Molecular Cell

Cells must be primed to rapidly induce inflammatory gene expression upon infection while also tuning the level of induction to avoid immunopathology. Here, we identify RNA polymerase III (Pol III), best known for transcribing noncoding RNAs, as a dual-function regulator of RNA polymerase II (Pol II)-dependent inflammatory gene expression. Pol III is selectively enriched at promoters of innate immune, pro-inflammatory, and stress-response genes, where it maintains chromatin accessibility and supports basal transcription. Upon infection with murine gammaher-pesvirus 68 (MHV68), Pol III redistributes from these promoters to retrotransposon loci, coinciding with enhanced expression of inflammatory genes. Depletion of the Pol III transcription factor Brf1 further amplifies inflammatory transcription during infection with MHV68, herpes simplex virus-1, and influenza A virus. Genes restrained by Pol III have TATA-box-enriched promoters and are functionally dependent on TATA-binding protein (TBP), suggesting that Pol III modulates inflammatory gene expression by competing with Pol II for shared transcriptional machinery. Thus, Pol III is a chromatin licensor in uninfected cells and an inflammation rheostat during viral infection. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/738346v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@d99325org.highwire.dtl.DTLVardef@4b781dorg.highwire.dtl.DTLVardef@bad1b6org.highwire.dtl.DTLVardef@11e41a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Human RNA Polymerase II (Pol II) is characterized by a dense layer of over 775 post-translational modifications (PTMs) that form a dynamic, rewritable regulatory architecture integrating large numbers of cellular signals to coordinate transcription initiation, elongation, termination, and co-transcriptional RNA processing. While genomic information has been extensively catalogued, the potential information capacity associated with Pol II PTM patterns has remained largely unquantified. Here, we analyze the PTM sites across the human Pol II complex (Rpb1-Rpb12) and estimate the state-space information capacity using Shannon entropy theory. We first provide a theoretical upper bound of ~707.98 bits per Pol II molecule (~88.50 bytes) corresponding to ~5.68 x 107 bits per nucleus (~7.10 MB), assuming ~80,200 Pol II molecules per cell. We distinguish this maximal capacity from a conservative, kinetically addressable estimate of ~114.88 bits per molecule (~1.15 MB per nucleus), reflecting physiological kinetic constraints and site coupling that restrict the simultaneously addressable PTM state space in vivo. Finally, we show that major PTM classes (phosphorylation, proline isomerization, O-GlcNAcylation and ubiquitination) operate over distinct lifetimes, from seconds to minutes and hours-scale processes, supporting a multi-timescale biochemical architecture of this enzyme. Together, these results provide a quantitative information framework that distinguishes maximal PTM state-space capacity from kinetically addressable physiological regulatory capacity, supporting a view of Pol II PTM patterning as a high-dimensional, dynamically reconfigurable, multi-timescale regulatory information layer. HighlightsA systems-level framework quantifies regulatory information in Pol II PTMs Known PTM modification sites provide 707.98 bits per Pol II as a regulatory upper bound Physiological kinetic constraints reduce accessible regulatory capacity to 114.88 bits Distinct PTM modification classes define fast, intermediate, and slow regulatory layers Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/734958v1_ufig1.gif" ALT="Figure 1"> View larger version (84K): org.highwire.dtl.DTLVardef@1cd7a8aorg.highwire.dtl.DTLVardef@128387forg.highwire.dtl.DTLVardef@1952adaorg.highwire.dtl.DTLVardef@307b22_HPS_FORMAT_FIGEXP M_FIG C_FIG

DNA replication forks frequently encounter obstacles and remodel into four-way junctions to actively slow DNA fork progression. Fork slowing can also spread to undamaged forks via an ATR-dependent mechanism that remained elusive. Here, using mild genotoxic stress, we show that fork slowing and reversal require full ATR activity, but no canonical ATR activators and signalling partners, defining a non-canonical ATR pathway distinct from origin firing control. Phospho-proteomics in S-phase cells under checkpoint-blind replication stress identified a subset of ATR-dependent phospho-sites, such as S343 on NBS1, the regulatory subunit of the MRN complex. This residue is essential to stimulate MRN exonuclease activity in vitro and required in cells for global fork slowing upon mild DNA damage. Ultimately, local UV-C micro-irradiation reveals that ATR-dependent MRN-stimulated resection dampens DNA synthesis at lesions and propagates fork slowing to undamaged chromatin, supporting MRN-mediated ssDNA exposure as a mean to coordinate replication slowdown across the nucleus.

