Single-molecule approaches to study G-quadruplex, R-loop, and protein interactions.

Cap-independent translation mediated by the 5' UTR of porcine epidemic diarrhea virus with IRES-like activity.

The formation of membraneless organelles via liquid-liquid phase separation (LLPS) of proteins and RNAs has emerged as a central mechanism of cellular compartmentalization to finely regulate biological processes. DDX3X, a member of the DEAD-box RNA helicase family, is one of the global regulators of RNA-containing phase-separated organelles. While the importance of DDX3X in organelle formation is well-recognized, the molecular mechanisms underlying its RNA-driven LLPS remain poorly understood. In this study, we focused on the dynamic interactions between the N-terminal intrinsically disordered region (N-IDR) of DDX3X and G-quadruplex (GQ) RNA, which is a key regulator of physiological membraneless organelle assembly owing to its unique ability to promote LLPS. Using solution nuclear magnetic resonance spectroscopy, we identified hotspot regions for self-assembly within the N-IDR. These regions comprise charged stretches interspersed with key aromatic residues, whose interactions drive LLPS through a combination of electrostatic and π-interactions. Binding of GQ RNA effectively strengthens intermolecular interactions involving the arginine-rich segments of the N-IDR, providing molecular insights into its RNA-driven LLPS. We further discuss the functional implications of GQ-specific granule formation under stress conditions, highlighting the potential roles of DDX3X-GQ RNA interplay in cellular translational regulation.

YBX1 is a DNA- and RNA-binding protein with multifaced roles in transcriptional, post-transcriptional, and translational regulation. Consequently, YBX1 controls many aspects of cellular function, including proliferation, differentiation, and apoptosis. In recent years, YBX1 emerged as an important player in the cardiovascular system, whose dysregulation underlies many forms of heart disease. Intriguingly, while reducing YBX1 levels in the myocardium confer protection against pathological cardiac remodeling, YBX1 knockdown in the heart also induces cardiac hypertrophy and fibrosis, raising safety concerns about targeting YBX1 therapeutically. Nevertheless, prior YBX1 loss-of-function studies used RNA interference (RNAi), which is susceptible to off-target effects and likely affected multiple cardiac cell types. Therefore, 'clean' YBX1 cardiac-specific loss-of-function genetic models are required to delineate YBX1's precise role in the heart. To that end, we constructed both global and cardiomyocyte-specific Ybx1 knockout (KO) mouse models. While Ybx1 global KO mice died in utero and exhibited severe cardiac defects, including noncompaction and delayed septal development, Ybx1 cardiomyocytes-specific KO mice (Ybx1cmKO) did not exhibit obvious morphological anomaly or cardiac dysfunction, suggesting that the myocardial YBX1 is dispensable for heart development and function. Although RNA-seq analysis revealed the upregulation of a few fibrosis-related genes, they did not drive cardiac fibrosis in Ybx1cmKO hearts. Our study provides compelling evidence that deleting YBX1 specifically in CMs would not cause unwanted adverse effects. However, caution is required to ensure the YBX1 ablation is restricted to CMs as loss of YBX1 in other cell types may lead to cardiac defects.

Cell cycle progression requires cells to continually remodel their gene expression programs as they transition through distinct functional states. Although transcriptional and post-translational mechanisms have long dominated our understanding of this regulation, recent work additionally highlights the essential contribution of cell cycle-specific mRNA decay and translational control. Across G1, S, G2, and mitosis, cells dynamically modulate global and transcript-specific mRNA stability and translation to coordinate processes including DNA replication, growth, checkpoint signaling, and chromosome segregation. Mitosis presents a particularly striking challenge: Transcription is reduced, necessitating that cells rely on post-transcriptional mechanisms to sustain mitotic functions and preserve viability. In this review, we highlight how these coordinated layers of post-transcriptional regulation collectively contribute to cell cycle control.

