The lethality of pancreatic ductal adenocarcinoma (PDAC) is driven, in part, by cellular plasticity that facilitates dedifferentiation and dissemination. Although transcriptional programs underlying these processes are well characterized, the contribution of translational control to PDAC cell-state regulation in vivo needs to be further understood to develop strategies to restrain malignant plasticity. Using a genome-wide CRISPR/Cas9 screen in immunocompetent hosts, we identified the noncanonical initiation factor eIF4G2 (DAP5/NAT1) as a translational checkpoint that restrains PDAC progression. Loss of eIF4G2 accelerated tumor growth, promoted poorly differentiated, basal-like histology, and triggered widespread metastasis. Ribosome profiling revealed that eIF4G2 supports the translation of a discrete cohort of mRNAs with long, guanine-cytosine (GC)-rich, structured 5' untranslated regions, including tumor suppressors such as Pten and transcriptional regulators such as Crebbp. Accordingly, loss of eIF4G2 was accompanied by secondary transcriptional enrichment of migration and wound-healing programs and induction of basal-like markers. In human PDAC, eIF4G2 expression was reduced in poorly differentiated lesions, and functional eIF4G2 perturbation in patient-derived PDAC cells increased clonogenic growth, whereas enforced eIF4G2 expression suppressed colony formation. Computational inference from human PDAC datasets revealed that reduced eIF4G2 activity correlated with increased metastasis, enhanced basal-like features, and poorer patient survival. Together, these findings establish noncanonical translation initiation as a determinant of PDAC cell-state control and identify eIF4G2 as a barrier to malignant plasticity and metastatic dissemination. SIGNIFICANCE:The translational checkpoint regulator eIF4G2 preserves epithelial identity and suppresses metastasis in pancreatic cancer, revealing selective translation as a determinant of subtype, prognosis, and therapeutic stratification.

During the switch from progenitor to differentiated cell, cellular physiology must change to accommodate increased translation and trafficking of membrane-bound proteins. We identify RNA-binding and E3 ubiquitin ligase Mex3a as a key driver of proper neuronal differentiation by regulating mRNA translation and trafficking of cell surface proteins in the context of Unfolded Protein Response (UPR) signaling. Loss of Mex3a in immature olfactory sensory neurons (OSNs) leads to defects in cilia structure, cell surface protein expression, and planar cell polarity in mature OSNs. Proteomics reveal a Mex3a-dependent decrease in proteins related to vesicle transport, lipid metabolism, and ribosome biogenesis. We identify RNA and ubiquitin targets of Mex3a and provide evidence that Mex3a can confer K27 ubiquitin linkage on substrates. Finally, modulating cellular levels of Mex3a changes the recruitment of translation factors Serbp1 and p-eEF2 to ribosomes with possible effects on translation. Our data reveal how a stemness factor regulates development post-transcriptionally and post-translationally to ensure robust differentiation.

Pancreatic ductal adenocarcinoma (PDA) is among the most lethal cancers, driven by cellular plasticity that fuels therapeutic resistance and early dissemination. The contribution of translational control to this plasticity remains poorly understood. Through an in vivo CRISPR/Cas9 screen, we identify the non-canonical initiation factor eIF4G2 (DAP5/NAT1) as a translational checkpoint restraining PDA progression. Loss of eIF4G2 accelerated tumor growth, induced poorly differentiated, basal-like histology, and triggered widespread metastasis. Ribosome profiling revealed that eIF4G2 loss does not alter bulk protein synthesis but instead impairs translation of a selective regulon, including tumor suppressors such as PTEN and CREBBP. Functional studies confirmed that PTEN loss was sufficient to drive dedifferentiation but insufficient to promote metastasis, implicating the broader eIF4G2-dependent program, including translational control of transcriptional regulators like CREBBP, in limiting dissemination. Consistently, eIF4G2-deficient tumors exhibited transcriptomic enrichment of programs related to migration and wound healing. Computational inference from human PDA datasets revealed reduced eIF4G2 activity in metastases, aligning with basal-like features and predicting poorer survival. These results support a model in which eIF4G2 maintains epithelial identity and restrains metastatic potential, highlighting selective translation as a determinant of PDA subtype and clinical outcome.