Electrophysiological studies have long treated the cardiac voltage-gated sodium channel Nav1.5 (SCN5A) as a monomeric pore-forming unit, consistent with all available cryo-EM structures, which show only monomeric architectures. In contrast, biochemical studies -- cross-linking, single-molecule pulldown, and native electrophoresis -- have reported [~]500 kDa Nav1.5 homodimers with coupled gating. To reconcile this apparent discrepancy, we combined selective labeling of the total (metabolic [35S]methionine) and plasma-membrane (membrane-impermeant IRDye 800CW) pools of hNav1.5 with high-resolution clear native electrophoresis (hrCNE) in Xenopus laevis oocytes. Total hNav1.5 migrated predominantly as a homodimer that dissociated into monomers upon denaturation, whereas surface-labeled hNav1.5 migrated exclusively as a monomer, confirming mature, Golgi-processed glycosylation by Endo H/PNGase F digestion. This monomer-dimer distribution was unaffected by co-expression with hNav{beta}1-{beta}4 subunits. Using an orthogonal SpyCatcher/SpyTag covalent tagging strategy, we captured the intracellular homodimer as an irreversible [~]500 kDa complex, and engineered TEV protease cleavage sites revealed that the two protomers associate in a previously unrecognized antiparallel, cyclic arrangement. AlphaFold2-Multimer confidently predicted a monomeric hNav1.5 fold but failed to generate a high-confidence homodimer interface, indicating that this arrangement is not strongly sequence-encoded. Together, our data resolve the electrophysiology-biochemistry discrepancy: hNav1.5 assembles as an antiparallel homodimer in intracellular compartments, likely subject to quality control, but is delivered to the plasma membrane -- the physiologically conducting compartment -- exclusively as a monomer, irrespective of {beta}-subunit association.

Epstein-Barr virus (EBV) causes over 200,000 cancers annually, including immunoblastic lymphomas in immunosuppressed hosts. Most transformed cells arrest, yet survive when deprived of the essential amino acid methionine. We instead find that EBV transformed lymphoblastoid cell lines (LCLs), which model the EBV latency III program-driven B-cell lymphoproliferative diseases of immunosuppressed hosts, rapidly die upon methionine restriction. Methionine restriction elevated LCL lipid reactive oxygen species and triggered ferroptosis. Whereas methionine restriction hypomethylates the EBV genome and triggers viral reactivation in latency I Burkitt cells by lowering the cellular methylation potential, the LCL latency III program instead redirected methionine toward redox defense, without altering the SAM/SAH ratio. Stable-isotope tracing revealed that latency III strongly induces transsulfuration, synthesizing cysteine de novo to support glutathione pools. The EBV oncoprotein LMP2A, which mimics B-cell receptor signaling, supported newly infected human B cell cystathionine-{beta}-synthase and cystathionine-{gamma}-lyase expression and methionine dependence, phenocopied by immunoglobulin crosslinking. In vivo, dietary methionine restriction impaired LCL xenograft outgrowth and depleted tumor cystine. Combined methioninase and cyst(e)inase administration blocked both cysteine sources, collapsed tumor glutathione levels, and triggered ferroptosis. Our results define methionine metabolism as a targetable ferroptosis vulnerability of EBV-transformed B cells. HighlightsO_LIMethionine restriction triggers EBV-transformed lymphoblastoid B cell ferroptosis C_LIO_LIEBV latency III induces transsulfuration to sustain LCL cysteine and glutathione C_LIO_LIMethioninase or dietary methionine restriction strongly impair LCL growth in vivo C_LIO_LIMethioninase plus cyst(e)inase collapses xenograft GSH levels and drives ferroptosis C_LI

A ubiquitous feature of higher order genome organization is the presence of topologically associating domains (TADs). The chromatin architectural proteins cohesin and CTCF are known critical organizers of TADs, but the mechanisms of TAD establishment and maintenance, including their accurate duplication during genome replication, are not well characterized. To address this gap, we used high-throughput imaging-based CRISPR/Cas9 knock-out screening to discover chromatin factors involved in maintenance and establishment of TADs. Among the cellular factors that affect TAD organization, we found enrichment for cell cycle proteins, especially components of the DNA replication machinery. Accordingly, we demonstrate that TADs undergo temporary unfolding during S-phase DNA replication and that interference with progression through replication impedes restoration of normal TAD structure. Mechanistically, inhibition of the RPA complex prevents cohesin and CTCF binding, delays post-replication TAD re-folding and affects TAD folding in non-cycling cells. These results provide novel insights into how TAD structures are re-established during genome duplication.