Nucleotide sequence can be translated in three reading frames producing distinct protein products. Many examples of RNA translation in two reading frames (dual coding) have been identified so far. We report translation of mRNA transcripts derived from SRD5A1 locus in all three reading frames that result in the synthesis of long polypeptides. This occurs due to initiation at three nearby AUG codons occurring in all three reading frames. Only one of the three proteoforms contains the conserved catalytical domain of SRD5A1 produced either from the second or the third AUG codon depending on the transcript. Paradoxically, ribosome profiling data and expression reporters indicate that the most efficient translation would produce catalytically inactive polypeptide. While phylogenetic analysis suggests that the long triple decoding region is specific to primates, occurrence of nearby AUGs in all three reading frames is ancestral to placental mammals. This suggests that their evolutionary significance belongs to regulation of translation rather than biological role of their products. By analysing multiple publicly available ribosome profiling data and with gene expression assays carried out in different cellular environments, we show that relative expression of these proteoforms is mutually dependent and varies across environments supporting this conjecture. We show that a remarkable feature of triple decoding is its resistance to frameshift causing variants with apparent implications to clinical interpretation of genomic sequence variants. We argue for the importance of identification, characterisation and annotation of productive RNA translation irrespective of the presumed biological roles of its products.

La-related protein 1 (LARP1) is an RNA-binding protein and downstream effector of mTOR and CDK9 signaling that regulates translation of mRNAs containing a 5'-terminal oligopyrimidine motif. While elevated LARP1 expression has been linked to poor prognosis in acute myeloid leukemia (AML), its mechanistic role remains unclear. Using CRISPR/Cas9-mediated LARP1 knockout and multi-omics analyses, we investigated LARP1's role in AML. LARP1 loss impaired proliferation, clonogenicity, and tumor growth in xenografts, and enhanced sensitivity of AML cells to 5-azacytidine and cytarabine. Polysome profiling and RNA sequencing revealed that LARP1 modulates a distinct set of transcripts involved in mitochondrial function, amino acid metabolism, and cell cycle regulation, independently of mTOR and CDK9. Proteomics analysis uncovered additional effects of LARP1 loss on immune signaling, lysosomal pathways, and protein stability, including changes not evident at the RNA level. Metabolomic profiling showed reprogramming of arginine/creatine metabolism and depletion of pyrimidine biosynthesis intermediates. Cytidine deaminase, a known resistance factor, was downregulated across omics layers upon LARP1 loss. These findings define LARP1 as a key integrator of translational regulation and metabolic control in AML, supporting leukemic cell survival and promoting drug resistance. Targeting LARP1 may uncover vulnerabilities in leukemia cells, not addressed by current therapies.

TRMT6/61A-mediated tRNA m1A methylation promotes codon-dependent TAB2 translation and drives AML progression.

The posttranslational modifications (PTMs) glutamylation and glycylation are primarily associated with tubulins and microtubule regulation, while their broader functional scope is largely underexplored. It is known that both PTMs also occur on other proteins, yet only a few non-tubulin substrates have been identified so far. To gain a broad view of the potential roles of these two PTMs, we established a SILAC-based quantitative proteomics approach to identify substrates in an unbiased manner. Expressing glutamylating and glycylating enzymes in cell lines strongly increased the PTM levels, from which modified proteins were purified with PTM-specific antibodies and identified with quantitative proteomics. We identified more than 100 putative substrates for both PTMs, including proteins involved in nucleocytoplasmic shuttling, RNA and chromatin-binding and translation regulation, out of which we validated a representative subset. Our work provides a reliable resource for substrates of glutamylation and glycylation that opens the opportunity to functionally explore the roles of these understudied PTMs in a variety of cellular processes.

Disuse, like several other pathological conditions, has specific effects on various skeletal muscles; the mechanisms underlying these responses remain unclear. We aimed to compare the disuse-induced changes in the phenotype and proteome of the calf and thigh muscles, and to assess the extent to which these proteomic changes are regulated at the mRNA and other levels. Twelve healthy young males participated in a 3-week bed rest. Disuse resulted in a greater decrease in lean mass, aerobic performance, and changes in the proteome and transcriptome of the calf muscles (m. soleus) than the thigh muscles (m. vastus lateralis). A greater decrease in calf muscle mass was associated with a decrease in the expression/deactivation of translation regulators, but not with the expression of the main sarcomeric proteins. At the same time, a significant decrease in aerobic performance of the ankle plantar flexors occurred without changing the expression of oxidative enzymes - a marker of mitochondrial density. That decrease was associated with dysregulation of mitochondrial biogenesis. Most large-scale changes in the transcriptome did not translate into changes in the proteome, indicating post-transcription protein buffering. However, changes in the RNA levels were revealed to play a dominant role in regulating specific proteins, whereas for others, this factor played little or no role. In conclusion, our findings partially explain why calf muscles with a strong postural function are more sensitive to short-term disuse. This provides a foundation for developing targeted approaches to counteract the negative effects of disuse on different muscles. KEY POINTS: Disuse, like several other pathological conditions, has specific effects on various skeletal muscles. The mechanisms underlying these responses remain unclear. Three-week bed rest resulted in a greater decrease in lean mass, endurance, and more pronounced changes in the proteome and transcriptome of the calf muscles than the thigh muscles. A greater decrease in calf muscle mass was associated with suppression of translation regulators. Meanwhile, a decrease in endurance of the calf muscles was associated with mitochondrial dysregulation. Most large-scale changes in the transcriptome did not translate into changes in the proteome. However, changes in the RNA levels played a dominant role in regulating expression of specific proteins, such as mitochondrial biogenesis regulators, whereas for others (proteins of ribosome and exosome, etc.) this factor played little or no role. Our findings provide a foundation for developing targeted approaches to counteract the negative effects of disuse on different muscles.

