OS, H1299, and H460 cells were lysed in polysome buffer (20 mM HEPES pH 7.6, 125 mM KCl, 5 mM MgCl₂, 2 mM DTT, DEPC-treated water). Lysates were incubated on ice for 15 min and clarified by centrifugation at 13,000 rpm for 15 min. Supernatants were layered onto 17.5–50% sucrose gradients prepared in polysome buffer and centrifuged for 2.4 h at 35,000 rpm in an SW41-Ti rotor (Beckman, Brea, CA, USA). Gradients were fractionated using a fraction collector (Brandel, Gaithersburg, MD, USA), and absorbance at 253 nm was monitored with a UA-6 detector (ISCO, Lincoln, NE, USA). m⁷GTP pulldown assay For m⁷GTP pulldown, cell extracts were pre-cleared with protein A–agarose beads (Santa Cruz Biotechnology, TX, USA) and then incubated with m⁷GTP-agarose beads (Jena Biosciences, Germany) or control protein A–agarose beads. m⁷GTP-agarose beads were equilibrated in m⁷GTP lysis buffer (50 mM HEPES pH 7.6, 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.5% NP-40, 10% glycerol, 1 mM Na₂VO₄, 10 mM NaF, 2 mM PMSF, 5 µg/ml leupeptin, 10 µg/ml aprotinin, 1 µg/ml pepstatin A) for 30 min before use. Bound m⁷GTP–protein complexes were washed and analyzed by immunoblotting. Antibodies are listed in Supplementary Table S5. Ribo-puromycylation assay HEK293T, H1299, and H460 cells were seeded in 60-mm dishes and grown for 2 days. Cells were pulsed with puromycin (10 µg/ml) for 10 min at 37 °C in a 5% CO₂ incubator, washed twice with cold PBS, and lysed in RIPA buffer. Lysates were subjected to Western blotting, and puromycylated nascent polypeptides were detected using an anti-puromycin antibody. SA-β-gal staining H1299, HCT116 (p53⁺/⁺), and HCT116 (p53⁻/⁻) cells were seeded in 6-well plates and transfected with siControl, siRBMX, or siYBX1 for 72 h. Senescence-associated β-galactosidase activity was assessed using a SA-β-gal staining kit (#9860, Cell Signaling Technology) according to the manufacturer’s instructions. The percentage of SA-β-gal–positive cells was calculated as the number of blue-stained (senescent) cells divided by the total number of cells counted. Identification of proteins by LC–MS/MS LC–MS/MS was performed using a nanoACQUITY UPLC system coupled to an LTQ-Orbitrap mass spectrometer (Thermo Electron, San Jose, CA, USA). Peptides were separated on a BEH C18 column (1.7 µm, 100 µm × 100 mm; Waters, Milford, MA, USA). Mobile phase A was 0.1% formic acid in water; mobile phase B was 0.1% formic acid in acetonitrile. The gradient was 10–40% B over 16 min, 40–95% B over 8 min, and 95–10% B over 11 min at a flow rate of 0.5 µl/min. Mass spectra were acquired in data-dependent mode with a full scan (m/z 300–2000) followed by MS/MS of selected precursors. The ion transfer tube was maintained at 275 °C, spray voltage at 2.3 kV, and normalized collision energy at 35%. MS/MS spectra were processed using SEQUEST (Thermo Quest), and peak lists were searched against an in-house database using MASCOT (Matrix Science, London, UK). Variable modifications included carbamidomethyl (C), deamidation (NQ), and oxidation (M). Peptide mass tolerance was set to 10 ppm, MS/MS ion tolerance to 0.8 Da, with up to two missed cleavages and charge states +2 and +3 considered. Only significant hits, as defined by MASCOT probability scores, were retained. Statistics Data are presented as mean ± SEM from at least three independent experiments unless otherwise indicated. Statistical significance between two groups was assessed using two-tailed paired Student’s t-tests, and multiple comparisons were analyzed by two-way ANOVA, using GraphPad Prism (GraphPad Software Inc.). P values < 0.05 were considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001). Declarations Conflict of interest: The authors have declared that no conflict of interest exists. AUTHOR CONTRIBUTIOMS JK, YJJ, JY, and HJY designed the experiments and analyzed data; JK, IC, and YJJ performed the experiments; JK, YJJ, JY, and HJY wrote the manuscript. COMPETING INTERESTS The authors declare no competing interests. FUNDING This work is supported by National Research Foundation of Korea (NRF), funded by the Ministry of Science and ICT (RS-2022-NR070848, RS-2022-NR072262 and RS-2024-00350874). ACKNOWLEDGEMENTS We thank all participants of this study for their invaluable devotion. References Glisovic T, Bachorik JL, Yong J, Dreyfuss G. RNA-binding proteins and post-transcriptional gene regulation. FEBS Lett 2008, 582 (14) : 1977-1986. Hentze MW, Castello A, Schwarzl T, Preiss T. A brave new world of RNA-binding proteins. Nat Rev Mol Cell Biol 2018, 19 (5) : 327-341. Geuens T, Bouhy D, Timmerman V. The hnRNP family: insights into their role in health and disease. Hum Genet 2016, 135 (8) : 851-867. Elliott DJ, Dalgliesh C, Hysenaj G, Ehrmann I. RBMX family proteins connect the fields of nuclear RNA processing, disease and sex chromosome biology. Int J Biochem Cell Biol 2019, 108: 1-6. Moursy A, Allain FH, Clery A. Characterization of the RNA recognition mode of hnRNP G extends its role in SMN2 splicing regulation. Nucleic Acids Res 2014, 42 (10) : 6659-6672. Sheng Y, Lei K, Sun C, Liu J, Tu Z, Zhu X , et al. Aberrant RBMX expression is relevant for cancer prognosis and immunotherapy response. Aging (Albany NY) 2024, 16 (1) : 226-245. Song Y, He S, Ma X, Zhang M, Zhuang J, Wang G , et al. RBMX contributes to hepatocellular carcinoma progression and sorafenib resistance by specifically binding and stabilizing BLACAT1. Am J Cancer Res 2020, 10 (11) : 3644-3665. Wang Y, Zhao Z, Guo T, Wu T, Zhang M, Luo D , et al. SOCS5-RBMX stimulates SREBP1-mediated lipogenesis to promote metastasis in steatotic HCC with HBV-related cirrhosis. NPJ Precis Oncol 2024, 8 (1) : 58. Qiu Y, Pu C, Wang C, Quan Z. The RNA-Binding Protein RBMX Mediates the Immunosuppressive Microenvironment of Osteosarcoma by Regulating CD8(+)T Cells. Cancers (Basel) 2025, 17 (17). Li A, Hong J, Ma X, Huang Y, Jiang Q, Zhang C , et al. Cancer-Derived Exosomal LINC01615 Induces M2 Polarization of Tumor-Associated Macrophages via RBMX-EZH2 Axis to Promote Colorectal Cancer Progression. Int J Nanomedicine 2025, 20: 7343-7358. Yan Q, Zeng P, Zhou X, Zhao X, Chen R, Qiao J , et al. RBMX suppresses tumorigenicity and progression of bladder cancer by interacting with the hnRNP A1 protein to regulate PKM alternative splicing. Oncogene 2021, 40 (15) : 2635-2650. Shin KH, Kang MK, Kim RH, Christensen R, Park NH. Heterogeneous nuclear ribonucleoprotein G shows tumor suppressive effect against oral squamous cell carcinoma cells. Clin Cancer Res 2006, 12 (10) : 3222-3228. Zheng T, Zhou H, Li X, Peng D, Yang Y, Zeng Y , et al. RBMX is required for activation of ATR on repetitive DNAs to maintain genome stability. Cell Death Differ 2020, 27 (11) : 3162-3176. Liu J, Zheng T, Chen D, Huang J, Zhao Y, Ma W , et al. RBMX involves in telomere stability maintenance by regulating TERRA expression. PLoS Genet 2023, 19 (9) : e1010937. Xu J, Richard S. Cellular pathways influenced by protein arginine methylation: Implications for cancer. Mol Cell 2021, 81 (21) : 4357-4368. Musiani D, Bok J, Massignani E, Wu L, Tabaglio T, Ippolito MR , et al. Proteomics profiling of arginine methylation defines PRMT5 substrate specificity. Sci Signal 2019, 12 (575). Hwang JW, Cho Y, Bae GU, Kim SN, Kim YK. Protein arginine methyltransferases: promising targets for cancer therapy. Exp Mol Med 2021, 53 (5) : 788-808. Blackwell E, Ceman S. Arginine methylation of RNA-binding proteins regulates cell function and differentiation. Mol Reprod Dev 2012, 79 (3) : 163-175. Cai T, Cinkornpumin JK, Yu Z, Villarreal OD, Pastor WA, Richard S. Deletion of RBMX RGG/RG motif in Shashi-XLID syndrome leads to aberrant p53 activation and neuronal differentiation defects. Cell Rep 2021, 36 (2) : 109337. Lorton BM, Shechter D. Cellular consequences of arginine methylation. Cell Mol Life Sci 2019, 76 (15) : 2933-2956. Wu K, Niu C, Liu H, Fu L. Research progress on PRMTs involved in epigenetic modification and tumour signalling pathway regulation (Review). Int J Oncol 2023, 62 (5). Wall ML, Lewis SM. Methylarginines within the RGG-Motif Region of hnRNP A1 Affect Its IRES Trans-Acting Factor Activity and Are Required for hnRNP A1 Stress Granule Localization and Formation. J Mol Biol 2017, 429 (2) : 295-307. Nichols RC, Wang XW, Tang J, Hamilton BJ, High FA, Herschman HR , et al. The RGG domain in hnRNP A2 affects subcellular localization. Exp Cell Res 2000, 256 (2) : 522-532. Clarke JP, Thibault PA, Salapa HE, Levin MC. A Comprehensive Analysis of the Role of hnRNP A1 Function and Dysfunction in the Pathogenesis of Neurodegenerative Disease. Front Mol Biosci 2021, 8: 659610. Martinez-Salas E, Lozano G, Fernandez-Chamorro J, Francisco-Velilla R, Galan A, Diaz R. RNA-binding proteins impacting on internal initiation of translation. Int J Mol Sci 2013, 14 (11) : 21705-21726. Szostak E, Gebauer F. Translational control by 3'-UTR-binding proteins. Brief Funct Genomics 2013, 12 (1) : 58-65. Moore KS, von Lindern M. RNA Binding Proteins and Regulation of mRNA Translation in Erythropoiesis. Front Physiol 2018, 9: 910. Marchione R, Leibovitch SA, Lenormand JL. The translational factor eIF3f: the ambivalent eIF3 subunit. Cell Mol Life Sci 2013, 70 (19) : 3603-3616. Yin Y, Long J, Sun Y, Li H, Jiang E, Zeng C , et al. The function and clinical significance of eIF3 in cancer. Gene 2018, 673: 130-133. Esteves P, Dard L, Brillac A, Hubert C, Sarlak S, Rousseau B , et al. Nuclear control of lung cancer cells migration, invasion and bioenergetics by eukaryotic translation initiation factor 3F. Oncogene 2020, 39 (3) : 617-636. Qi J, Dong Z, Liu J, Zhang JT. EIF3i promotes colon oncogenesis by regulating COX-2 protein synthesis and beta-catenin activation. Oncogene 2014, 33 (32) : 4156-4163. Ma S, Dong Z, Cui Q, Liu JY, Zhang JT. eIF3i regulation of protein synthesis, cell proliferation, cell cycle progression, and tumorigenesis. Cancer Lett 2021, 500: 11-20. Zhang Y, Wan X, Yang X, Liu X, Huang Q, Zhou L , et al. eIF3i promotes colorectal cancer cell survival via augmenting PHGDH translation. J Biol Chem 2023, 299 (9) : 105177. Jackson RJ, Hellen CU, Pestova TV. The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Biol 2010, 11 (2) : 113-127. Hernandez-Segura A, Nehme J, Demaria M. Hallmarks of Cellular Senescence. Trends Cell Biol 2018, 28 (6) : 436-453. van der Houven van Oordt W, Diaz-Meco MT, Lozano J, Krainer AR, Moscat J, Caceres JF. The MKK(3/6)-p38-signaling cascade alters the subcellular distribution of hnRNP A1 and modulates alternative splicing regulation. J Cell Biol 2000, 149 (2) : 307-316. Doller A, Huwiler A, Muller R, Radeke HH, Pfeilschifter J, Eberhardt W. Protein kinase C alpha-dependent phosphorylation of the mRNA-stabilizing factor HuR: implications for posttranscriptional regulation of cyclooxygenase-2. Mol Biol Cell 2007, 18 (6) : 2137-2148. Doller A, Pfeilschifter J, Eberhardt W. Signalling pathways regulating nucleo-cytoplasmic shuttling of the mRNA-binding protein HuR. Cell Signal 2008, 20 (12) : 2165-2173. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011, 144 (5) : 646-674. Griner EM, Kazanietz MG. Protein kinase C and other diacylglycerol effectors in cancer. Nat Rev Cancer 2007, 7 (4) : 281-294. Liu N, Zhou KI, Parisien M, Dai Q, Diatchenko L, Pan T. N6-methyladenosine alters RNA structure to regulate binding of a low-complexity protein. Nucleic Acids Res 2017, 45 (10) : 6051-6063. Larsen SC, Sylvestersen KB, Mund A, Lyon D, Mullari M, Madsen MV , et al. Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells. Sci Signal 2016, 9 (443) : rs9. Dormann D, Madl T, Valori CF, Bentmann E, Tahirovic S, Abou-Ajram C , et al. Arginine methylation next to the PY-NLS modulates Transportin binding and nuclear import of FUS. EMBO J 2012, 31 (22) : 4258-4275. Kumari R, Jat P. Mechanisms of Cellular Senescence: Cell Cycle Arrest and Senescence Associated Secretory Phenotype. Front Cell Dev Biol 2021, 9: 645593. Lu ZH, Books JT, Ley TJ. YB-1 is important for late-stage embryonic development, optimal cellular stress responses, and the prevention of premature senescence. Mol Cell Biol 2005, 25 (11) : 4625-4637. Zhu B, Zhang Z, Pardeshi L, Chen Y, Ge W. Y box-binding protein 1 regulates zebrafish folliculogenesis partly through p21-mediated control of follicle cell proliferation. Development 2024, 151 (21). Kwon E, Todorova K, Wang J, Horos R, Lee KK, Neel VA , et al. The RNA-binding protein YBX1 regulates epidermal progenitors at a posttranscriptional level. Nat Commun 2018, 9 (1) : 1734. Beckerman R, Prives C. Transcriptional regulation by p53. Cold Spring Harb Perspect Biol 2010, 2 (8) : a000935. Engeland K. Cell cycle regulation: p53-p21-RB signaling. Cell Death Differ 2022, 29 (5) : 946-960. Sama RR, Ward CL, Kaushansky LJ, Lemay N, Ishigaki S, Urano F , et al. FUS/TLS assembles into stress granules and is a prosurvival factor during hyperosmolar stress. J Cell Physiol 2013, 228 (11) : 2222-2231. Chen J. The Cell-Cycle Arrest and Apoptotic Functions of p53 in Tumor Initiation and Progression. Cold Spring Harb Perspect Med 2016, 6 (3) : a026104. Stopa N, Krebs JE, Shechter D. The PRMT5 arginine methyltransferase: many roles in development, cancer and beyond. Cell Mol Life Sci 2015, 72 (11) : 2041-2059. Kim H, Ronai ZA. PRMT5 function and targeting in cancer. Cell Stress 2020, 4 (8) : 199-215. Kim J, Chang IY, Lee JH, Yong J, Jeon YJ, You HJ. The role of Ephexin1 in translation and mTOR-targeted cancer therapy. Exp Mol Med 2025, 57 (8) : 1847-1860. Kim J, Park RY, Kee Y, Jeong S, Ohn T. Splicing factor SRSF3 represses translation of p21(cip1/waf1) mRNA. Cell Death Dis 2022, 13 (11) : 933. Additional Declarations There is no duality of interest Supplementary Files SupTableRBMXtranslation.xlsx Supplementary tables SupFigureRBMXtranslationCDDFinal.docx Supplementary Figures and their legends Cite Share Download PDF Status: Under Review Version 1 posted Review # 1 received at journal 03 Mar, 2026 Reviewer # 2 agreed at journal 22 Feb, 2026 Reviewer # 1 agreed at journal 19 Feb, 2026 Reviewers invited by journal 19 Feb, 2026 Submission checks completed at journal 16 Feb, 2026 First submitted to journal 13 Feb, 2026 Unknown event 13 Feb, 2026 Editor assigned by journal 12 Feb, 2026 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8859405","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":593946403,"identity":"e39aeff0-56a5-47ae-8333-4ff3e632f0fe","order_by":0,"name":"Ho Jin You","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYBACA4YDQLKCIQEuIkGcljOkaQECxjZStJgznjF8zDvPLo+f/4zZA4YaOwbJ2Qfwa7FsOGNszLstuVhyRo65AcOxZAZpvgT8WgwOnDGT5t12IHHDDR4zCQa2AwxyPAQcBtEyB6jl/Bmgln9Ea2kAajmQYybB2HaAQZqwlmPFhnOOJSfOnJFWJpHYl8wj2UNIy43DGx+8qbFL7Oc/vE3iwzc7OYkzBLQwSJwwQHASGBgIOQsI+NsfEFY0CkbBKBgFIxsAAP65QFsoUlpBAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-0530-4017","institution":"Chosun University","correspondingAuthor":true,"prefix":"","firstName":"Ho","middleName":"Jin","lastName":"You","suffix":""},{"id":593946404,"identity":"3a3d2f21-1e45-401c-a8e8-81f9311b3607","order_by":1,"name":"Jeeho Kim","email":"","orcid":"https://orcid.org/0000-0001-6869-3606","institution":"Chosun University","correspondingAuthor":false,"prefix":"","firstName":"Jeeho","middleName":"","lastName":"Kim","suffix":""},{"id":593946405,"identity":"c6ab1c71-40d6-4f70-9b2e-04a3752ecccf","order_by":2,"name":"In-Youb Chang","email":"","orcid":"https://orcid.org/0000-0002-8911-7541","institution":"Chosun university college of medicine","correspondingAuthor":false,"prefix":"","firstName":"In-Youb","middleName":"","lastName":"Chang","suffix":""},{"id":593946407,"identity":"9cdbf026-1c0a-49de-baad-3c61e7ebf848","order_by":3,"name":"Young Jin Jeon","email":"","orcid":"","institution":"Chosun University","correspondingAuthor":false,"prefix":"","firstName":"Young","middleName":"Jin","lastName":"Jeon","suffix":""},{"id":593946410,"identity":"dc470ef0-82ce-44c8-9fb4-fb1c12699742","order_by":4,"name":"Jeongsik Yong","email":"","orcid":"https://orcid.org/0000-0002-2758-0450","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Jeongsik","middleName":"","lastName":"Yong","suffix":""}],"badges":[],"createdAt":"2026-02-12 08:20:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8859405/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8859405/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103329479,"identity":"8170a36d-e2d2-4a24-81a8-7589f4935361","added_by":"auto","created_at":"2026-02-24 13:30:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1400342,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRBMX localizes to the cytoplasm of lung (LC) and colorectal cancer (CRC) cells, and its cytoplasmic accumulation is promoted by growth factor signaling. A, B\u003c/strong\u003e Schematic overview of the bioinformatic pipeline used to predict RBMX-interacting proteins with comPPI (\u003ca href=\"https://comppi.linkgroup.hu/\"\u003ehttps://comppi.linkgroup.hu/\u003c/a\u003e) and STRING (\u003ca href=\"https://string-db.org/\"\u003ehttps://string-db.org/\u003c/a\u003e) (A), and classification of these predicted interactors according to their subcellular localization (B). \u003cstrong\u003eC\u003c/strong\u003e IHC staining of RBMX in normal, grade I/II, grade III/IV, and metastatic LC and CRC tissues and their matched normal counterparts. Hematoxylin was used as a counterstain. Scale bar, 100 μm. \u003cstrong\u003eD\u003c/strong\u003eQuantification of cytoplasmic RBMX expression scores in LC and CRC tissue microarrays (TMAs). Data are presented as mean ± SEM. ns, not significant; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 (two-tailed Student’s t-test). \u003cstrong\u003eE\u003c/strong\u003eKaplan–Meier survival curves of LC and CRC patients stratified by cytoplasmic RBMX expression. LC: high (n = 22) vs low (n = 36); CRC: high (n = 46) vs low (n = 57). P values were determined by log-rank test. \u003cstrong\u003eF \u003c/strong\u003eImmunoblot analysis of RBMX in whole-cell, cytoplasmic, and nuclear extracts from H1299, HCT116, U2OS, MDA-MB-231, H23, H358, and SW480 cells. eIF3b and Histone H3 were used as cytoplasmic and nuclear markers, respectively. \u003cstrong\u003eG\u003c/strong\u003e Confocal microscopy of RBMX in H1299 and HCT116 cells. Fluorescence intensity was quantified using Zeiss ZEN software (v3.10). Scale bar, 10 μm. \u003cstrong\u003eH, I\u003c/strong\u003eConfocal analysis of RBMX localization in H1299 cells following 12 h serum starvation and treatment with EGF (100 ng/ml, 20 min) or TPA (200 nM, 20 min). Scale bar, 10 μm. \u003cstrong\u003eJ, K\u003c/strong\u003e Immunoblot analysis of RBMX in whole-cell, cytoplasmic, and nuclear extracts from H1299 cells treated with EGF (100 ng/ml, 20 min) or TPA (200 nM, 20 min). Protein levels were quantified using ImageJ (\u003ca href=\"https://imagej.net/ij/\"\u003ehttps://imagej.net/ij/\u003c/a\u003e). eIF3b and Histone H3 served as cytoplasmic and nuclear markers, respectively.\u003c/p\u003e","description":"","filename":"mainFigure161.png","url":"https://assets-eu.researchsquare.com/files/rs-8859405/v1/24ae9127b8df151adbd5d070.png"},{"id":103329480,"identity":"1f4282ce-4571-40bd-9938-4b1c463723da","added_by":"auto","created_at":"2026-02-24 13:30:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1177009,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytoplasmic RBMX interacts with translation initiation factors and promotes m7G-cap–dependent translation initiation. A \u003c/strong\u003eSchematic workflow for identifying cytoplasmic RBMX-interacting proteins by Flag-RBMX immunoprecipitation and mass spectrometry in HEK293T cytoplasmic extracts. \u003cstrong\u003eB\u003c/strong\u003eSilver-stained gel of Flag immunoprecipitates from cytoplasmic extracts of HEK293T cells overexpressing Flag-RBMX. Bands identified by mass spectrometry are indicated. \u003cstrong\u003eC\u003c/strong\u003e STRING-based protein–protein interaction network of cytoplasmic RBMX-interacting proteins identified by mass spectrometry. \u003cstrong\u003eD\u003c/strong\u003eValidation of the cytoplasmic RBMX interactome. HEK293T cells overexpressing Flag-RBMX for 36 h were lysed and subjected to anti-Flag immunoprecipitation followed by mock or RNase A (10 μg/ml) treatment. Interacting proteins were analyzed by immunoblotting. RNase A activity was confirmed by electrophoresis and EtBr staining of RNA. \u003cstrong\u003eE\u003c/strong\u003e Polysome profiling of HEK293T cells treated with mock or EDTA (30 mM, 30 min). Individual fractions were analyzed by immunoblotting using the indicated antibodies. \u003cstrong\u003eF\u003c/strong\u003e Ribo-puromycylation assay to assess de novo protein synthesis in H1299 cells transfected with control or RBMX siRNA. Puromycylated nascent polypeptides were detected by anti-puromycin immunoblot, and total protein loading was verified by Ponceau S staining. \u003cstrong\u003eG, H\u003c/strong\u003e Distribution of translation-related proteins in polysome fractions from control or RBMX-depleted H1299 cells (G), and quantification of immunoblot signals using ImageJ (H). \u003cstrong\u003ei\u003c/strong\u003e m7G-cap pulldown assay in H1299 cells treated with control or RBMX siRNA. Lysates were incubated with m7G-agarose beads, and associated initiation factors and RBMX were analyzed by Western blotting. \u003cstrong\u003eJ\u003c/strong\u003e m7G-cap pulldown analysis in H1299 cells serum-starved for 12 h and treated with control or EGF (100 ng/ml, 20 min). Recruitment of translation initiation factors to the m7G cap was examined by Western blotting. \u003cstrong\u003eK, L \u003c/strong\u003ePolysome profiling of shControl and shRBMX H1299 cells treated with mock or EGF (100 ng/ml, 20 min) (K), and quantification of initiation factor distribution using ImageJ (L). \u003cstrong\u003eM\u003c/strong\u003e m7G-cap pulldown assay in shControl and shRBMX H1299 cells after 12 h serum starvation and treatment with control or EGF (100 ng/ml, 20 min). Recruitment of initiation factors to the m7G cap was analyzed by Western blotting.\u003c/p\u003e","description":"","filename":"mainFigure162.png","url":"https://assets-eu.researchsquare.com/files/rs-8859405/v1/b86fbb0574e609b9c554de76.png"},{"id":103506807,"identity":"a1469640-af5c-46c3-a286-6d9f165c37de","added_by":"auto","created_at":"2026-02-26 13:39:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":821664,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytoplasmic RBMX selectively regulates the translation of oncogenic transcripts, including YBX1. A\u003c/strong\u003e Schematic of cytoplasmic RNA immunoprecipitation (RIP) followed by RNA-seq to identify RBMX-bound transcripts in HEK293T cells expressing Flag-RBMX. \u003cstrong\u003eB, C\u003c/strong\u003eValidation of cytoplasmic RIP specificity. Western blotting of immunoprecipitates (B) and peak-calling analysis of RIP-seq data (C). \u003cstrong\u003eD, E\u003c/strong\u003eProportion of transcript classes (mRNAs, lncRNAs, pseudogenes) bound by cytoplasmic RBMX (D) and functional classification of RBMX-bound mRNAs (E) based on RIP/Input ≥ 1.5 and p \u0026lt; 0.05. \u003cstrong\u003eF\u003c/strong\u003e KEGG pathway analysis of cytoplasmic RBMX-bound mRNAs (RIP/Input ≥ 1.5, p \u0026lt; 0.05, FDR \u0026lt; 0.05). \u003cstrong\u003eG\u003c/strong\u003eIntegrated analysis of whole-cell RBMX PAR-CLIP data (GSE74085) and cytoplasmic Flag-RBMX RIP-seq. Bubble plot showing the top 20 overlapping transcripts ranked by RIP/Input ratio and –log10 (p value), visualized using Python (Matplotlib v3.9.0, Pandas v2.2.2). \u003cstrong\u003eH\u003c/strong\u003e qRT-PCR validation of RBMX-bound and non-bound transcripts in cytoplasmic RIPs from HEK293T cells expressing Flag-RBMX. Data are shown as RIP/Input ratios. \u003cstrong\u003eI\u003c/strong\u003e Western blot analysis of H1299 cells expressing shControl (#1, #2) or shRBMX (#1, #2). β-actin served as a loading control. \u003cstrong\u003eJ\u003c/strong\u003e qRT-PCR analysis of YBX1, PARG, FKBP3, and BACH1 mRNAs in shControl and shRBMX H1299 cells (#1, #2). \u003cstrong\u003eK, L\u003c/strong\u003e Polysome profiling of H1299 cells following RBMX knockdown. Polysome fractions were collected (K) and analyzed by qRT-PCR for RBMX-bound and non-bound transcripts, presented as a cumulative distribution (L). Based on the above analyses of multiple RBMX-bound and translationally regulated transcripts, YBX1 was selected for subsequent functional and clinical analyses as a representative target linking selective translation to senescence regulation. \u003cstrong\u003eM\u003c/strong\u003eAnalysis of RBMX and YBX1 target gene expression in the TCGA lung cancer cohort (LUSC, LUAD). Samples are ordered from high to low RBMX expression. \u003cstrong\u003eN\u003c/strong\u003eqRT-PCR analysis of canonical YBX1 target genes (SRSF7, hnRNPL, CDK2, eIF5) in H1299 cells treated with siControl, siYBX1, or siRBMX (#1, #2). \u003cstrong\u003eO-Q\u003c/strong\u003e IHC analysis of RBMX and YBX1 in lung cancer tissues. Representative images arranged by low-to-high cytoplasmic RBMX expression (O). Staining intensities were quantified using ImageJ (Fiji) and used for relative expression (P) and correlation analyses (Q). ns, not significant; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"mainFigure163.png","url":"https://assets-eu.researchsquare.com/files/rs-8859405/v1/5a4aa589b5bc6306e37c4c9a.png"},{"id":103329483,"identity":"24a9a305-e6b8-404e-b197-87d7669a3b93","added_by":"auto","created_at":"2026-02-24 13:30:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":757639,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRBMX directly binds eIF3i/eIF3F, and these interactions are linked to translational activation.