The Wood-Ljungdahl pathway is one of biology's most ancient routes for carbon fixation and energy metabolism, used by organisms such as methanogenic archaea. One of its central metabolic complexes is the acetyl-CoA decarbonylase/synthase (ACDS) complex, catalyzing acetyl-CoA synthesis and cleavage through the coordinated action of carbon monoxide dehydrogenase (CODH), acetyl-CoA synthase (ACS), and corrinoid iron-sulfur protein (CoFeSP). Unlike bacterial CODH/ACS, archaeal ACDS lacks a stable bifunctional CODH-ACS architecture, raising the question of how reactive CO and methyl intermediates are efficiently transferred between catalytic modules. Using cryo-electron microscopy, crosslinking mass spectrometry, small-angle X-ray scattering, and biophysical analyses, we resolved the organization and dynamics of the ~2 MDa archaeal ACDS supercomplex from Methanosarcina acetivorans. We identified CoFeSP as a central architectural scaffold that self-assembles into hexa- to octameric oligomers via a conserved N-terminal region of the CdhD subunit. This scaffold likely tethers CODH and ACS through conserved disordered terminal regions, positioning the catalytic modules in the complex's periphery. We propose a mechanism in which ACS transiently alternates between CODH and CoFeSP, enabling efficient CO and methyl-group transfer without stable binary complexes. This dynamic organization represents a fundamental difference to the stable bifunctional CODH/ACS in bacteria, highlighting how transient interactions enable efficient acetyl-CoA metabolism in archaea.

Cohesin-mediated loop extrusion and chromatin state-dependent compartmentalization are major drivers of three-dimensional (3D) genome organization. Although epigenomic perturbations are widely assumed to reshape chromatin architecture, the mechanisms that determine how changes in chromatin state are translated into structural reorganization remain poorly understood. Here, we identify cohesin residence time as a key regulator of the genome's architectural response to histone hyperacetylation induced by histone deacetylase inhibition (HDACi). Acute depletion of RAD21 or CTCF weakens chromatin loops but preserves HDACi-induced changes in compartmentalization, contact-scaling behavior, and loop density. In contrast, perturbation of cohesin loading or release produces opposing effects: NIPBL depletion sensitizes and amplifies architectural responses to HDACi, whereas WAPL loss renders the genome largely refractory to HDACi-induced remodeling, suppressing changes in compartments and loop density while stabilizing CTCF-anchored loops. These distinct architectural outcomes occur despite comparable levels of HDACi-induced histone hyperacetylation across genotypes, indicating that differential epigenomic input is not responsible for the observed effects. Together, our findings demonstrate that dynamic cohesin turnover, rather than cohesin chromatin association alone, governs whether epigenomic perturbations are converted into higher-order genome reorganization. These results establish cohesin residence time as a molecular gate linking chromatin state to 3D genome architecture and reveal a previously unrecognized principle underlying chromatin architecture plasticity.

G-quadruplexes (G4s) are non-canonical DNA structures that regulate transcription, replication, and DNA repair but, when unresolved, hinder replication fork progression and compromise genome stability. Here, we identify a WRNIP1-dependent mechanism that promotes the resolution of R-loop-associated G4 structures. Loss of WRNIP1 or disruption of its ubiquitin-binding zinc finger (UBZ) domain causes persistent G4/R-loop accumulation, leading to transcription-replication conflicts, DNA damage, and genome instability. We further show that WRNIP1 interacts with the G4 helicase FANCJ and that mutation of the UBZ domain disrupts this interaction, impairing FANCJ stability and recruitment to G4 sites. Mechanistically, WRNIP1 acts upstream of FANCJ by promoting ATM-dependent signaling required for FANCJ stabilization and chromatin association. ATM-mediated phosphorylation of FANCJ at Ser990 protects the helicase from ubiquitin-dependent proteasomal degradation. Together, these findings define a WRNIP1-ATM-FANCJ regulatory axis that promotes G4 resolution during DNA replication and preserves genome stability.