Post-transcriptional regulation of gene expression is orchestrated by RNA-binding proteins (RBPs), which regulate key aspects of the RNA life cycle including splicing, localization, translation, and decay. Although RBPs have been initially considered as isolated regulators, it is becoming clear that RNA molecules are commonly bound by several RBPs whose coordination directs their fate. These combinatorial interactions produce complex, context-dependent post-transcriptional regulatory networks (PTRNs) whose outcomes are difficult to predict. RBPs may also switch function depending on cell state, subcellular localization, or post-translational modification, adding further complexity to RNA regulation. This review focuses on recent technological advances expanding our ability to map and interpret PTRNs. Multiplexed methods allow profiling of the RNA-binding patterns of several RBPs in parallel, whereas deeper interaction proteomics studies reveal protein-protein connections and changes in distinct biological settings. Complementary RNA-targeting pulldown and single-molecule imaging strategies enable real-time and single-cell-resolution visualization of ribonucleoprotein assembly and dynamics, while functional high-throughput screens allow assignment of first order functions for these RBPs. Overall, these approaches set the stage for comprehensive decoding of the spatiotemporal structure of PTRNs and reveal how RBP interactions coordinate sets of RNAs to collectively regulate them in response to physiological demands. In addition to describing these systems-level approaches, we outline key future analytical and experimental innovations that could transform our understanding of RBP function. We believe that a systems-level understanding of RBPs as dynamic, integrated components of multiscale regulatory regimes is required to fully understand the complexity of gene expression control and its disruption in disease.

Metabolic syndrome and excessive alcohol consumption (MetALD) result in liver injury and fibrosis, which are driven by increased collagen production by activated hepatic stellate cells (HSCs). Our previous studies demonstrated that LARP6, an RNA-binding protein, may facilitate collagen production. However, the expression and function of LARP6 as a regulator of fibrosis development in a disease-relevant model remain poorly understood. We demonstrated that LARP6 was upregulated in human activated HSCs in metabolic dysfunction-associated steatohepatitis (MASH) and MetALD. By using single-nucleus RNA-seq and assay for transposase-accessible chromatin sequencing, we showed that JUNB upregulated LARP6 expression in activated HSCs. Moreover, LARP6 knockdown in human HSCs suppressed fibrogenic gene expression. By integrating enhanced crosslinking and IP analysis and ribosome profiling in HSCs, we showed that LARP6 interacted with mature mRNAs comprising more than 300 genes, including RNA structural elements within COL1A1, COL1A2, and COL3A1 to regulate mRNA expression and translation. IP-mass spectrometry analysis demonstrated LARP6 protein-protein interactions with mRNA translation components and the actin cytoskeleton. Furthermore, Dicer substrate siRNA-based HSC-specific gene knockdown or pharmacological inhibition of LARP6 attenuated fibrosis development in human MASH and MetALD liver spheroids. Our results suggest LARP6 plays a key role in fibrogenic gene regulation and that targeting LARP6 in human HSCs may represent a therapeutic approach for liver fibrosis.

Translationally downregulated transcripts with matching protein decreases in sgEif4g2 vs sgRosa PDAC cells.

RNA-binding proteins (RBPs) are key regulators of gene expression that shape cellular function in health and disease. However, the roles of RBPs in immune cells within the central nervous system (CNS) remain poorly understood. Here, we identify ARID5A as an RBP highly expressed in microglia and uncover its RNA-mediated regulatory functions using integrated multi-omics analyses of its RNA, DNA, and protein interactions. ARID5A regulates the splicing and translation of its RNA targets, many of which are integral to lysosomal, immune, and iron metabolism pathways. We confirm the functional relevance of this ARID5A-dependent RNA regulatory network by demonstrating that ARID5A modulates lysosomal activity, cytokine secretion, iron accumulation, and ferroptosis in iPSC-derived microglia. We further demonstrate that knockdown of microglial ARID5A reduces neuronal ferroptosis in co-cultures, underscoring the interconnected nature of these pathways. Moreover, in microglia harboring the TREM2-T66M mutation, ARID5A depletion restores dysregulated lysosomal and metabolic functions. Our results highlight the importance of protein-RNA interactions in regulating microglial cell biology. RNA-binding proteins shape gene expression, but their roles in brain immune cells are unclear. Here, the authors show that ARID5A regulates RNA processing in microglia, linking immune and lysosomal pathways to iron-driven death in neurodegeneration.