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003eCo-immunoprecipitation (Co-IP) analysis of HEK293T cells transfected with Flag-tagged initiation factors (eIF2α, eIF3F, eIF3i, eIF3h, eIF3g, eIF4A1, eIF5A, eIF4G1) and eEF1α1. Anti-Flag immunoprecipitates were analyzed for RBMX binding by Western blotting. \u003cstrong\u003eB\u003c/strong\u003e In vitro pulldown assays showing interactions between Flag-tagged RBMX and Hisx6-tagged translation factors (eIF3e, eIF3F, eIF3g, eIF3h, eIF3i, eIF3m, eIF2α, eIF4A1). \u003cstrong\u003eC\u003c/strong\u003e Schematic of full-length RBMX and deletion mutants, and summary of their interaction strengths with eIF3F and eIF3i. In vitro GST pulldown assays were performed using GST-tagged RBMX fragments (RRM, NTD, and RGG domains) and recombinant Hisx6-eIF3i. GST alone served as a negative control. \u003cstrong\u003eD\u003c/strong\u003e Proximity ligation assay (PLA) detecting RBMX–eIF3F and RBMX–eIF3i interactions (red puncta) in normal lung tissues (n = 9) and LC tissues of grade I (n = 13), grade II (n = 14), and grade III/IV (n = 13). Nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. \u003cstrong\u003eE\u003c/strong\u003e Quantification of PLA signals shown in (D). Data are presented as mean ± SEM; p values were determined by two-tailed Student’s t-test. ns, not significant; *P \u0026lt; 0.05. \u003cstrong\u003eF\u003c/strong\u003e Kaplan–Meier survival curves of LC patients stratified by RBMX–eIF3F/eIF3i interaction levels. P values were determined by log-rank test. *P \u0026lt; 0.05, **P \u0026lt; 0.01. \u003cstrong\u003eG\u003c/strong\u003e Schematic representation of the RBMX RGG/RG motif as a PRMT5 methylation site. \u003cstrong\u003eH, I\u003c/strong\u003e Methylation-specific immunoblot showing that cytoplasmic RBMX is hypomethylated relative to nuclear RBMX. RBMX was immunoprecipitated from whole-cell, cytoplasmic, and nuclear extracts of H1299 cells using an anti-RBMX antibody, and symmetric dimethyl-arginine (sdme-RG) was detected by anti-sdme-RG immunoblot. Methylation levels were quantified using ImageJ (I). \u003cstrong\u003eJ\u003c/strong\u003e Confocal microscopy of H1299 cells treated with PRMT5 inhibitor (5 μM, 12 h). RBMX localization and fluorescence intensity were quantified using Zeiss ZEN software (v3.10). Scale bar, 10 μm. \u003cstrong\u003eK, L\u003c/strong\u003eCo-IP analysis of Flag-RBMX in HEK293T cells treated with vehicle or PRMT5 inhibitor (GSK3235025, 5 μM, 12 h). Interactions with the indicated translation initiation factors were examined by immunoblotting.\u003c/p\u003e","description":"","filename":"mainFigure164.png","url":"https://assets-eu.researchsquare.com/files/rs-8859405/v1/d59d88257d7e577ad92f7996.png"},{"id":103506550,"identity":"fefe352f-c472-49ab-98f0-91f9cd6450a0","added_by":"auto","created_at":"2026-02-26 13:37:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":405750,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePRMT5-dependent methylation modulates RBMX localization and interaction with translation initiation factors. A, B\u003c/strong\u003e Co-IP analysis of HEK293T cells co-expressing Flag-eIF3i and V5-tagged RBMX (WT or 4RK). RBMX–eIF3i interactions were assessed by immunoblotting, and signal intensities were quantified using ImageJ.\u003cstrong\u003e C\u003c/strong\u003e Confocal microscopy of H1299 cells expressing V5-empty vector, V5-RBMX_WT, or V5-RBMX_4RK. RBMX subcellular localization and fluorescence intensity were quantified using Zeiss ZEN software (v3.10). Scale bar, 10 μm.\u003cstrong\u003e D\u003c/strong\u003e Immunoblot analysis of whole-cell, cytoplasmic, and nuclear extracts from HEK293T cells expressing V5-vector, V5-RBMX_WT, or V5-RBMX_4RK. eIF3b and Lamin B1 served as cytoplasmic and nuclear markers, respectively.\u003cstrong\u003e E, F\u003c/strong\u003e Polysome profiling of HEK293T cells expressing V5-empty vector, V5-RBMX_WT, or V5-RBMX_4RK. Fractions were analyzed by immunoblotting for the indicated factors, and signals were quantified using ImageJ. \u003cstrong\u003eG\u003c/strong\u003e m7G-cap pulldown analysis in HEK293T cells expressing V5-vector, V5-RBMX_WT, or V5-RBMX_4RK. Lysates were incubated with m7G-agarose beads, and associated initiation factors were examined by Western blotting.\u003c/p\u003e","description":"","filename":"mainFigure165.png","url":"https://assets-eu.researchsquare.com/files/rs-8859405/v1/9609d4fb42805c22045d32a8.png"},{"id":103329485,"identity":"eb55cb53-5e54-4fae-bbd1-143b156a6724","added_by":"auto","created_at":"2026-02-24 13:30:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1067370,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRBMX depletion induces p21-dependent, p53-independent cellular senescence. A\u003c/strong\u003e Phase-contrast images of H1299 and H460 cells treated with siControl or siRBMX (#1, #2) for 72 h, showing increased cell size and multinucleation upon RBMX loss. Scale bar, 100 μm. RBMX knockdown was verified by Western blot.\u003cstrong\u003e B\u003c/strong\u003e RNA-seq analysis following treatment with siControl or siRBMX (#1, #2) for 72 h. Differentially expressed genes (DEGs) with \u0026gt;2-fold change and p \u0026lt; 0.05 are displayed as a heatmap.\u003cstrong\u003e C\u003c/strong\u003e ClueGO KEGG pathway analysis of DEGs induced by RBMX knockdown. Node size reflects statistical significance, with the most significant terms highlighted. Analysis was performed using ClueGO in Cytoscape (v3.10.1).\u003cstrong\u003e D\u003c/strong\u003e Bubble plot summarizing ClueGO KEGG pathway results, visualized using Python and ranked by gene number per term and –log\u003csub\u003e10\u003c/sub\u003e (p value). The top 20 genes from the Cell cycle and Cellular senescence categories are shown.\u003cstrong\u003e E \u003c/strong\u003e\u0026nbsp;Human Cell Stress Array analysis of lysates from control and RBMX-depleted H1299 cells. Relative protein levels are shown as fold change versus control. Error bars indicate mean ± SEM of spot intensities.\u003cstrong\u003e F\u003c/strong\u003e Immunoblot analysis of H1299, HCT116, and SW480 cells treated with siControl or siRBMX for 72 h, using the indicated antibodies. \u003cstrong\u003eG\u003c/strong\u003e Schematic summary of the RBMX–eIF3–YBX1–translation axis based on Figures 1–5. \u003cstrong\u003eH, I\u003c/strong\u003e Immunoblot (H) and qRT-PCR (I) analyses of HCT116 cells with RBMX knockdown and/or Myc-YBX1 overexpression. p21\u003csup\u003ecip/waf1\u003c/sup\u003e mRNA levels were normalized to β-actin. \u003cstrong\u003eJ\u003c/strong\u003e SA-β-gal staining of HCT116 cells with RBMX depletion and Myc-YBX1 overexpression. Representative images and quantification of SA-β-gal–positive cells are shown. Scale bar, 100 μm. Data are mean ± SEM; p values were determined by two-tailed Student’s t-test. ns, not significant; ***P \u0026lt; 0.001. \u003cstrong\u003eK\u003c/strong\u003e Immunoblot analysis of H1299 cells expressing V5-empty vector, V5-RBMX_WT, or V5-RBMX_4RK, probed with the indicated antibodies. \u003cstrong\u003eL \u003c/strong\u003eProposed model illustrating how cytoplasmic RBMX, regulated by PRMT5-dependent methylation, promotes tumor malignancy in LC and CRC by enhancing translation of cancer-related transcripts and suppressing p21\u003csup\u003ecip/waf1\u003c/sup\u003e-mediated senescence.\u003c/p\u003e","description":"","filename":"mainFigure166.png","url":"https://assets-eu.researchsquare.com/files/rs-8859405/v1/98c70c43085d0a9e2594a140.png"},{"id":103511453,"identity":"e32204ca-4e3d-46d7-b4c5-4943005d528b","added_by":"auto","created_at":"2026-02-26 14:09:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6682055,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8859405/v1/91a1ccf3-2bdb-4d9c-a5d8-420935f68d42.pdf"},{"id":103329481,"identity":"6c654fbe-def9-4ef3-8165-bd896e49b6c2","added_by":"auto","created_at":"2026-02-24 13:30:26","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":241019,"visible":true,"origin":"","legend":"Supplementary tables","description":"","filename":"SupTableRBMXtranslation.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8859405/v1/c79d5f2a12e667b371e56b2c.xlsx"},{"id":103329486,"identity":"36431386-9596-4d14-99d1-a116e48527db","added_by":"auto","created_at":"2026-02-24 13:30:26","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":9769178,"visible":true,"origin":"","legend":"Supplementary Figures and their legends","description":"","filename":"SupFigureRBMXtranslationCDDFinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-8859405/v1/60c67fa54d0c7ad063cb32bd.docx"}],"financialInterests":"There is no duality of interest","formattedTitle":"Cytoplasmic RBMX coordinates selective mRNA translation to suppress senescence in cancer","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eRNA-binding proteins (RBPs) are central regulators of post-transcriptional gene expression, coordinating alternative splicing, mRNA stability, localization, and translation\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Within this broad class, the heterogeneous nuclear ribonucleoprotein (hnRNP) family comprises a major group of RBPs that control nuclear RNA metabolism in development and disease \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. RBMX (RNA-binding motif protein X-linked, also known as hnRNPG) is one of the most structurally conserved hnRNPs and occupies a key position at the interface of RNA processing and genome maintenance \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAcross human cancers, RBMX has emerged as a context-dependent regulator whose function can be either oncogenic or tumor suppressive. RBMX is frequently dysregulated in malignancies, and changes in its expression have been linked to tumor progression, remodeling of the immune microenvironment, therapy resistance, and clinical outcome \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In hepatocellular carcinoma, RBMX reprograms oncogenic metabolic and transcriptional pathways to promote proliferation, invasion, metastasis, and sorafenib resistance; in osteosarcoma, it shapes an immunosuppressive tumor microenvironment by modulating CD8⁺ T-cell infiltration and effector function; and in colorectal cancer, tumor-derived exosomal LINC01615 recruits RBMX to an EZH2 regulatory axis that enhances EZH2 expression and drives M2 polarization of tumor-associated macrophages \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In contrast, in bladder cancer, RBMX restrains tumorigenicity and invasion by regulating hnRNPA1-dependent PKM splicing and cancer metabolism \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, and in oral squamous cell carcinoma, RBMX acts as a tumor suppressor that limits proliferation and transformation \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Together, these observations position RBMX as a lineage- and context-dependent regulatory node whose biological output\u0026mdash;pro-tumorigenic or tumor-suppressive\u0026mdash;is shaped by tissue identity, genetic background, and upstream signaling \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAt the molecular level, RBMX contains a single RNA recognition motif (RRM) and a C-terminal RGG/RG-rich domain, enabling interactions with both structured and intrinsically disordered RNA elements as well as core spliceosomal components \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Consistent with this domain architecture, RBMX predominantly localizes to the nucleus, where it regulates exon definition, splice-site choice, and alternative splicing programs \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Beyond canonical splicing control, RBMX participates in co-transcriptional RNA processing and transcription\u0026ndash;splicing coupling, thereby enhancing the fidelity of nascent mRNA production \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. RBMX also safeguards genome integrity by suppressing harmful R-loops, coordinating ATR-dependent DNA damage responses, mitigating replication stress, and promoting accurate processing of pre-mRNAs containing ultra-long introns and repetitive sequences \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. These activities collectively underscore RBMX as a multifunctional nuclear regulator that couples RNA metabolism to genomic stability \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eProtein arginine methyltransferase 5 (PRMT5) is the major type II arginine methyltransferase that catalyzes symmetric dimethylation within RGG/RG motifs of RBPs and other nuclear proteins \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Through methylation of splicing factors, transcriptional regulators, chromatin-associated proteins, and components of the RNA-processing and translational machinery, PRMT5 integrates signals that control transcription, pre-mRNA splicing, translation, stemness, and DNA damage repair \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In cancer, PRMT5 rewires RNA-metabolic networks by modifying multiple RBPs\u0026mdash;including hnRNPA1, FUS, and TDP-43\u0026mdash;thereby promoting tumor-specific RNA processing and stress-adaptation programs \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRBMX is a bona fide PRMT5 substrate: symmetric dimethylation of its C-terminal RGG/RG motif modulates its interaction landscape, nuclear complex assembly, and functional specificity\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Studies of Shashi X-linked intellectual disability have shown that deletion of the RBMX RGG/RG region perturbs p53 signaling, neuronal differentiation, and nuclear RBMX behavior, while biochemical and proteomic analyses have identified RBMX among PRMT5-modified substrates within nuclear ribonucleoprotein assemblies \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. More broadly, arginine methylation of RGG/RG-containing RBPs operates as a molecular switch that regulates protein\u0026ndash;RNA affinity, phase separation, and subcellular organization \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. For several RBPs, including hnRNPA1 and related hnRNPs, inhibition or loss of arginine methylation promotes nuclear-to-cytoplasmic translocation and reshapes their cytoplasmic activities, linking PRMT5-mediated modification to nucleo-cytoplasmic trafficking and translational control \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Despite extensive work on the nuclear functions of RBMX, however, it has remained unclear whether PRMT5 regulates RBMX subcellular distribution or licenses a transition toward cytoplasmic, translation-related functions\u0026mdash;representing a critical gap in understanding how RBMX integrates post-translational regulation with cancer phenotypes \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecent work has established that specific RBPs can relocalize to the cytoplasm and directly engage the translation initiation machinery to drive tumor-selective translational reprogramming \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The eukaryotic initiation factor 3 (eIF3) complex is a central orchestrator of such control; its subunits are frequently altered in tumors and regulate mRNA selection, proteome remodeling, and stress adaptation \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Within this complex, eIF3F and eIF3i have emerged as particularly important subunits. eIF3F displays context-dependent activities, acting as a negative regulator of translation and tumor growth in some settings, while promoting migration, invasion, and metastasis in lung adenocarcinoma through interaction with STAT3 in others \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. eIF3i is a proto-oncogenic factor overexpressed in multiple cancers, including colorectal cancer, where it enhances COX-2 and PHGDH translation, supports proliferation and survival, and drives oncogenesis and metastasis \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Although interactions between RBPs and eIF3 subunits have been implicated in establishing malignant translational networks\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, whether RBMX participates in such mechanisms\u0026mdash;and how PRMT5-dependent methylation might tune these interactions\u0026mdash;has remained unknown \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Importantly, Dysregulated translational control has emerged as a critical determinant of cell fate decisions, including senescence, during tumor progression \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e .\u003c/p\u003e \u003cp\u003eIn this study, we identify a previously unrecognized cytoplasmic role of RBMX as a PRMT5-regulated translational regulator that governs entry into senescence in cancer cells. We show that RBMX directly interacts with eIF3i and eIF3F through its RGG/RG motif, that this interaction is markedly enhanced in a demethylation-mimetic RBMX mutant, and that cytoplasmic RBMX promotes cap-dependent translation initiation in cancer cells. Using cytoplasmic RIP-seq, we further demonstrate that RBMX selectively regulates the translation of cancer-associated mRNAs, including YBX1. RBMX depletion sharply reduces YBX1 protein levels without altering its mRNA, leading to p21\u003csup\u003eCip/Waf1\u003c/sup\u003e induction and robust, p53-independent cellular senescence. These findings define a cytoplasmic RBMX\u0026ndash;YBX1\u0026ndash;p21\u003csup\u003eCip/Waf1\u003c/sup\u003e translational axis that dictates senescence fate and reveal a methylation-sensitive mechanism linking PRMT5-dependent control of RBMX to cell fate regulation in cancer. Collectively, our work expands the functional landscape of RBMX beyond its canonical nuclear roles and uncovers a PRMT5-RBMX-YBX1-p21 pathway through which selective translation shapes senescence-associated cancer phenotypes.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eCytoplasmic localization of nuclear RBMX is context dependent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRBMX has been reported to exert both oncogenic and tumor-suppressive roles, with its functional impact varying across cancer types \u003csup\u003e4, 6\u003c/sup\u003e. To better define its role in tumorigenesis, we first analyzed RBMX expression patterns across diverse human cancers. TCGA datasets and immunohistochemical (IHC) staining of patient-derived tissues consistently showed that RBMX expression is significantly elevated in multiple tumor types\u0026mdash;including lung, colorectal, and liver cancers\u0026mdash;compared with corresponding normal tissues. RBMX protein levels were also markedly higher across 11 lung cancer (LC) and 11 colorectal cancer (CRC) cell lines relative to normal lung and colon epithelial cell lines (Supplementary Fig. S1A\u0026ndash;D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunctionally, RBMX appeared critical for lung cancer progression. In H1299 cells with stable RBMX knockdown, we observed a substantial reduction in proliferation accompanied by decreased Ki67 expression. RBMX depletion also impaired clonogenic potential and significantly suppressed tumor growth and Ki67 staining in xenograft models (Supplementary Fig. S1E-J). These results collectively identify RBMX as a key driver of cancer cell proliferation and tumorigenic growth.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlthough RBMX has been predominantly studied for its nuclear roles \u003csup\u003e4, 6\u003c/sup\u003e, its mechanistic contribution to oncogenesis remains poorly defined. To explore potential functions beyond the nucleus, we first analyzed its predicted protein interaction landscape. Surprisingly, interrogation of the compartmentalized protein\u0026ndash;protein interaction database (comPPI), followed by functional enrichment and STRING analyses, revealed that a substantial proportion of predicted RBMX interactors\u0026mdash;66.82%\u0026mdash;were nucleocytoplasmic shuttling proteins, and an additional 9.18% were exclusively cytoplasmic (Fig. 1A-B and Supplementary Fig. S1K-L). Contrary to expectations based on prior nuclear-focused studies, these findings suggest that RBMX may also localize to and function within the cytoplasm, prompting us to examine its context-dependent distribution in cancer cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBecause cancer cells often display altered signaling states that drive the cytoplasmic translocation of various RBPs \u003csup\u003e36, 37, 38\u003c/sup\u003e, we hypothesized that RBMX may exhibit context-dependent cytoplasmic localization in tumors. To test this, we performed IHC on tissue microarrays from lung and colorectal cancer patients and quantified RBMX staining specifically within the cytoplasm. Cytoplasmic RBMX levels were significantly higher in tumor tissues than in normal tissues and increased progressively with tumor grade (Fig. 1C and D). Elevated cytoplasmic RBMX expression was also associated with poorer patient prognosis (Fig. 1E).\u003c/p\u003e\n\u003cp\u003eConsistent with these patient data, RBMX protein was detectable in the cytoplasmic fraction of seven cancer cell lines, including H1299 and HCT116 (Fig. 1F and G). Because hyperactive growth factor signaling is a hallmark of cancer progression \u003csup\u003e39, 40\u003c/sup\u003e, we examined whether external stimuli promote cytoplasmic RBMX accumulation. Indeed, treatment with EGF or TPA after serum starvation markedly increased cytoplasmic RBMX levels (Fig. 1H-K and Supplementary Fig. S1M-N).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTaken together, these findings demonstrate that RBMX, although classically considered a nuclear RBP, can localize to the cytoplasm in cancer cells. This cytoplasmic localization is enhanced by growth factor signaling and correlates with tumor malignancy and poor clinical outcomes, suggesting that RBMX may exert important cytoplasmic functions during cancer progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytoplasmic RBMX promotes cap-dependent translation initiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo define the physiological relevance of RBMX in the cytoplasm, we first profiled RBMX-associated cytoplasmic proteins. Immunoprecipitation followed by mass spectrometry using cytoplasmic extracts from HEK293T cells overexpressing Flag-tagged RBMX identified 22 proteins that selectively interacted with cytoplasmic RBMX (Fig. 2A-B and Supplementary Table S1). STRING network analysis revealed that these interactors were enriched for factors involved in translation and methylation pathways (Fig. 2C and Supplementary Fig. S2A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo validate the proteomic findings, we performed co-immunoprecipitation and Western blot analyses in HEK293T cells expressing Flag-RBMX. RBMX interacted with the identified protein candidates even after RNase A treatment, indicating that these associations are RNA-independent (Fig. 2D). Moreover, upon arsenite or thapsigargin treatment\u0026mdash;conditions that induce stress granule (SG) formation by causing translation initiation factors to aggregate\u0026mdash;RBMX co-localized with eIF3b within SGs and co-immunoprecipitated with eIF3b in cytoplasmic extracts (Supplementary Fig. S2B and C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further assess the involvement of RBMX in translation, we examined its distribution across ribosomal fractions. Polysome profiling showed that RBMX localized to the 40S, 60S, and 80S ribosomal subunits as well as light polysomes (Supplementary Fig. S2D). EDTA-mediated polysome disassembly caused RBMX, translation initiation factors, and ribosomal proteins to shift in parallel patterns (Fig. 2E and Supplementary Fig. S2E-H), supporting a functional association of RBMX with translational complexes.\u003c/p\u003e\n\u003cp\u003eIn line with these observations, knockdown of RBMX in H1299 and H460 cells significantly reduced de novo protein synthesis (Fig. 2F and Supplementary Fig. S2I). Polysome profiling after RBMX depletion demonstrated a pronounced loss of polysomes accompanied by enhanced 40S and 80S peaks. Several translation-related proteins\u0026mdash;including eEF2, eIF4A1, eIF3b, eIF3F, eIF3i, rpS3, rpL26, and rpL36a\u0026mdash;shifted toward monosome fractions upon RBMX loss (Fig. 2G-H and Supplementary Fig. S2J-K), underscoring the essential role of RBMX in sustaining active protein synthesis.\u003c/p\u003e\n\u003cp\u003eTranslation initiation represents a highly regulated rate-limiting step in protein synthesis \u003csup\u003e34\u003c/sup\u003e. To evaluate the functional relevance of RBMX\u0026ndash;initiation factor interactions, we examined recruitment of translation initiation components to the mRNA 7-methylguanosine (m7G) cap. In H1299 cells, m7G-cap pulldown assays revealed that RBMX depletion markedly reduced the association of eIF4A1, eIF3b, and eIF3i with the cap structure. Importantly, RBMX itself was detected within the cap-bound initiation complex (Fig. 2I).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConversely, treatment with EGF\u0026mdash;a potent stimulator of global protein synthesis\u0026mdash;significantly enhanced the recruitment of RBMX, eIF4G1, eIF3b, and eIF4A1 to the m7G cap (Fig. 2J). Consistent with increased translational activity, EGF treatment elevated polysome abundance and shifted RBMX, eIF3b, eIF3F, eIF3i, rpS6, rpS3, and rpL26 into heavy polysome fractions. These effects were entirely absent in RBMX-depleted cells (Fig. 2K and L). m7G-cap pulldown assays further demonstrated that EGF-induced recruitment of eIF3b, eIF3i, eIF3F, and eIF4A1 to the initiation complex required RBMX, as this enhancement was abolished upon RBMX knockdown (Fig. 2M).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCollectively, these results demonstrate that cytoplasmic RBMX facilitates protein synthesis by engaging translation initiation factors and promoting assembly of the cap-dependent translation initiation complex.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytoplasmic RBMX is a specificity factor for oncogenic mRNA translation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeveral RNA-binding proteins (RBPs) that relocalize to the cytoplasm can selectively engage specific transcripts and modulate their stability or translation \u003csup\u003e15, 18, 24\u003c/sup\u003e. Although global mapping of RBP-bound RNAs has provided important insights into such mechanisms, previous studies examining the RBMX interactome relied on whole-cell extracts without distinguishing between nuclear and cytoplasmic RNA pools \u003csup\u003e41\u003c/sup\u003e. Given that RBMX is predominantly nuclear, its cytoplasmic RNA targets were likely obscured in these earlier analyses.\u003c/p\u003e\n\u003cp\u003eTo define the cytoplasmic RNA interactome of RBMX, we expressed Flag-RBMX in HEK293T cells, isolated cytoplasmic fractions, and performed RNA immunoprecipitation followed by RNA sequencing. This approach identified a distinct set of RBMX-bound RNAs compared with controls (Fig. 3A-C and Supplementary Fig. S3A-B). Most cytoplasmic RBMX-associated transcripts were mRNAs (74.48%), with additional binding to lncRNAs (19.27%) and pseudogenes (5.04%) (Fig. 3D and Supplementary Table S2). Functional annotation of significantly enriched mRNAs (RIP/Input \u0026ge; 1.5-fold, p \u0026lt; 0.05) highlighted genes involved in transcription (18.08%), membrane protein trafficking (15.88%), and metabolic processes (15.72%). KEGG pathway analysis further revealed enrichment of cancer-associated pathways, including ribosome biogenesis and MAPK, Notch, and mTOR signaling (Fig. 3E-F and Supplementary Fig. S3C and Supplementary Tables S3 and S4).