Co-transcriptional splicing is a conserved feature of eukaryotic gene expression. However, establishing the functional nature of this process has been difficult. Here using high throughput CRISPR/Cas9 screens we surprisingly find that SF3B3, the third largest subunit of the U2 snRNP complex, is a major regulator of RNA Pol II pause release and processivity. Remarkably, the absence of SF3B3 dramatically perturbs transcription but U2 snRNP assembly and RNA splicing remains unaffected. Mechanistically, SF3B3 coordinates the chromatin occupancy of transcriptional kinases (CDK9/12/13) alongside the PAF1c and Integrator complexes to regulate Pol II. Structure / function analyses of SF3B3 revealed that a metazoan specific 18aa sequence within its disordered tail phenocopies its loss and mediates the physical association and stability of SF3B5. We show that loss of SF3B5 mirrors SF3B3 deficiency suggesting this submodule, although resident within the U2 snRNP complex, evolved to primarily coordinate RNA Pol II in a splicing-independent manner.

Topoisomerase II (TOP2) resolves DNA topological constraints through a tightly regulated cycle of DNA double-strand cleavage and religation. Nearby DNA damage or chemotherapeutic agents such as etoposide block the DNA religation step, stabilizing TOP2-DNA cleavage complexes (TOP2ccs) at DNA double-strand breaks (DSBs). The SUMO E3 ligase ZATT (ZNF451) has recently emerged as a key effector of TOP2cc repair, but its mechanism of action remains poorly understood. Here, we show that ZATT is sufficient to resolve TOP2ccs independently of TDP2, TOP2 proteolysis, and canonical DSB repair pathways. Using Xenopus egg extracts and biochemical reconstitution, we find that ZATT salvages trapped TOP2 by promoting TOP2 release from its stalled cleavage complex. Structural modeling and targeted mutagenesis in Xenopus egg extracts and human cells identify a highly conserved hydrophobic pocket in the tower domain of TOP2 where the ZATT coiled-coil "hooks on" to promote TOP2cc resolution. Our findings reveal a new strategy to resolve TOP2ccs that bypasses the exposure of dangerous DNA breaks.

Radiation and chemotherapy rely on an intact DNA damage response (DDR) to halt cell-cycle progression and eliminate damaged cells, yet many tumors evade these outcomes and develop resistance. Adenoviruses remodel host signaling networks in ways that mirror tumor evolution, providing a powerful system to dissect how DDR pathways are subverted. Here, we identify two scenarios in which the central DDR kinases ATM and ATR are reprogrammed from enforcing CHK1/CHK2-dependent checkpoint arrest to activating a NEMO-NF-{kappa}B survival pathway. This rewiring induces transcriptional programs associated with stress tolerance, anti-apoptotic signaling, and chemoresistance, and promotes the accumulation of cells with abnormal DNA content. These findings reveal a previously unrecognized mode of DDR plasticity that generates a pro-survival state reminiscent of early tumor evolution and suggest how ATM- and ATR-dependent pathways can be co-opted to promote therapeutic resistance.

Faithful chromosome segregation during meiosis I requires tight control of interhomolog recombination. In budding yeast, the meiotic chromosome axis, built on Rec8-containing cohesin together with Red1 and Hop1, acts as a central platform regulating meiotic recombination from programmed DNA double-strand break (DSB) formation to checkpoint signaling and chromosome segregation, yet how cohesin recruits axis proteins remains unclear. Here, we identified a conserved cohesin-interacting motif (CIM) in Red1 that directly binds Rec8. AlphaFold3 modeling predicted that Red1-CIM forms a short -helix that docks into a conserved hydrophobic pocket within the Rec8 C-terminal winged-helix domain, which we confirmed biochemically. Disruption of the Red1-CIM preferentially impaired Red1 recruitment to Rec8-dependent chromosomal regions, while relative enrichment in Rec8-independent domains was preserved, leading to reduced DSB formation in Rec8-dependent domains. The Red1-CIM mutation also reduced crossover formation, increased chromosome missegregation, and reduced spore viability. Notably, this spore lethality exceeded that predicted by the reduction in DSB formation. Consistently, red1-CIM mutants failed to activate the meiotic checkpoint kinase Mek1. Finally, we provide evolutionary, structural, and biochemical evidence that this Red1-Rec8 interaction is conserved across fungi and plants. Together, these findings define a direct molecular bridge linking cohesin to chromosome-axis organization, spatial DSB regulation, and checkpoint signaling during meiosis.