List of differentially expressed transcripts in sgEif4g2 PDAC cells compared to sgRosa control PDAC cells.

Quality assessment and validation of the genome-wide CRISPR screen.

Recent studies have revealed many alternative exons differentially spliced across diverse neuron types in the mammalian brain, but their links to neuronal physiology remain unclear. Here we characterize a deeply conserved microexon E35a in Ank3 encoding ankyrin-G (AnkG), a multifaceted adaptor protein best known as a master organizer of the axon initial segment (AIS) and as a leading genetic risk factor for bipolar disorder. E35a is predominantly skipped in cortical glutamatergic neurons but included in cortical GABAergic neurons and cerebellar neurons, which is dictated by multiple neuronal splicing factors. In E35a-deletion mice we generated, interneurons show increased excitability and somatic Ca2+ activity, without disruption in AIS. Biochemical analyses suggest that E35a inclusion facilitates AnkG interaction with a protein complex involving inositol trisphosphate receptors (InsP3Rs) important for intracellular Ca2+ signaling. Alternative splicing therefore allows AnkG to modulate neuron type-specific excitability in addition to its ubiquitous pan-neuronal role in organizing the AIS.

RNA granules are essential regulators of post-transcriptional gene expression, enabling mRNA transport, localization, and local translation in neurons. The localized transcriptome is diverse; however, how different mRNAs are organized into granules for efficient localization and translation remains unknown. Here, we combine real-time endogenous single RNA imaging with protein and RNA proximity labeling to investigate two distinct endogenous neuronal mRNA granule populations, Actb and Arc , in stimulated primary hippocampal neurons. Using orthogonal RNA labeling systems in a dual knock-in mouse model, we show that Actb and Arc mRNAs are packaged into spatially segregated granules with distinct trafficking dynamics, localization kinetics, and responses to synaptic stimulation. Actb granules displayed rapid and sustained localization, whereas Arc granules showed delayed, transient recruitment, consistent with their respective roles in structural and activity-dependent plasticity. Proximity labeling reveals that these granules are distinct in their mRNA composition, despite sharing core RNA-binding proteins, suggesting that shared cis-regulatory elements within mRNA 3'UTR regions drive selective co-packaging of mRNAs into unique granules. Together, these findings demonstrate that neuronal mRNAs are differentially sorted into molecularly and functionally distinct granules, providing a framework for understanding how precise spatio-temporal control of mRNA localization and translation is achieved across complex neuronal arbors.

Alternative polyadenylation (APA) generates transcript isoforms with distinct 3' ends, yet the repertoire of its protein regulators remains poorly defined. Using a large-scale tethered function screen, we profiled 879 human RNA-binding proteins (RBPs) and identified 63 high-confidence activators of poly(A) site (PAS) selection, most of which were not previously linked to APA. We validated these factors by knockdown PAS-seq, RNA sequencing (RNA-seq), and enhanced cross-linking and immunoprecipitation (eCLIP) analyses and developed a fine-tuned protein language model that predicts PAS selection activators and their key functional domains. We then mechanistically dissected two unexpected hits: GRB2, a signaling adaptor protein, and RNPS1, a peripheral component of the exon junction complex (EJC). Both regulate APA, at least in part, through direct interactions with distinct subunits of the cleavage and polyadenylation (CPA) machinery. Together, our study provides a comprehensive resource of APA-regulating RBPs and uncovers unexpected roles of signaling and EJC factors in APA regulation.