\u003c/p\u003e\n\u003cp\u003eComparison with previously published PAR-CLIP datasets generated from whole-cell extracts identified 539 overlapping transcripts. Among these, YBX1 mRNA ranked among the most enriched shared targets (Fig. 3G and Supplementary Table S5). To validate these interactions, we performed qRT-PCR and RT-PCR analyses, confirming that RBMX specifically bound YBX1, PPAR\u0026gamma;, PARG, MAN1A2, FKBP3, PPDPF, EphA8, and BACH1 mRNAs, but not YTHDF1, ARFGAP1, \u0026beta;-actin, or GMEB2 (Fig. 3H and Supplementary Fig. S3D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe next examined the functional consequences of RBMX binding on gene expression. In H1299, HCT116, and SW480 cells, RBMX depletion markedly reduced the protein levels\u0026mdash;but not the mRNA abundance\u0026mdash;of YBX1, PARG, BACH1, and FKBP3 (Fig. 3I-J and Supplementary Fig. S3E-F). These observations suggest that RBMX enhances the translation of its bound mRNAs. Supporting this conclusion, polysome profiling in RBMX-depleted H1299 cells showed that YBX1, PARG, FKBP3, and BACH1 transcripts shifted from heavy to light polysomes, consistent with reduced translational efficiency. In contrast, non-bound transcripts such as \u0026beta;-actin, ARFGAP1, and YTHDF1 displayed unchanged polysome distributions (Fig. 3K and L).\u003c/p\u003e\n\u003cp\u003eAmong the cytoplasmic RBMX targets, YBX1 was of particular interest due to its strong enrichment and established oncogenic function. Analysis of TCGA lung cancer RNA-seq datasets revealed a significant positive correlation between RBMX expression and the expression of YBX1 target genes (Fig. 3M and Supplementary Fig. S3G), whereas no such correlation was observed for non-target genes (Supplementary Fig. S3H). Consistent with these findings, knockdown of either RBMX or YBX1 in H1299, HCT116, and SW480 cells reduced the expression of canonical YBX1 targets, including SRSF7, hnRNPL, CDK2, eIF5, eEF1A1, and C11orf58 (Fig. 3N and Supplementary Fig. S3I-J). Importantly, overexpression of Myc-tagged YBX1 restored the expression of these targets in RBMX-depleted cells (Supplementary Fig. S3K-L), demonstrating that impaired YBX1 translation mediates the downstream effects of RBMX loss.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, clinical tissue analyses revealed that cytoplasmic RBMX expression positively correlated with YBX1 protein levels in lung cancer patient samples (Fig. 3O-Q). Similar correlations were observed for PARG, FKBP3, and BACH1 (Supplementary Fig. S3M-O), extending the relevance of RBMX-mediated translational regulation to human tumors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCollectively, these findings establish cytoplasmic RBMX as a specificity factor that selectively enhances the translation of oncogenic mRNAs, thereby contributing to cancer progression through targeted modulation of the translational landscape.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eeIF3i and eIF3F mediate cytoplasmic RBMX function in translational regulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTranslation initiation is the rate-limiting step of protein synthesis and a key regulatory node in translational control \u003csup\u003e34\u003c/sup\u003e. To elucidate how RBMX promotes translation, we first examined its interactions with major components of the translation initiation machinery. HEK293T cells were transfected with a panel of Flag-tagged initiation factors\u0026mdash;including eIF2\u0026alpha;, eIF3F, eIF3i, eIF3h, eIF3g, eIF4A1, eIF5A, eIF4G1, and eEF1\u0026alpha;1\u0026mdash;followed by anti-Flag immunoprecipitation. Immunoblot analysis revealed that RBMX associates with eIF3F, eIF3i, eIF3g, eIF4A1, and eIF4G1 (Fig. 4A).\u003c/p\u003e\n\u003cp\u003eTo determine whether these interactions are direct, we generated recombinant full-length RBMX using a cell-free protein synthesis (CFPS) system and purified His\u003csub\u003ex6\u003c/sub\u003e-tagged recombinant initiation factors (eIF3e, eIF3F, eIF3g, eIF3h, eIF3i, eIF3m, eIF2\u0026alpha;, and eIF4A1). Direct binding assays demonstrated that only eIF3i and eIF3F directly interact with RBMX (Fig. 4B). We next mapped the RBMX domains required for these interactions. Using GST-fused deletion mutants of RBMX in direct pull-down assays with His\u003csub\u003ex6\u003c/sub\u003e-tagged eIF3i or eIF3F, we found that only the RGG domain of RBMX was capable of binding both proteins (Fig. 4C and Supplementary Fig. S4A). These findings indicate that the RGG domain serves as the primary interface through which RBMX engages eIF3i and eIF3F.\u003c/p\u003e\n\u003cp\u003eTo explore the clinical relevance of these interactions, we performed proximity ligation assays (PLA) on tissue microarrays containing normal, primary, and various tumor-grade samples from lung cancer and colorectal cancer patients. PLA signals corresponding to RBMX\u0026ndash;eIF3i and RBMX\u0026ndash;eIF3F interactions were markedly elevated in tumor tissues relative to normal tissues and increased progressively with tumor grade (Fig. 4D-E and Supplementary Fig. S4B\u0026ndash;C). Importantly, patients with higher RBMX\u0026ndash;eIF3i/eIF3F interaction levels exhibited significantly poorer prognosis in both lung cancer and colorectal cancer cohorts (Fig. 4F and Supplementary Fig. S4D), underscoring the clinical significance of these interactions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBecause the RGG domain of RBMX is known to be methylated by PRMT5 (Fig.4G) \u003csup\u003e19, 42\u003c/sup\u003e, and because PRMT5\u0026ndash;MEP50\u0026ndash;pICln methylosome components were identified among RBMX-interacting proteins in cytoplasmic extracts (Fig. 2A-C), we next examined the methylation status of RBMX. Fractionation analyses revealed that RBMX in the cytoplasm predominantly existed in a demethylated form, whereas methylated RBMX was enriched in the nucleus (Fig. 4H-I). We then asked how PRMT5-mediated methylation influences RBMX localization and function. Pharmacological inhibition of PRMT5 promoted cytoplasmic translocation of RBMX, as observed by confocal microscopy and immunoblotting (Fig. 4J and Supplementary Fig. S4E). Consistent with this shift, treatment of Flag-RBMX\u0026ndash;expressing HEK293T cells with a PRMT5 inhibitor substantially enhanced the interaction between RBMX and multiple translation initiation factors, including eIF4G, eIF3b, eIF3i, and eIF3F (Fig. 4K-L).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCollectively, these findings demonstrate that eIF3i and eIF3F directly engage the RGG domain of RBMX to facilitate translation initiation, and that PRMT5-mediated methylation modulates RBMX cytoplasmic localization and interaction with the initiation machinery. These results highlight the mechanistic and clinical significance of RBMX\u0026ndash;eIF3 interactions in translational regulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDemethylation of the RBMX RGG motif drives its cytoplasmic localization and control of translation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBecause PRMT5 methylates a broad spectrum of substrates \u003csup\u003e15, 16, 17\u003c/sup\u003e, the effects of PRMT5 inhibition on RBMX function could include indirect contributions. To directly assess how RBMX demethylation influences its activity, we generated a methylation-deficient mutant by substituting four arginine residues within the RGG domain (R369K, R373K, R377K, and R384K) with lysines (RBMX_4RK). Consistent with our hypothesis, the demethylation-mimetic RBMX_4RK mutant exhibited markedly enhanced interactions with translation initiation factors compared with wild-type RBMX (Fig. 5A-B and Supplementary Fig. S5A), along with a more pronounced cytoplasmic localization (Fig. 5C-D). These findings align with prior reports showing that demethylation of RGG-containing RBPs promotes their cytoplasmic translocation \u003csup\u003e18, 24, 43\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe next examined whether demethylated RBMX more actively stimulates translation. Overexpression of V5-tagged RBMX_WT or RBMX_4RK in HEK293T cells followed by polysome profiling revealed that RBMX_WT increased polysome abundance and shifted eIF3b, eIF3i, eIF3F, and ribosomal protein L26 toward heavy polysome fractions. Notably, these effects were significantly amplified in RBMX_4RK-expressing cells (Fig. 5E-F). Consistently, m7G-cap pulldown analyses showed that RBMX_4RK enhanced the recruitment of multiple initiation factors to the cap structure more effectively than RBMX_WT (Fig. 5G).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunctionally, RBMX_4RK promoted cell migration and anchorage-independent growth in H1299 cells to a considerably greater extent than wild-type RBMX (Supplementary Fig. S5B and C). Together, these findings demonstrate that demethylation of the RBMX RGG domain strengthens its interactions with translation initiation factors, enhances translational output, and contributes to tumorigenic phenotypes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytoplasmic RBMX\u0026ndash;YBX1\u0026ndash;\u0026nbsp;p21\u003csup\u003ecip/waf1\u003c/sup\u003e axis prevents cellular senescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLoss of RBMX markedly impaired proliferative capacity and tumor growth in vitro and in vivo (Supplementary Fig. S1E-I). During these experiments, we observed that RBMX knockdown in H1299 and H460 cells induced pronounced increases in cell size and multinucleation (Fig. 6A), morphological features strongly associated with cellular senescence \u003csup\u003e35, 44\u003c/sup\u003e. As shown in Figure 3, RBMX-bound cytoplasmic transcripts were significantly enriched for genes regulating transcription. To characterize downstream changes in gene expression following RBMX loss, we performed ClueGO-based KEGG pathway enrichment using genes altered more than twofold (p \u0026lt; 0.05) after RBMX knockdown. The most prominently affected pathways were related to the cell cycle and cellular senescence (Fig. 6B and C). Among these genes, CDKN1A (p21\u003csup\u003ecip/waf1\u003c/sup\u003e) showed the strongest induction within both categories (Fig. 6D).\u003c/p\u003e\n\u003cp\u003eTo validate these transcriptomic findings, we used a cell stress protein array, which confirmed upregulation of multiple senescence-associated proteins\u0026mdash;including p21\u003csup\u003ecip/waf1\u003c/sup\u003e, HSP70, SOD2, Thioredoxin1, and SIRT2\u0026mdash;with p21\u003csup\u003ecip/waf1\u003c/sup\u003e showing the most robust increase (Fig. 6E). Consistent with this, depletion of RBMX or YBX1 in H1299 cells greatly increased SA-\u0026beta;-galactosidase staining (Supplementary Fig. S6A) and induced strong p21 expression at both mRNA and protein levels (Fig. 6F and Supplementary Fig. S6B-D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBecause YBX1 is a direct target of RBMX-mediated translation and is known to repress p21 expression \u003csup\u003e45, 46, 47\u003c/sup\u003e, we examined whether RBMX loss induces senescence via YBX1 downregulation. Indeed, RBMX knockdown decreased YBX1 levels and increased p21\u003csup\u003ecip/waf1\u003c/sup\u003e expression (Fig. 6F and Supplementary Fig. S6C), consistent with a model in which impaired YBX1 translation drives p21 upregulation. Given that p21\u003csup\u003ecip/waf1\u003c/sup\u003e is a canonical transcriptional target of p53 (Fig. 6G) \u003csup\u003e48, 49\u003c/sup\u003e, we tested whether RBMX regulates p21 in a p53-dependent manner. In p53-null H1299 cells, HCT116 p53⁻/⁻ cells, and HCT116 cells treated with p53 siRNA, RBMX depletion continued to induce p21 expression (Fig. 6F and Supplementary Fig. S6E-H), demonstrating that this effect occurs independently of p53.\u003c/p\u003e\n\u003cp\u003eTo further test whether YBX1 mediates RBMX-dependent p21 regulation, we performed rescue experiments. In HCT116 cells, RBMX knockdown increased p21 expression and senescence-associated SA-\u0026beta;-gal positivity; co-expression of Myc-tagged YBX1 abolished both effects (Fig. 6H-J). Importantly, RBMX-mediated regulation of YBX1 and p21 was substantially more pronounced in cells expressing the demethylation-mimetic RBMX_4RK mutant than in those expressing wild-type RBMX (Fig. 6K), linking RBMX post-translational modification to its control of senescence pathways.\u003c/p\u003e\n\u003cp\u003eCollectively, these findings demonstrate that cytoplasmic RBMX prevents cellular senescence by sustaining YBX1 protein expression and repressing p21\u003csup\u003eCip/Waf1\u003c/sup\u003e expression, thereby promoting tumor cell fitness and malignancy (Fig. 6L).