Resolution of topological stress is crucial for genome integrity. Vertebrate topoisomerase II (TOP2) resolves catenanes to relieve topological stress and unlink daughter molecules during DNA replication. Topoisomerase III (TOP3) can also resolve catenanes, but its direct role during DNA replication, substrate specificity, and relevant binding partners remain unclear. Here we show that TOP3 becomes crucial when TOP2 function is compromised. We find that in Xenopus egg extracts, TOP3 promotes replication fork progression and daughter strand unlinking specifically during replication termination. TOP3 can carry out this role independently of its binding partners RMI1-RMI2 and the BLM helicase by acting on lagging-strand single-stranded DNA (ssDNA). Strikingly, elevated lagging-strand ssDNA drives formation of intramolecular ssDNA intertwines, which are ordinarily resolved by TOP3. Thus, TOP3 resolves intermolecular linkages to promote fork progression during termination and resolves intramolecular linkages that arise when high levels of ssDNA are present during DNA replication.

RNA dysregulation is a recognized contributor to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), the most common motor neuron (MN) disease. However, the molecular mechanisms linking defects in RNA metabolism to selective neuronal vulnerability remain poorly understood. Alterations in the cellular levels of R-loops -structures forming by reannealing of the nascent RNA with the template DNA during transcription- have been observed in neurodegeneration, but it is unclear how perturbations in R-loop homeostasis contribute to neuronal dysfunction. Here we investigate the molecular basis of a juvenile form of ALS dubbed ALS4 that is caused by mutations in the helicase SETX, which plays important roles in the resolution of R-loops and transcription termination. Using isogenic human induced pluripotent stem cell-derived MNs, we show that ALS4-associated SETX mutations induce progressive axonal defects and widespread transcriptomic alterations, including reduced expression or altered splicing of transcripts critical for neuronal function. ALS4 MNs exhibit a transcriptional signature marked by cellular stress, aberrant cell cycle re-entry, and compromised neuronal identity that is partially shared by other forms of ALS. Mechanistically, these defects are partly driven by downstream aberrant activation of the TGF-{beta} signaling pathway, whose pharmacological inhibition ameliorates axonal defects. Finally, our analyses support a link between mutant SETX ectopic activity at R-loops and the observed alterations in RNA expression and splicing, providing new insights into how RNA dysregulation can drive neuronal dysfunction Altogether, our work reveals how perturbations at the interface of transcription and R-loop metabolism can reshape neuronal identity and drive disease. TeaserDeregulation of TGF-{beta} signaling drives axonal defects and compromised motor neuron identity in senataxin-mediated ALS

Translation fidelity is generally viewed as a constitutive process that deteriorates under stress and aging. Here we show that the fidelity of amino acid incorporation is instead dynamically regulated by the circadian clock. In Neurospora crassa, methionine (Met) misincorporation into proteins exhibits robust daily rhythms, peaking at night coincident with elevated reactive oxygen species (ROS). Rhythmic Met misincorporation requires the circadian clock, the ERK-family MAPK MAK1, and MAK1-dependent phosphorylation of methionyl-tRNA synthetase (MetRS), linking circadian signaling to regulated mistranslation associated with oxidative stress resistance. Preventing MetRS phosphorylation abolishes rhythmic Met misincorporation, impairs growth, and increases sensitivity to oxidative stress, whereas a phosphomimetic MetRS mutant enhances oxidative stress survival. Proteome-wide analyses identified thousands of Met misincorporation events, including a rhythmic subset that oscillates independently of corresponding protein abundance, suggesting that mistranslation dynamically remodels proteome composition across the day. Together, these findings establish translation fidelity as a regulated circadian output and support a model in which the circadian clock temporally regulates mistranslation to enhance oxidative stress resilience. Significance StatementBiological clocks regulate translation termination fidelity, but whether they also control the accuracy of amino acid incorporation during protein synthesis was unknown. We show that the circadian clock drives rhythmic methionine misincorporation into proteins through ERK-family MAPK signaling and phosphorylation of methionyl-tRNA synthetase. Methionine misincorporation peaks during periods of elevated oxidative stress, and disrupting this regulation compromises oxidative stress survival, whereas constitutive activation enhances resistance. Together with previous work on translation termination fidelity, these findings reveal that biological clocks regulate multiple layers of translation fidelity and identify adaptive mistranslation as a mechanism that promotes cellular resilience.