Inosine pranobex (IP) is a long-used antiviral drug whose mechanism of action remains incompletely understood. However, molecular efficacy has been attributed mainly to immunomodulatory effects. There are data which suggest that the cellular activity of IP stems from its major constituent compound, inosine, known for its pleiotropic roles in purine metabolism, RNA modification, and translation regulation. We investigated whether IP acts as a stable complex or a mixture of its components and compared the biological effects of inosine itself and IP in vitro. The structural composition of IP was analyzed using compositional and microscopic methods. Cytotoxicity, antiviral activity against coxsackievirus B3 (CVB-3), and global DNA methylation changes were evaluated in A549 and HeLa cell lines using MTT, colony formation, plaque reduction, and post-labeling methods, respectively. We found that IP is physically heterogeneous and function as a mixture of components rather than a stable complex. In cell-based assays, inosine exhibited higher antiviral activity than IP, particularly under pre-treatment conditions, where it provided stronger protection against CVB-3-induced cytopathic effects. Neither compound showed significant cytotoxicity within the tested concentration ranges. Both inosine and IP influenced global DNA methylation levels, but inosine induced more pronounced and concentration-dependent changes. The superior antiviral and epigenetic activity of inosine compared with IP suggests that inosine is the main principal active component responsible for IP's biological effects. While IP's immunomodulatory functions were not evaluated here, our findings strongly suggest that inosine contributes substantially to its antiviral efficacy. Further studies, including in vivo models, are warranted to clarify the epigenetic mechanism underlying these observations.

Oocyte developmental competence relies on the coordinated progression of nuclear and cytoplasmic maturation, driven by the precise translational regulation of stored maternal mRNAs. Using an integrative transcriptomic and translatomic approach, we characterized the dynamic translational landscape of porcine oocytes during maturation. Through cross-species analysis with human and mouse data, we discovered conserved and species-specific translational programs, highlighting a greater translational resemblance between porcine and human oocytes. Comparative profiling further revealed aberrant maternal mRNA translational activation and degradation during in vitro maturation (IVM) vs. in vivo conditions, including defective translational activation of GPLD1 and HNRNPK, which were pinpointed as mechanisms compromising oocyte quality and embryonic development. To further dissect how translational regulation coordinates oocyte nuclear and cytoplasmic maturation, we employed a dbcAMP-induced cell cycle-synchronized model to identify gene sets with cell cycle-dependent and -independent translational activation. Characterization of these groups identified cytoplasmic polyadenylation element binding protein 1 (CPEB1) as a key orchestrator within the translational regulatory network, where it specifically activates the translation of cell cycle-dependent maternal factors through the cytoplasmic polyadenylation elements (CPEs) within 3'UTRs. Collectively, these findings elucidate key translational mechanisms during porcine oocyte maturation and offer a molecular basis for improving in vitro maturation and reproductive efficiency in livestock.