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study uncovers a regulatory mechanism in which post-translational modification and growth factor signaling cooperate to convert RBMX from a nuclear splicing factor into a cytoplasmic, translation-selective effector of cancer progression. Although RBMX has been associated primarily with genome maintenance and co-transcriptional RNA processing \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, our data show that its oncogenic activity arises through a mechanistically distinct program controlled by PRMT5-dependent arginine methylation of the C-terminal RGG/RG motif \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. This methylation event functions as a molecular gate: methylated RBMX remains nuclear, whereas demethylated RBMX\u0026mdash;promoted by growth factor signaling\u0026mdash;translocates to the cytoplasm, where it engages the translation initiation machinery.\u003c/p\u003e \u003cp\u003eMechanistically, demethylation promotes an interaction-competent state of RBMX that enables its association with eIF3i and eIF3F, two subunits critical for mRNA recruitment and start-codon scanning. Our proteomic, biochemical, and ribosomal-engagement analyses demonstrate that cytoplasmic RBMX facilitates productive assembly of the cap-dependent initiation complex, thereby enabling translation of specific mRNAs. Importantly, RBMX does not act as a global translational enhancer. Instead, demethylation unmasks a role for RBMX as a translation-specificity factor, preferentially promoting the translation of a restricted gene set enriched for oncogenic regulators. This property distinguishes RBMX from shuttling RBPs such as hnRNPA1, FUS, HUR, and TDP-43 whose cytoplasmic mislocalization drives broader perturbations of RNA metabolism \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Our findings introduce the concept that the translational landscape of cancer cells includes PTM-tuned, transcript-selective RBPs that act as regulatory nodes downstream of growth and stress signals.\u003c/p\u003e \u003cp\u003eWithin this selective network, YBX1 emerges as the principal functional target. YBX1 controls oncogenic transcriptional and translational programs and represses p21-mediated senescence \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. We show that RBMX is required for YBX1 protein synthesis without altering its mRNA abundance, defining a translational dependency. RBMX depletion sharply reduces YBX1 protein levels and triggers p53-independent induction of p21, resulting in a robust senescence response. The demethylation-mimetic RBMX mutant (4RK) strongly amplifies YBX1 translation and suppresses senescence, demonstrating that RGG demethylation is the switch that determines whether RBMX exerts nuclear or cytoplasmic functions. This establishes a mechanistic RBMX\u0026ndash;YBX1\u0026ndash;p21 axis, linking extracellular cues and PTM control to cell-cycle arrest and proliferative escape.\u003c/p\u003e \u003cp\u003eThe mechanistic insights uncovered here hold major translational implications. Cytoplasmic RBMX abundance, RBMX\u0026ndash;eIF3 engagement, and YBX1 expression correlate strongly across patient tumors and align with poor survival outcomes in lung and colorectal cancers. These findings suggest that tumors with high RBMX cytoplasmic activity may represent a distinct subclass driven by PTM-controlled translational plasticity. Our data also carry substantial relevance for cancer therapies targeting PRMT5. While PRMT5 is widely regarded as oncogenic \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, our results reveal a paradox: PRMT5-dependent methylation restrains RBMX cytoplasmic activity, and PRMT5 inhibition inadvertently enhances RBMX-driven selective translation, thereby potentiating tumor aggressiveness. This mechanistic duality mirrors observations in other methylation-sensitive RBPs, where loss of arginine methylation promotes cytoplasmic accumulation and pathological translation \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. These insights raise the possibility that PRMT5 inhibitors\u0026mdash;currently advancing in clinical trials\u0026mdash;may carry unintended risks in tumors with high RBMX expression or dependency.\u003c/p\u003e \u003cp\u003eCollectively, this study positions RBMX as a methylation- and signal-tuned regulator of selective translation whose cytoplasmic activation promotes YBX1 synthesis, suppresses p21-driven senescence, and accelerates tumor progression. By defining demethylation as a key activating PTM and revealing RBMX\u0026rsquo;s function as a translation-specificity factor, we provide a mechanistic framework that connects extracellular signaling, PTM remodeling, translational selectivity, and cancer cell fate. These findings suggest that therapeutic strategies targeting RBMX\u0026rsquo;s methylation state, cytoplasmic trafficking, or translational interactions may offer new opportunities to disrupt oncogenic translational circuits\u0026mdash;particularly in RBMX-high tumors. Future work will be required to delineate the complete RBMX-dependent translational portfolio, to resolve how RBMX integrates with other RBPs and stress-response networks, and to evaluate whether dual modulation of RBMX and PRMT5 can yield tumor-selective vulnerabilities.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eCell culture and transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH1299 and H460 cells were cultured in RPMI-1640 medium (Invitrogen), and HEK293T, SW480, U2OS, HCT116, and HCT116 p53⁻/⁻ cells were maintained in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; Invitrogen). All cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, and cells were maintained at 37 \u0026deg;C in a humidified incubator with 5% CO₂. Plasmid transfections were performed using TurboFect (Thermo Scientific, Waltham, MA, USA) according to the manufacturer\u0026rsquo;s instructions. Sodium arsenite, EGF, and TPA were purchased from Sigma-Aldrich (St. Louis, MO, USA), and the PRMT5 inhibitor GSK3235025 was obtained from Selleckchem (Houston, TX, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid constructs and cloning\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman RBMX cDNA was amplified by RT-PCR and cloned into pCI-neo-Flag or V5 expression vectors (Promega). Serial deletion constructs of RBMX were generated by PCR amplification of individual fragments using pCI-neo-Flag-RBMX as a template, followed by insertion into XhoI and NotI sites of the pCI-neo-Flag vector. For in vitro GST pulldown assays, RBMX fragments were subcloned into pGEX4T-1 (GE Healthcare). The pCI-neo-Flag or V5-RBMX_4RK constructs (R369K, R373K, R377K, and R384K) were generated by site-directed mutagenesis using the QuikChange II Site-Directed Mutagenesis kit (Agilent Technologies). His\u003csub\u003ex6\u003c/sub\u003e-tagged eIF3e, eIF3F, eIF3g, eIF3h, eIF3i, eIF3m, eIF2\u0026alpha;, and eIF4A1 constructs used for recombinant protein isolation have been described previously\u003csup\u003e54\u003c/sup\u003e. The pDEST-myc-YBX1 plasmid (#19878) was obtained from Addgene (Watertown, MA, USA). All PCR primer sequences are listed in Supplementary Table S6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time PCR (RT-qPCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using TRIzol reagent (Invitrogen), and 2 \u0026micro;g of RNA was reverse transcribed with oligo(dT) primers and M-MuLV Reverse Transcriptase (Invitrogen). RT-qPCR was performed using gene-specific primers and the SYBR Premix Ex Taq\u0026trade; kit (TaKaRa Bio, Shiga, Japan) on a CFX96 Real-Time PCR Detection System (Bio-Rad, CA, USA). Target transcripts included RBMX, YBX1, PPAR\u0026gamma;, MAN1A2, FKBP3, PPDPF, EphA8, BACH1, YTHDF1, ARFGAP1, GMEB2, SRSF7, hnRNPL, CDK2, eIF5, ZNF207, UBXN4, C11orf58, eIF1\u0026alpha;1, SSRP1, PRPF40, IPO5, and \u0026beta;-actin. Each sample was analyzed in triplicate. Ct values for each gene were normalized to \u0026beta;-actin, and relative expression was calculated using the comparative Ct method (\u0026Delta;Ct = Ct(\u0026beta;-actin) \u0026ndash; Ct(target)). Fold changes in expression relative to control were calculated as 2^\u0026minus;\u0026Delta;\u0026Delta;Ct. Primer sequences used for RT-qPCR are listed in Supplementary Table S6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA interference\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor transient knockdown, cells were transfected with siRNAs (40 nM) using Lipofectamine RNAiMAX (Invitrogen). After 36 h, cells were trypsinized, re-plated, and transfected a second time for an additional 36 h. Knockdown efficiency was confirmed by Western blotting. For stable knockdown of RBMX, cells were transfected with pSilencer2.1-U6-hygro control shRNA or pSilencer2.1-U6-hygro RBMX shRNA using TurboFect and selected in medium containing 500 \u0026micro;g/ml hygromycin for 4\u0026ndash;5 weeks. siRNA and shRNA sequences are listed in Supplementary Table S7.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoblot and immunoprecipitation analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were lysed in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitors (1 mM Na₂VO₄, 10 mM NaF, 2 mM PMSF, 5 \u0026micro;g/ml leupeptin, 10 \u0026micro;g/ml aprotinin, 1 \u0026micro;g/ml pepstatin A; Roche). Equal amounts of protein were resolved by SDS-PAGE and transferred to PVDF membranes (Pall Life Sciences, USA). Membranes were incubated with primary antibodies overnight at 4 \u0026deg;C, followed by HRP-conjugated secondary antibodies for 1 h at room temperature. Signals were detected using ECL chemiluminescent reagents (iNtRON Biotechnology). For immunoprecipitation, lysates were pre-cleared with protein G\u0026ndash;Sepharose beads (GE Healthcare) and then incubated with the appropriate antibodies. Immune complexes were captured with protein G\u0026ndash;Sepharose, washed, and analyzed by immunoblotting. Antibodies used in this study are listed in Supplementary Table S8.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry (IHC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIHC was performed on tissue microarrays (TMAs) containing lung and colorectal cancer samples of various grades and adjacent normal tissues (Super Bio Chips; CDN4, CD4, CDA3, CCN5, CC5, CCA4; Seoul, South Korea). Heat-induced antigen retrieval was carried out in 1\u0026times; antigen retrieval buffer (pH 9.0; Abcam) at 95 \u0026deg;C for 15 min. After quenching endogenous peroxidase activity and blocking in 3% H₂O₂, sections were incubated with primary antibodies overnight at 4 \u0026deg;C, followed by HRP-conjugated secondary antibodies for 1 h at room temperature. DAB (3,3\u0026prime;-diaminobenzidine) was used as the chromogen (2 min), and slides were counterstained with Harris\u0026rsquo;s hematoxylin. Staining intensity was scored from 0 to 4, and the extent of staining from 0% to 100%; final scores were obtained by multiplying intensity and extent. Slides were evaluated independently by two pathologists. For RBMX, cytoplasmic staining intensity was quantified using ImageJ (Fiji) after excluding nuclear regions. Antibodies are listed in Supplementary Table S8.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTotal RNA sequencing analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated from cells using TRIzol reagent (1 ml per 60-mm dish) and treated with DNase I (Invitrogen). RNA-seq libraries were prepared and sequenced on an Illumina NovaSeq 6000 platform (DNA Link\u0026trade;, Seoul, Korea). Reads were mapped to the human reference genome (GRCh37/hg19) using TopHat v2.0.13 (http://ccb.jhu.edu/software/tophat/). Expression levels and differentially expressed genes (DEGs) were determined using Cuffdiff v2.2.1 (http://cole-trapnell-lab.github.io/cufflinks/papers/), generating FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values. Geometric and pooled normalization methods were applied for library normalization and dispersion estimation. Heatmaps were generated in R 3.4.1 using the heatmap.2 function (gplots package). DEGs between control and RBMX-deficient cells were subjected to KEGG pathway enrichment using ClueGO (Cytoscape v3.10.1). Bubble plots summarizing pathway significance and gene-level parameters were generated using Python (Matplotlib v3.9.0, Pandas v2.2.2). RNA-seq data have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession number GSE310912.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA immunoprecipitation (RIP), sequencing, and data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRIP was performed as described previously \u003csup\u003e55\u003c/sup\u003e with modifications. HEK293T cells were transfected with either empty vector or Flag-RBMX expression plasmid and harvested 48 h later. Cytoplasmic fractions were prepared by removing nuclear components. Cytoplasmic lysates were incubated with anti-Flag M2 antibody (Sigma-Aldrich) coupled to protein G magnetic beads. RNA\u0026ndash;protein complexes were extracted in 1 ml TRIzol (Invitrogen), and co-precipitated RNA was purified according to the manufacturer\u0026rsquo;s protocol and treated with DNase I (Invitrogen). RIP RNA libraries were sequenced on an Illumina NovaSeq 6000 platform (DNA Link). Peak calling and read density visualization were performed using the UCSC Genome Browser. Functional enrichment of RBMX-associated transcripts was carried out using ClueGO (Cytoscape v3.10.1) based on KEGG annotations. Bubble plots were generated in Python (Matplotlib v3.9.0, Pandas v2.2.2), with the x-axis representing the Flag-RBMX RIP/Input ratio, bubble size indicating the percentage of associated genes, and color reflecting \u0026ndash;log₁₀(p value) using a viridis_r color scale. RIP-seq data have been deposited in GEO under accession number GSE311136.