Poly (ADP-ribose) Polymerase 1 (PARP1) is an abundant nuclear enzyme that dynamically engages chromatin in diverse cellular scenarios. In the context of DNA repair, PARP1 becomes enzymatically activated and subsequently attaches ADP-ribose units onto various proteins, including histones, to signal and coordinate the DNA damage response. In the absence of DNA damage, PARP1 modulates chromatin structure by directly binding to nucleosomes, however, the molecular basis of this interaction is unknown. Here, we define a distinct, enzymatically inactive mode of PARP1 chromatin binding, in which the Zn1, Zn2, Zn3, and BRCT domains cooperatively bind nucleosomal linker DNA and drive compaction of undamaged chromatin. This binding mode does not trigger catalytic activation and therefore is insensitive to PARP inhibitors (PARPi). Together, our results support a model in which PARP1 associates with the genome in an inactive state to compact chromatin and to surveil for DNA lesions. SummaryPARP1 engages undamaged chromatin in a distinct binding mode that results in chromatin compaction but does not lead to enzymatic activation.

Although transcription and translation are spatially separated in eukaryotic cells, gene expression requires coordination between the nucleus and cytoplasm. Whether translation rapidly influences nuclear transcription remains unclear. Here we show that ongoing translation sustains productive RNA polymerase II (Pol II) elongation in mouse embryonic stem cells. Translation inhibition reduces nascent transcription within 15 minutes, impairs Pol II pause release, and preferentially represses long genes before substantial loss of chromatin-associated Pol II. Unexpectedly, despite reduced transcription, nuclear RNA transiently accumulates owing impaired in RNA turnover. Mechanistically, translational arrest redistributes RNA-processing and RNA-surveillance factors from the nucleus to stalled ribosome-associated complexes in the cytoplasm, reducing their nuclear availability. Acute depletion of RNA-surveillance components phenocopies elongation defects caused by translational inhibition, particularly in long genes. Together, our findings identify ongoing translation as a regulator of nuclear RNA surveillance capacity and reveal a feedback mechanism sustaining productive Pol II elongation and nuclear RNA homeostasis.

The family of human G-patch proteins comprises more than 20 members, each characterized by a glycine-rich G-patch implicated in mediating interactions with RNA helicases. Here, we systematically identify the cognate RNA helicase of each G-patch protein, highlighting the association of DHX15 with a network of 20 G-patch cofactors. DHX35 and GPATCH1 represent a unique G-patch protein-RNA helicase pair, and we uncover a regulatory circuit between these partners. Comprehensive in vitro analyses of ATPase activity and RNA binding identify distinguishing features of DHX15- and non-DHX15-associated G-patch proteins, and demonstrate the roles of most G-patch proteins as bona fide stimulatory cofactors of DHX15. RNA interactome analyses of each G-patch protein and complementary transcriptome-wide alternative splicing analyses in cells lacking a G-patch protein reveal distinct modes of regulation of mRNA maturation by different G-patch proteins. For example, ZGPAT affects splicing indirectly through its requirement for efficient 2'-O-methylation of snRNAs, GPATCH8 exemplifies DHX15-associated alternative splicing modulation, whereas SUGP2 suppresses splicing in an RNA helicase-independent manner via direct binding to pre-mRNA introns.

The protein-coding sequence has long been considered the primary determinant of protein function. Alternative splicing within 3'UTRs (AS-3'UTRs) generates multiple transcript isoforms from a single gene, yet their roles in protein function and disease relevance remain largely unexplored. Through systematic transcriptome-wide identification of cancer-associated AS-3'UTRs, we uncover that AS-3'UTRs of {beta}-catenin mRNA direct distinct subcellular localization of {beta}-catenin mRNA isoforms. Specifically, an aberrantly spliced 3'UTR isoform promotes cytoplasmic mRNA condensate formation through FUS binding to a cancer-associated alternative exon (Exon 16A). Because {beta}-catenin function is exquisitely dependent on its subcellular distribution between adherens junctions and the nucleus, this aberrant 3'UTR isoform reprograms {beta}-catenin localization. By sequestering {beta}-catenin in the cytoplasm, the aberrant 3'UTR isoform prevents its incorporation into E-cadherin-based adherens junctions, thereby inducing epithelial-mesenchymal transition (EMT)-associated transcriptional programs. Notably, the expression signature of the aberrant 3'UTR isoform robustly correlates with poor clinical outcomes in colorectal cancer patients. Together, our findings reveal that AS-3'UTRs operate as a previously unrecognized post-transcriptional regulatory mechanism through which the untranslated region of mRNA, without altering a single amino acid, reprograms protein subcellular fate to drive oncogenic phenotypes.