Sleep plays a critical role in animal physiology, primarily governed by the brain, and its disruption is prevalent in various brain disorders. Mettl5 is associated with intellectual disability (ID), which often includes sleep disturbances. However, the mechanism underlying these sleep disruptions in ID remains poorly understood. In this study, we investigated the sleep phenotypes resulting from Drosophila Mettl5 mutations. Rescue experiments revealed that Mettl5 functions predominantly within neurons and glia marked by Mettl5-Gal4 to regulate sleep. Previous work established that Mettl5 forms a complex with Trmt112 to influence rRNA methylation. Notably, a mutation in Trmt112 recapitulated these sleep disturbances, implicating translational regulation by the Mettl5/Trmt112 complex. Subsequent RNA-seq and Ribo-seq analyses of Mettl51bp mutants uncovered downstream effects, including altered expression of proteasome components and clock genes. Rescue experiments confirmed that the net increase in PERIOD protein underlies the sleep phenotype. This study illuminates the interplay between ribosome function, clock genes, and the proteasome in sleep regulation, highlighting the integrated roles of protein synthesis and degradation. These findings could potentially provide an example for in vivo study of rRNA methylation function, expand our understanding of protein homeostasis in sleep, and offer insights into the sleep phenotypes associated with ID.

Enhancing soluble sugar accumulation in banana (Musa acuminata) through 5-azacytidine-mediated reinforcement of starch degradation involving DNA methylation-dependent and independent pathways.

Spatiotemporal regulation of mRNA translation is central to gene expression. Over the past decade, translation has become directly observable in live cells at single-mRNA resolution by tagging nascent chains with tandem arrays of short epitope tags recognized by genetically encodable fluorescent intracellular antibodies (intrabodies). While this technology has revolutionized our understanding of translation regulation, the current toolbox of tagging systems remains limited. Here, we developed a novel and tight-binding intrabody against a short (11-amino acid) HIV protease epitope (named UTag). To ensure robust intracellular folding of the anti-UTag intrabody, we further engineered a cysteine-free variant that folds and functions independently of disulfide-bond formation, as validated by X-ray crystallography. The cysteine-free anti-UTag intrabody retains high binding affinity comparable to the parental intrabody while exhibiting significantly improved thermostability (∼80 °C). Importantly, the cysteine-free UTag system enables real-time tracking of single-mRNA translation in live cells with performance on par with the parental UTag system as well as the established SunTag and ALFA-tag. Collectively, these results demonstrate that the newly developed UTag system expands the toolbox for live-cell translation tracking and provides complementary tools for multiplexed applications.

Neuroinflammation, particularly that involving reactive microglia, the brain's resident immune cells, is implicated in the pathogenesis of major neurodegenerative diseases (NDs). Multiple studies have reported changes in ribosomal protein (RP) expression during neurodegeneration, but the significance of these changes remains unclear. Ribosomes are evolutionarily conserved protein-synthesizing machines, and although commonly viewed as invariant, accumulating evidence suggests functional ribosome specialization through variation in their protein composition. Among RPs, S24, encoded by RPS24 in humans and Rps24 in mice, is unique as its transcripts undergo alternative splicing to produce protein variants with different C-terminal sequences that are differentially expressed across tissues and cell types. Understanding heterogeneous RP expression patterns across brain regions and cell types could reveal mechanisms underlying selective vulnerability in NDs and provide new biomarkers for neuroinflammatory responses. To identify RP expression patterns across brain regions in neurons, astrocytes, and microglia we analyzed cell type-specific translating mRNAs from mice. To investigate Rps24 isoform-specific expression, we performed cell type-resolved transcript analysis and developed antibodies specific for the S24-PKE protein variant encoded by mRNA isoform Rps24c. We examined Rps24c/S24-PKE expression in brains from mouse models of aging and neurodegeneration, as well as in human postmortem tissue from patients with Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). This work revealed distinct RP expression patterns across brain regions and between neurons, astrocytes, and microglia, including neuron-enriched RPs Rpl13a and Rps10. Analysis of RP paralogs revealed complex expression relationships with their canonical counterparts, suggesting regulated mechanisms for generating heterogeneous ribosomes. Across brain regions and cell types, Rplp0 and Rpl13a, commonly used normalization references, showed heterogeneous expression, raising important methodological considerations for gene expression studies. Rps24 isoforms exhibited striking cell type-specific expression patterns. Rps24c was predominantly expressed in microglia and was increased by neuroinflammation caused by aging, neurodegeneration, or inflammatory chemicals. Using S24-PKE-specific antibodies, we verified increased expression of this protein variant in brains with AD, PD, and HD, and in relevant mouse models. These findings establish heterogeneous RP expression as a feature of brain cell types which may enable cell type-specific translation regulation via specialized ribosomes. This work also identifies Rps24c/S24-PKE as a potential novel marker for neuroinflammation and neurodegeneration and provides new tools for monitoring these responses.