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman Cell Stress Array\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell lysates from cells treated with siControl or siRBMX for 72 h were analyzed using the Human Cell Stress Array (ARY018; R\u0026amp;D Systems). Two hundred micrograms of protein were incubated with the array membrane according to the manufacturer\u0026rsquo;s instructions. Bound proteins were detected with a cocktail of biotinylated detection antibodies, streptavidin-HRP (1:2000), and chemiluminescent substrate. Membranes were scanned, and dot intensities were quantified using ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePolysome profiling analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK293T, U2OS, H1299, and H460 cells were lysed in polysome buffer (20 mM HEPES pH 7.6, 125 mM KCl, 5 mM MgCl₂, 2 mM DTT, DEPC-treated water). Lysates were incubated on ice for 15 min and clarified by centrifugation at 13,000 rpm for 15 min. Supernatants were layered onto 17.5\u0026ndash;50% sucrose gradients prepared in polysome buffer and centrifuged for 2.4 h at 35,000 rpm in an SW41-Ti rotor (Beckman, Brea, CA, USA). Gradients were fractionated using a fraction collector (Brandel, Gaithersburg, MD, USA), and absorbance at 253 nm was monitored with a UA-6 detector (ISCO, Lincoln, NE, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003em⁷GTP pulldown assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor m⁷GTP pulldown, cell extracts were pre-cleared with protein A\u0026ndash;agarose beads (Santa Cruz Biotechnology, TX, USA) and then incubated with m⁷GTP-agarose beads (Jena Biosciences, Germany) or control protein A\u0026ndash;agarose beads. m⁷GTP-agarose beads were equilibrated in m⁷GTP lysis buffer (50 mM HEPES pH 7.6, 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.5% NP-40, 10% glycerol, 1 mM Na₂VO₄, 10 mM NaF, 2 mM PMSF, 5 \u0026micro;g/ml leupeptin, 10 \u0026micro;g/ml aprotinin, 1 \u0026micro;g/ml pepstatin A) for 30 min before use. Bound m⁷GTP\u0026ndash;protein complexes were washed and analyzed by immunoblotting. Antibodies are listed in Supplementary Table S5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRibo-puromycylation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK293T, H1299, and H460 cells were seeded in 60-mm dishes and grown for 2 days. Cells were pulsed with puromycin (10 \u0026micro;g/ml) for 10 min at 37 \u0026deg;C in a 5% CO₂ incubator, washed twice with cold PBS, and lysed in RIPA buffer. Lysates were subjected to Western blotting, and puromycylated nascent polypeptides were detected using an anti-puromycin antibody.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSA-\u0026beta;-gal staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH1299, HCT116 (p53⁺/⁺), and HCT116 (p53⁻/⁻) cells were seeded in 6-well plates and transfected with siControl, siRBMX, or siYBX1 for 72 h. Senescence-associated \u0026beta;-galactosidase activity was assessed using a SA-\u0026beta;-gal staining kit (#9860, Cell Signaling Technology) according to the manufacturer\u0026rsquo;s instructions. The percentage of SA-\u0026beta;-gal\u0026ndash;positive cells was calculated as the number of blue-stained (senescent) cells divided by the total number of cells counted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of proteins by LC\u0026ndash;MS/MS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLC\u0026ndash;MS/MS was performed using a nanoACQUITY UPLC system coupled to an LTQ-Orbitrap mass spectrometer (Thermo Electron, San Jose, CA, USA). Peptides were separated on a BEH C18 column (1.7 \u0026micro;m, 100 \u0026micro;m \u0026times; 100 mm; Waters, Milford, MA, USA). Mobile phase A was 0.1% formic acid in water; mobile phase B was 0.1% formic acid in acetonitrile. The gradient was 10\u0026ndash;40% B over 16 min, 40\u0026ndash;95% B over 8 min, and 95\u0026ndash;10% B over 11 min at a flow rate of 0.5 \u0026micro;l/min. Mass spectra were acquired in data-dependent mode with a full scan (m/z 300\u0026ndash;2000) followed by MS/MS of selected precursors. The ion transfer tube was maintained at 275 \u0026deg;C, spray voltage at 2.3 kV, and normalized collision energy at 35%. MS/MS spectra were processed using SEQUEST (Thermo Quest), and peak lists were searched against an in-house database using MASCOT (Matrix Science, London, UK). Variable modifications included carbamidomethyl (C), deamidation (NQ), and oxidation (M). Peptide mass tolerance was set to 10 ppm, MS/MS ion tolerance to 0.8 Da, with up to two missed cleavages and charge states +2 and +3 considered. Only significant hits, as defined by MASCOT probability scores, were retained.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are presented as mean \u0026plusmn; SEM from at least three independent experiments unless otherwise indicated. Statistical significance between two groups was assessed using two-tailed paired Student\u0026rsquo;s t-tests, and multiple comparisons were analyzed by two-way ANOVA, using GraphPad Prism (GraphPad Software Inc.). P values \u0026lt; 0.05 were considered statistically significant (*P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest:\u003c/h2\u003e \u003cp\u003eThe authors have declared that no conflict of interest exists.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eAUTHOR CONTRIBUTIOMS\u003c/h2\u003e \u003cp\u003eJK, YJJ, JY, and HJY designed the experiments and analyzed data; JK, IC, and YJJ performed the experiments; JK, YJJ, JY, and HJY wrote the manuscript.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCOMPETING INTERESTS\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eThis work is supported by National Research Foundation of Korea (NRF), funded by the Ministry of Science and ICT (RS-2022-NR070848, RS-2022-NR072262 and RS-2024-00350874).\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eWe thank all participants of this study for their invaluable devotion.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGlisovic T, Bachorik JL, Yong J, Dreyfuss G. RNA-binding proteins and post-transcriptional gene regulation. \u003cem\u003eFEBS Lett\u003c/em\u003e 2008, \u003cstrong\u003e582\u003c/strong\u003e(14)\u003cstrong\u003e:\u003c/strong\u003e 1977-1986.\u003c/li\u003e\n\u003cli\u003eHentze MW, Castello A, Schwarzl T, Preiss T. A brave new world of RNA-binding proteins. \u003cem\u003eNat Rev Mol Cell Biol\u003c/em\u003e 2018, \u003cstrong\u003e19\u003c/strong\u003e(5)\u003cstrong\u003e:\u003c/strong\u003e 327-341.\u003c/li\u003e\n\u003cli\u003eGeuens T, Bouhy D, Timmerman V. The hnRNP family: insights into their role in health and disease. \u003cem\u003eHum Genet\u003c/em\u003e 2016, \u003cstrong\u003e135\u003c/strong\u003e(8)\u003cstrong\u003e:\u003c/strong\u003e 851-867.\u003c/li\u003e\n\u003cli\u003eElliott DJ, Dalgliesh C, Hysenaj G, Ehrmann I. RBMX family proteins connect the fields of nuclear RNA processing, disease and sex chromosome biology. \u003cem\u003eInt J Biochem Cell Biol\u003c/em\u003e 2019, \u003cstrong\u003e108:\u003c/strong\u003e 1-6.\u003c/li\u003e\n\u003cli\u003eMoursy A, Allain FH, Clery A. Characterization of the RNA recognition mode of hnRNP G extends its role in SMN2 splicing regulation. \u003cem\u003eNucleic Acids Res\u003c/em\u003e 2014, \u003cstrong\u003e42\u003c/strong\u003e(10)\u003cstrong\u003e:\u003c/strong\u003e 6659-6672.\u003c/li\u003e\n\u003cli\u003eSheng Y, Lei K, Sun C, Liu J, Tu Z, Zhu X\u003cem\u003e, et al.\u003c/em\u003e Aberrant RBMX expression is relevant for cancer prognosis and immunotherapy response. \u003cem\u003eAging (Albany NY)\u003c/em\u003e 2024, \u003cstrong\u003e16\u003c/strong\u003e(1)\u003cstrong\u003e:\u003c/strong\u003e 226-245.\u003c/li\u003e\n\u003cli\u003eSong Y, He S, Ma X, Zhang M, Zhuang J, Wang G\u003cem\u003e, et al.\u003c/em\u003e RBMX contributes to hepatocellular carcinoma progression and sorafenib resistance by specifically binding and stabilizing BLACAT1. \u003cem\u003eAm J Cancer Res\u003c/em\u003e 2020, \u003cstrong\u003e10\u003c/strong\u003e(11)\u003cstrong\u003e:\u003c/strong\u003e 3644-3665.\u003c/li\u003e\n\u003cli\u003eWang Y, Zhao Z, Guo T, Wu T, Zhang M, Luo D\u003cem\u003e, et al.\u003c/em\u003e SOCS5-RBMX stimulates SREBP1-mediated lipogenesis to promote metastasis in steatotic HCC with HBV-related cirrhosis. \u003cem\u003eNPJ Precis Oncol\u003c/em\u003e 2024, \u003cstrong\u003e8\u003c/strong\u003e(1)\u003cstrong\u003e:\u003c/strong\u003e 58.\u003c/li\u003e\n\u003cli\u003eQiu Y, Pu C, Wang C, Quan Z. The RNA-Binding Protein RBMX Mediates the Immunosuppressive Microenvironment of Osteosarcoma by Regulating CD8(+)T Cells. \u003cem\u003eCancers (Basel)\u003c/em\u003e 2025, \u003cstrong\u003e17\u003c/strong\u003e(17).\u003c/li\u003e\n\u003cli\u003eLi A, Hong J, Ma X, Huang Y, Jiang Q, Zhang C\u003cem\u003e, et al.\u003c/em\u003e Cancer-Derived Exosomal LINC01615 Induces M2 Polarization of Tumor-Associated Macrophages via RBMX-EZH2 Axis to Promote Colorectal Cancer Progression. \u003cem\u003eInt J Nanomedicine\u003c/em\u003e 2025, \u003cstrong\u003e20:\u003c/strong\u003e 7343-7358.\u003c/li\u003e\n\u003cli\u003eYan Q, Zeng P, Zhou X, Zhao X, Chen R, Qiao J\u003cem\u003e, et al.\u003c/em\u003e RBMX suppresses tumorigenicity and progression of bladder cancer by interacting with the hnRNP A1 protein to regulate PKM alternative splicing. \u003cem\u003eOncogene\u003c/em\u003e 2021, \u003cstrong\u003e40\u003c/strong\u003e(15)\u003cstrong\u003e:\u003c/strong\u003e 2635-2650.\u003c/li\u003e\n\u003cli\u003eShin KH, Kang MK, Kim RH, Christensen R, Park NH. Heterogeneous nuclear ribonucleoprotein G shows tumor suppressive effect against oral squamous cell carcinoma cells. \u003cem\u003eClin Cancer Res\u003c/em\u003e 2006, \u003cstrong\u003e12\u003c/strong\u003e(10)\u003cstrong\u003e:\u003c/strong\u003e 3222-3228.\u003c/li\u003e\n\u003cli\u003eZheng T, Zhou H, Li X, Peng D, Yang Y, Zeng Y\u003cem\u003e, et al.\u003c/em\u003e RBMX is required for activation of ATR on repetitive DNAs to maintain genome stability. \u003cem\u003eCell Death Differ\u003c/em\u003e 2020, \u003cstrong\u003e27\u003c/strong\u003e(11)\u003cstrong\u003e:\u003c/strong\u003e 3162-3176.\u003c/li\u003e\n\u003cli\u003eLiu J, Zheng T, Chen D, Huang J, Zhao Y, Ma W\u003cem\u003e, et al.\u003c/em\u003e RBMX involves in telomere stability maintenance by regulating TERRA expression. \u003cem\u003ePLoS Genet\u003c/em\u003e 2023, \u003cstrong\u003e19\u003c/strong\u003e(9)\u003cstrong\u003e:\u003c/strong\u003e e1010937.\u003c/li\u003e\n\u003cli\u003eXu J, Richard S. Cellular pathways influenced by protein arginine methylation: Implications for cancer. \u003cem\u003eMol Cell\u003c/em\u003e 2021, \u003cstrong\u003e81\u003c/strong\u003e(21)\u003cstrong\u003e:\u003c/strong\u003e 4357-4368.\u003c/li\u003e\n\u003cli\u003eMusiani D, Bok J, Massignani E, Wu L, Tabaglio T, Ippolito MR\u003cem\u003e, et al.\u003c/em\u003e Proteomics profiling of arginine methylation defines PRMT5 substrate specificity. \u003cem\u003eSci Signal\u003c/em\u003e 2019, \u003cstrong\u003e12\u003c/strong\u003e(575).\u003c/li\u003e\n\u003cli\u003eHwang JW, Cho Y, Bae GU, Kim SN, Kim YK. Protein arginine methyltransferases: promising targets for cancer therapy. \u003cem\u003eExp Mol Med\u003c/em\u003e 2021, \u003cstrong\u003e53\u003c/strong\u003e(5)\u003cstrong\u003e:\u003c/strong\u003e 788-808.\u003c/li\u003e\n\u003cli\u003eBlackwell E, Ceman S. Arginine methylation of RNA-binding proteins regulates cell function and differentiation. \u003cem\u003eMol Reprod Dev\u003c/em\u003e 2012, \u003cstrong\u003e79\u003c/strong\u003e(3)\u003cstrong\u003e:\u003c/strong\u003e 163-175.\u003c/li\u003e\n\u003cli\u003eCai T, Cinkornpumin JK, Yu Z, Villarreal OD, Pastor WA, Richard S. Deletion of RBMX RGG/RG motif in Shashi-XLID syndrome leads to aberrant p53 activation and neuronal differentiation defects. \u003cem\u003eCell Rep\u003c/em\u003e 2021, \u003cstrong\u003e36\u003c/strong\u003e(2)\u003cstrong\u003e:\u003c/strong\u003e 109337.\u003c/li\u003e\n\u003cli\u003eLorton BM, Shechter D. Cellular consequences of arginine methylation. \u003cem\u003eCell Mol Life Sci\u003c/em\u003e 2019, \u003cstrong\u003e76\u003c/strong\u003e(15)\u003cstrong\u003e:\u003c/strong\u003e 2933-2956.\u003c/li\u003e\n\u003cli\u003eWu K, Niu C, Liu H, Fu L. Research progress on PRMTs involved in epigenetic modification and tumour signalling pathway regulation (Review). \u003cem\u003eInt J Oncol\u003c/em\u003e 2023, \u003cstrong\u003e62\u003c/strong\u003e(5).\u003c/li\u003e\n\u003cli\u003eWall ML, Lewis SM. Methylarginines within the RGG-Motif Region of hnRNP A1 Affect Its IRES Trans-Acting Factor Activity and Are Required for hnRNP A1 Stress Granule Localization and Formation. \u003cem\u003eJ Mol Biol\u003c/em\u003e 2017, \u003cstrong\u003e429\u003c/strong\u003e(2)\u003cstrong\u003e:\u003c/strong\u003e 295-307.