Stress responses, including the unfolded protein response (UPR), are commonly studied via induction with harsh exogenous stressors, leaving endogenous functions of these pathways less well understood. We found that the endogenous UPR that precedes meiosis in budding yeast is required for gamete production but diverges dramatically from previously defined UPR outputs, with only a few characterized UPR targets induced, and mildly. The role of this UPR can be replaced by increasing ER chaperones, reducing bulk translation, or impairing the machinery for protein translocation into the ER. ER integrity appears compromised in pre-meiotic cells lacking the UPR, as foci of reticulon proteins are seen and correlate strongly with an inability of cells to enter meiosis. These findings indicate that physiological UPR activation supports proteostasis and normal ER structure, preparing cells for meiotic entry by reducing the load of proteins that enter the ER. Overall, our study reveals surprising features of a physiological UPR induction that enables a cell fate decision.

Defense-associated reverse transcriptase (DRT) systems mediate antiviral immunity through distinct modes of cDNA synthesis: class 1 DRTs catalyze untemplated synthesis, whereas class 2 DRTs polymerize non-coding RNA-templated products. However, how these distinct modes drive defense remains unclear. Here, we report that DRT3 immunity arises when class 1 and class 2 RT activities cooperate to produce self-complementary double-stranded DNA (dsDNA). DRT3a uses a 5'-ACACAC-3' RNA template to synthesize poly-(dTdG) repeats, whereas DRT3b synthesizes poly-(dCdA) repeats without any nucleic acid template. Cryo-electron microscopy reveals that DRT3b forms a hexamer and uses active-site-adjacent residues as deoxyadenosine and deoxycytidine gates to enforce alternating nucleotide addition, representing a unique example of amino-acid-templated DNA polymerization. DRT3 is toxic in cells lacking RecBCD, implicating host recombination machinery in limiting dsDNA accumulation, and the phage-encoded RecBCD inhibitor Gam triggers DRT3-mediated abortive infection. These findings reveal how two polymerases with distinct templating strategies generate complementary DNA for antiviral defense.

Protein tyrosine phosphorylation is critical for cellular function, and aberrant phosphorylation is tied to a wide range of human diseases. Identifying the substrates of protein tyrosine phosphatases, the enzymes that erase this modification, is critical to understanding human biology and disease states. The state-of-the-art method for tyrosine phosphatase substrate identification requires the use of mutations that modestly increase the lifetime of enzyme-substrate complexes by kill catalytic activity. While these "substrate-trapping" mutants are useful tools, they work best for high-affinity or abundant substrates that remain phosphatase-bound through cell lysis and enrichment. Here, we use site-specific photo-crosslinking to covalently capture the substrates of tyrosine phosphatases in situ . We identify eight different positions around the active site of the phosphatase PTP1B where photo-crosslinker amino acids can be incorporated via amber codon suppression without dramatically disrupting catalytic activity. We then conduct photo-crosslinking experiments in mammalian cells and identify crosslinked proteins by mass spectrometry proteomics, revealing that our approach can capture known PTP1B interactors and substrates. We then show that PTP1B photo-crosslinking in situ is sensitive to enzyme localization and identify new PTP1B substrates that regulate contacts between the endoplasmic reticulum and plasma membrane. We also demonstrate that photo-crosslinking can capture signal-dependent interactions. For example, we observe PTP1B crosslinking to the epidermal growth factor (EGF) receptor, a known substrate, in an EGF-dependent manner, and we identify other potential EGF-dependent substrates. Overall, our approach reveals previously unknown roles of PTP1B in signaling systems and could be readily extended to other tyrosine phosphatases in the same family.