Dysregulated selective translation in cancer: The pivotal functions of RNA-binding proteins and emerging therapeutic avenues.

Scalable genetic circuits are essential for implementing complex functions in living cells. Toward this goal, RNA regulators can provide a much-needed parts library with added benefits of low metabolic load, design flexibility, and logic capacity. However, despite the great potential of synthetic RNA circuits, constructing such circuits with wide dynamic ranges and multiplexed regulatory cascades remains a challenge. To address this, we introduce RATEX (Ribosome-Assisted Transcriptional EXpression controller) by integrating a translation-to-transcription converter with synthetic RNA regulators, enabling a compact and scalable RNA-programmed circuit architecture. The RATEX platform repurposes a large library of well-characterized translation regulators with up to 1,492-fold gene regulation, while leveraging natural ribosome-mediated sensing of diverse environmental inputs, such as metabolites. We demonstrated multi-input logic processing with up to a 6-input OR logic gate for RNA inputs and hybrid 3-input logic gates to sense diverse metabolite and small-molecule inputs alongside RNA signals. Signal amplification with multiplexed combinatorial control of RNA outputs was achieved through multiplexed signaling cascades. Finally, the RNA- and metabolite-sensing 3-input AND gates were used to control cellular morphology and intracellular spatial organization. Together, the RATEX platform, with its scalable and modular architecture, offers a broad potential design space for synthetic biology and biotechnology.

During final cell division, the cleaved midbody is either released or asymmetrically retained as a midbody remnant (MBR). MBRs play critical roles in cell communication, signal transduction, and translation regulation, influencing cellular fate. Here, we synthesize their functions as RNA-processing granules, polarity regulators, and signaling platforms, emphasizing their role in primary cilia formation. In polarized epithelial cells, the MBR moves along the apical surface to the centrosome, delivering membrane components to permit ciliogenesis. In ductal carcinoma cells, MBR-localized Shc1-binding protein (SHCBP1) interacts with TBC1 domain family member 30 (TBC1D30 to antagonize Ras-related protein Rab-8 (Rab8) GTPase activity, blocking MBR-centrosome proximity and silencing ciliogenesis. Beyond ciliary regulation, MBRs integrate Wnt, PDGF, TGF-β, and genomic stability networks, acting as dynamic signaling hubs during cancer development. Regarding therapeutic strategies targeting MBRs, High SHCBP1 expression correlates with ciliary loss and poor prognosis in breast, pancreatic, and cholangiocarcinoma. Targeting the SHCBP1/Rab8 axis to restore ciliogenesis by reestablishing MBR-centrosome proximity offers a potential therapeutic strategy. In addition, secreted MBRs are enriched in signaling components and transcripts, serving as intercellular carriers of oncogenic cargoes and promising liquid biopsy biomarkers. In summary, by tracing MBRs from their postmitotic origin to their pathogenic roles, we highlight vulnerabilities within MBR regulatory networks and provide novel insights for cancer therapeutics.

Prostate cancer (PC) is one of the most common malignancies in men, and the emergence of androgen receptor-low/negative castration-resistant PC (ARL/- CRPC) following androgen receptor signaling inhibitor (ARSI) therapy remains a critical clinical challenge. The RNA-binding protein DEAD-box helicase 3 X-linked (DDX3X) has been implicated in the translational regulation of androgen receptor (AR) mRNA; however, the underlying binding mechanisms are not well defined. Here, we show that DDX3X colocalizes with AR mRNA in ARL/- CRPC cells and selectively recognizes non-canonical RNA G-quadruplex (rG4) motifs within the sequence of AR mRNA. RNA immunoprecipitation sequencing (RIP-seq) revealed enrichment of DDX3X-AR mRNA interactions in ARL/- CRPC cells. Fluorescence imaging confirmed the colocalization of DDX3X and AR mRNA within cytoplasmic granules, and biochemical assays confirmed the ability of selected AR mRNA fragments to form rG4 structures bound by DDX3X. Proteomic profiling of DDX3X-Ras GTPase-activating protein-binding protein 1 (G3BP1) complexes identified several RNA-binding proteins, including IGF2BP1, PUM2, and UBAP2, which may act as candidate cofactors. Together, these findings shed light on the interaction between AR mRNA and DDX3X and identify putative protein partners, offering insights into future therapeutic strategies.