\u003c/li\u003e\n\u003cli\u003eNichols RC, Wang XW, Tang J, Hamilton BJ, High FA, Herschman HR\u003cem\u003e, et al.\u003c/em\u003e The RGG domain in hnRNP A2 affects subcellular localization. \u003cem\u003eExp Cell Res\u003c/em\u003e 2000, \u003cstrong\u003e256\u003c/strong\u003e(2)\u003cstrong\u003e:\u003c/strong\u003e 522-532.\u003c/li\u003e\n\u003cli\u003eClarke JP, Thibault PA, Salapa HE, Levin MC. A Comprehensive Analysis of the Role of hnRNP A1 Function and Dysfunction in the Pathogenesis of Neurodegenerative Disease. \u003cem\u003eFront Mol Biosci\u003c/em\u003e 2021, \u003cstrong\u003e8:\u003c/strong\u003e 659610.\u003c/li\u003e\n\u003cli\u003eMartinez-Salas E, Lozano G, Fernandez-Chamorro J, Francisco-Velilla R, Galan A, Diaz R. RNA-binding proteins impacting on internal initiation of translation. \u003cem\u003eInt J Mol Sci\u003c/em\u003e 2013, \u003cstrong\u003e14\u003c/strong\u003e(11)\u003cstrong\u003e:\u003c/strong\u003e 21705-21726.\u003c/li\u003e\n\u003cli\u003eSzostak E, Gebauer F. Translational control by 3\u0026apos;-UTR-binding proteins. \u003cem\u003eBrief Funct Genomics\u003c/em\u003e 2013, \u003cstrong\u003e12\u003c/strong\u003e(1)\u003cstrong\u003e:\u003c/strong\u003e 58-65.\u003c/li\u003e\n\u003cli\u003eMoore KS, von Lindern M. RNA Binding Proteins and Regulation of mRNA Translation in Erythropoiesis. \u003cem\u003eFront Physiol\u003c/em\u003e 2018, \u003cstrong\u003e9:\u003c/strong\u003e 910.\u003c/li\u003e\n\u003cli\u003eMarchione R, Leibovitch SA, Lenormand JL. The translational factor eIF3f: the ambivalent eIF3 subunit. \u003cem\u003eCell Mol Life Sci\u003c/em\u003e 2013, \u003cstrong\u003e70\u003c/strong\u003e(19)\u003cstrong\u003e:\u003c/strong\u003e 3603-3616.\u003c/li\u003e\n\u003cli\u003eYin Y, Long J, Sun Y, Li H, Jiang E, Zeng C\u003cem\u003e, et al.\u003c/em\u003e The function and clinical significance of eIF3 in cancer. \u003cem\u003eGene\u003c/em\u003e 2018, \u003cstrong\u003e673:\u003c/strong\u003e 130-133.\u003c/li\u003e\n\u003cli\u003eEsteves P, Dard L, Brillac A, Hubert C, Sarlak S, Rousseau B\u003cem\u003e, et al.\u003c/em\u003e Nuclear control of lung cancer cells migration, invasion and bioenergetics by eukaryotic translation initiation factor 3F. \u003cem\u003eOncogene\u003c/em\u003e 2020, \u003cstrong\u003e39\u003c/strong\u003e(3)\u003cstrong\u003e:\u003c/strong\u003e 617-636.\u003c/li\u003e\n\u003cli\u003eQi J, Dong Z, Liu J, Zhang JT. EIF3i promotes colon oncogenesis by regulating COX-2 protein synthesis and beta-catenin activation. \u003cem\u003eOncogene\u003c/em\u003e 2014, \u003cstrong\u003e33\u003c/strong\u003e(32)\u003cstrong\u003e:\u003c/strong\u003e 4156-4163.\u003c/li\u003e\n\u003cli\u003eMa S, Dong Z, Cui Q, Liu JY, Zhang JT. eIF3i regulation of protein synthesis, cell proliferation, cell cycle progression, and tumorigenesis. \u003cem\u003eCancer Lett\u003c/em\u003e 2021, \u003cstrong\u003e500:\u003c/strong\u003e 11-20.\u003c/li\u003e\n\u003cli\u003eZhang Y, Wan X, Yang X, Liu X, Huang Q, Zhou L\u003cem\u003e, et al.\u003c/em\u003e eIF3i promotes colorectal cancer cell survival via augmenting PHGDH translation. \u003cem\u003eJ Biol Chem\u003c/em\u003e 2023, \u003cstrong\u003e299\u003c/strong\u003e(9)\u003cstrong\u003e:\u003c/strong\u003e 105177.\u003c/li\u003e\n\u003cli\u003eJackson RJ, Hellen CU, Pestova TV. The mechanism of eukaryotic translation initiation and principles of its regulation. \u003cem\u003eNat Rev Mol Cell Biol\u003c/em\u003e 2010, \u003cstrong\u003e11\u003c/strong\u003e(2)\u003cstrong\u003e:\u003c/strong\u003e 113-127.\u003c/li\u003e\n\u003cli\u003eHernandez-Segura A, Nehme J, Demaria M. Hallmarks of Cellular Senescence. \u003cem\u003eTrends Cell Biol\u003c/em\u003e 2018, \u003cstrong\u003e28\u003c/strong\u003e(6)\u003cstrong\u003e:\u003c/strong\u003e 436-453.\u003c/li\u003e\n\u003cli\u003evan der Houven van Oordt W, Diaz-Meco MT, Lozano J, Krainer AR, Moscat J, Caceres JF. The MKK(3/6)-p38-signaling cascade alters the subcellular distribution of hnRNP A1 and modulates alternative splicing regulation. \u003cem\u003eJ Cell Biol\u003c/em\u003e 2000, \u003cstrong\u003e149\u003c/strong\u003e(2)\u003cstrong\u003e:\u003c/strong\u003e 307-316.\u003c/li\u003e\n\u003cli\u003eDoller A, Huwiler A, Muller R, Radeke HH, Pfeilschifter J, Eberhardt W. Protein kinase C alpha-dependent phosphorylation of the mRNA-stabilizing factor HuR: implications for posttranscriptional regulation of cyclooxygenase-2. \u003cem\u003eMol Biol Cell\u003c/em\u003e 2007, \u003cstrong\u003e18\u003c/strong\u003e(6)\u003cstrong\u003e:\u003c/strong\u003e 2137-2148.\u003c/li\u003e\n\u003cli\u003eDoller A, Pfeilschifter J, Eberhardt W. Signalling pathways regulating nucleo-cytoplasmic shuttling of the mRNA-binding protein HuR. \u003cem\u003eCell Signal\u003c/em\u003e 2008, \u003cstrong\u003e20\u003c/strong\u003e(12)\u003cstrong\u003e:\u003c/strong\u003e 2165-2173.\u003c/li\u003e\n\u003cli\u003eHanahan D, Weinberg RA. Hallmarks of cancer: the next generation. \u003cem\u003eCell\u003c/em\u003e 2011, \u003cstrong\u003e144\u003c/strong\u003e(5)\u003cstrong\u003e:\u003c/strong\u003e 646-674.\u003c/li\u003e\n\u003cli\u003eGriner EM, Kazanietz MG. Protein kinase C and other diacylglycerol effectors in cancer. \u003cem\u003eNat Rev Cancer\u003c/em\u003e 2007, \u003cstrong\u003e7\u003c/strong\u003e(4)\u003cstrong\u003e:\u003c/strong\u003e 281-294.\u003c/li\u003e\n\u003cli\u003eLiu N, Zhou KI, Parisien M, Dai Q, Diatchenko L, Pan T. N6-methyladenosine alters RNA structure to regulate binding of a low-complexity protein. \u003cem\u003eNucleic Acids Res\u003c/em\u003e 2017, \u003cstrong\u003e45\u003c/strong\u003e(10)\u003cstrong\u003e:\u003c/strong\u003e 6051-6063.\u003c/li\u003e\n\u003cli\u003eLarsen SC, Sylvestersen KB, Mund A, Lyon D, Mullari M, Madsen MV\u003cem\u003e, et al.\u003c/em\u003e Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells. \u003cem\u003eSci Signal\u003c/em\u003e 2016, \u003cstrong\u003e9\u003c/strong\u003e(443)\u003cstrong\u003e:\u003c/strong\u003e rs9.\u003c/li\u003e\n\u003cli\u003eDormann D, Madl T, Valori CF, Bentmann E, Tahirovic S, Abou-Ajram C\u003cem\u003e, et al.\u003c/em\u003e Arginine methylation next to the PY-NLS modulates Transportin binding and nuclear import of FUS. \u003cem\u003eEMBO J\u003c/em\u003e 2012, \u003cstrong\u003e31\u003c/strong\u003e(22)\u003cstrong\u003e:\u003c/strong\u003e 4258-4275.\u003c/li\u003e\n\u003cli\u003eKumari R, Jat P. Mechanisms of Cellular Senescence: Cell Cycle Arrest and Senescence Associated Secretory Phenotype. \u003cem\u003eFront Cell Dev Biol\u003c/em\u003e 2021, \u003cstrong\u003e9:\u003c/strong\u003e 645593.\u003c/li\u003e\n\u003cli\u003eLu ZH, Books JT, Ley TJ. YB-1 is important for late-stage embryonic development, optimal cellular stress responses, and the prevention of premature senescence. \u003cem\u003eMol Cell Biol\u003c/em\u003e 2005, \u003cstrong\u003e25\u003c/strong\u003e(11)\u003cstrong\u003e:\u003c/strong\u003e 4625-4637.\u003c/li\u003e\n\u003cli\u003eZhu B, Zhang Z, Pardeshi L, Chen Y, Ge W. Y box-binding protein 1 regulates zebrafish folliculogenesis partly through p21-mediated control of follicle cell proliferation. \u003cem\u003eDevelopment\u003c/em\u003e 2024, \u003cstrong\u003e151\u003c/strong\u003e(21).\u003c/li\u003e\n\u003cli\u003eKwon E, Todorova K, Wang J, Horos R, Lee KK, Neel VA\u003cem\u003e, et al.\u003c/em\u003e The RNA-binding protein YBX1 regulates epidermal progenitors at a posttranscriptional level. \u003cem\u003eNat Commun\u003c/em\u003e 2018, \u003cstrong\u003e9\u003c/strong\u003e(1)\u003cstrong\u003e:\u003c/strong\u003e 1734.\u003c/li\u003e\n\u003cli\u003eBeckerman R, Prives C. Transcriptional regulation by p53. \u003cem\u003eCold Spring Harb Perspect Biol\u003c/em\u003e 2010, \u003cstrong\u003e2\u003c/strong\u003e(8)\u003cstrong\u003e:\u003c/strong\u003e a000935.\u003c/li\u003e\n\u003cli\u003eEngeland K. Cell cycle regulation: p53-p21-RB signaling. \u003cem\u003eCell Death Differ\u003c/em\u003e 2022, \u003cstrong\u003e29\u003c/strong\u003e(5)\u003cstrong\u003e:\u003c/strong\u003e 946-960.\u003c/li\u003e\n\u003cli\u003eSama RR, Ward CL, Kaushansky LJ, Lemay N, Ishigaki S, Urano F\u003cem\u003e, et al.\u003c/em\u003e FUS/TLS assembles into stress granules and is a prosurvival factor during hyperosmolar stress. \u003cem\u003eJ Cell Physiol\u003c/em\u003e 2013, \u003cstrong\u003e228\u003c/strong\u003e(11)\u003cstrong\u003e:\u003c/strong\u003e 2222-2231.\u003c/li\u003e\n\u003cli\u003eChen J. The Cell-Cycle Arrest and Apoptotic Functions of p53 in Tumor Initiation and Progression. \u003cem\u003eCold Spring Harb Perspect Med\u003c/em\u003e 2016, \u003cstrong\u003e6\u003c/strong\u003e(3)\u003cstrong\u003e:\u003c/strong\u003e a026104.\u003c/li\u003e\n\u003cli\u003eStopa N, Krebs JE, Shechter D. The PRMT5 arginine methyltransferase: many roles in development, cancer and beyond. \u003cem\u003eCell Mol Life Sci\u003c/em\u003e 2015, \u003cstrong\u003e72\u003c/strong\u003e(11)\u003cstrong\u003e:\u003c/strong\u003e 2041-2059.\u003c/li\u003e\n\u003cli\u003eKim H, Ronai ZA. PRMT5 function and targeting in cancer. \u003cem\u003eCell Stress\u003c/em\u003e 2020, \u003cstrong\u003e4\u003c/strong\u003e(8)\u003cstrong\u003e:\u003c/strong\u003e 199-215.\u003c/li\u003e\n\u003cli\u003eKim J, Chang IY, Lee JH, Yong J, Jeon YJ, You HJ. The role of Ephexin1 in translation and mTOR-targeted cancer therapy. \u003cem\u003eExp Mol Med\u003c/em\u003e 2025, \u003cstrong\u003e57\u003c/strong\u003e(8)\u003cstrong\u003e:\u003c/strong\u003e 1847-1860.\u003c/li\u003e\n\u003cli\u003eKim J, Park RY, Kee Y, Jeong S, Ohn T. Splicing factor SRSF3 represses translation of p21(cip1/waf1) mRNA. \u003cem\u003eCell Death Dis\u003c/em\u003e 2022, \u003cstrong\u003e13\u003c/strong\u003e(11)\u003cstrong\u003e:\u003c/strong\u003e 933.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-differentiation","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cdd","sideBox":"Learn more about [Cell Death \u0026 Differentiation](http://www.nature.com/cdd/)","snPcode":"41418","submissionUrl":"https://mts-cdd.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Differentiation","twitterHandle":"@cddpress","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8859405/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8859405/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlthough RBMX has been studied primarily as a nuclear RNA-binding protein involved in splicing and genome maintenance, we identify a previously unrecognized cytoplasmic role for RBMX in regulating senescence entry through selective mRNA translation in cancer cells. We show that a reversible methylation-dependent switch promotes RBMX relocalization to the cytoplasm, where RBMX directly engages the eIF3i/eIF3F initiation module to regulate translation initiation. Cytoplasmic RBMX does not globally enhance protein synthesis; instead, it functions as a translation-specificity factor that preferentially promotes translation of oncogenic mRNAs, including YBX1. Disruption of this translational program reduces YBX1 protein output without affecting mRNA abundance, leading to p53-independent induction of p21 and a robust senescence response. These findings establish selective translational control by cytoplasmic RBMX as a decisive upstream mechanism governing senescence fate in cancer cells.\u003c/p\u003e","manuscriptTitle":"Cytoplasmic RBMX coordinates selective mRNA translation to suppress senescence in cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-24 13:30:17","doi":"10.21203/rs.3.rs-8859405/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-04T00:37:10+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-22T22:45:38+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-19T18:10:24+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-02-19T13:55:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-16T10:15:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Differentiation","date":"2026-02-14T01:20:53+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2026-02-13T09:48:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-12T08:06:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-differentiation","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cdd","sideBox":"Learn more about [Cell Death \u0026 Differentiation](http://www.nature.com/cdd/)","snPcode":"41418","submissionUrl":"https://mts-cdd.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Differentiation","twitterHandle":"@cddpress","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"48deecf8-4a01-4f2a-9dcb-68d2395c358d","owner":[],"postedDate":"February 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":63204899,"name":"Biological sciences/Cancer/Cancer models"},{"id":63204900,"name":"Biological sciences/Biochemistry/Proteins/Transcription factors"}],"tags":[],"updatedAt":"2026-02-24T13:30:17+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-24 13:30:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8859405","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8859405","identity":"rs-8859405","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}