Diverse epigenetic regulatory mechanisms ensure and regulate cellular diversity. Among others, the histone 3 lysine 9 me3 (H3K9me3) post translational modification participates in silencing lineage-inappropriate genes. H3K9me3 restricts access of transcription factors and other regulatory proteins to cell-fate controlled genes. In mice, olfactory sensory neurons (OSN) express one olfactory receptor (OR) gene out of 2,600 possibilities. This monoallelic and stochastic OR choice happens as OSNs differentiate and undergo dramatic changes in nuclear architecture. OR genes from different chromosomes converge into specialized nuclear bodies and chromatin compartments as H3K9me3 and chromatin binding proteins including heterochromatin protein 1 (HP1) are incorporated. In this work, we have uncovered an unexpected role for HP1β in OR choice and neuronal identity that cannot be rescued by HP1α in vivo. With the use of a conditional knock-in mouse model that replaces HP1β for HP1α, we observe changes in H3K9me3 levels, DNA accessibility, and Hi-C contacts over OR gene clusters. These changes alter the expression patterns that partition the mouse olfactory epithelium into five OR expression zones, which results in a reduced OR repertoire leading to a loss of olfactory sensory neuron diversity. We propose that HP1β modulates the competition of OR-promoters for enhancers to promote receptor diversity, by establishing repression gradients in a zonal fashion.

Human embryonic stem cells (hESCs) provide a powerful in vitro model to study lineage specification and the regulatory programs underlying early human development. Here, we present a high-resolution, temporal multi-omics dataset tracking mRNA, translation, and protein expression dynamics during hESC differentiation into definitive endoderm and subsequent polyhormonal (PH) cells, a key pancreatic lineage. RNA-seq, ribosome profiling, and quantitative mass spectrometry-based proteomics were performed on matched samples collected at ten time points in biological duplicates, allowing detailed characterization of transcriptional, translational, and protein abundance changes over the differentiation timeline. The dataset exhibits high technical quality, with strong reproducibility between replicates and rigorous quality control metrics across all omics platforms. This extensive dataset provides critical insights into the complex regulatory mechanisms driving polyhormonal cell differentiation and serves as a valuable resource for the research community, enabling deeper exploration of mammalian development, endodermal lineage specification, and gene regulation.

Ribosome profiling to characterize eIF4G2-dependent translatome.

Faithful chromosome segregation is essential for producing viable gametes during meiosis, a specialized type of cell division relative to mitosis. Here we identify Gim3, a subunit of the ubiquitously expressed and conserved prefoldin complex, as a critical regulator of meiotic but not mitotic chromosome segregation in budding yeast. Loss of Gim3 causes profound defects in chromosome segregation and gamete viability through reduced tubulin protein levels, which are also associated with reduced spindle length. In mitosis, GIM3 deletion minimally affects spindle length and chromosome segregation, despite similarly reduced tubulin levels in both contexts, highlighting an intriguing difference between the sensitivity of meiotic and mitotic spindles to tubulin abundance. Beyond chromosome segregation defects, gim3 ∆ cells exhibit aberrant meiotic cellular remodeling, including defects in exclusion of age-associated protein aggregates from newly forming gametes. Independently induced meiotic chromosome missegregation similarly disturbs cellular remodeling, pointing to a fundamental coupling between these aspects of gamete production. Together, our findings identify Gim3 as a key factor required for maintaining meiotic chromosome segregation integrity, and reveal an exciting and previously unrecognized link between chromosome segregation and meiotic cellular remodeling.

Missense mutations in PTPN11, which encodes the protein tyrosine phosphatase SHP2, are common in several developmental disorders and cancers. While many mutations disrupt auto-inhibition and hyperactivate SHP2, several do not enhance catalytic activity. Both activating and non-activating mutations could potentially drive pathogenic signaling by altering SHP2 interactions or localization. We employed proximity-labeling proteomics to map the interaction networks of wild-type SHP2, ten clinically relevant mutants, and SHP2 bound to an inhibitor that stabilizes its auto-inhibited state. Our analyses reveal mutation- and inhibitor-dependent alterations in the SHP2 interactome, with several mutations also changing localization. Some mutants show increased mitochondrial localization and impact mitochondrial function. This study provides a resource for exploring SHP2 signaling and offers new insights into the molecular basis of SHP2-driven diseases. Furthermore, this work highlights the capacity for proximity-labeling proteomics to detect missense-mutation-dependent changes in protein interactions and localization.