Translational Regulation Research Articles

Boosting the toolbox for live imaging of translation.

Live imaging of translation based on tag recognition by a single-chain antibody is a powerful technique to assess translation regulation in living cells. However, this approach is challenging and requires optimization in terms of expression level and detection sensitivity of the system, especially in a multicellular organism. Here, we improved existing fluorescent tools and developed new ones to image and quantify nascent translation in the living Drosophila embryo and in mammalian cells. We tested and characterized five different green fluorescent protein variants fused to the single-chain fragment variable (scFv) and uncovered photobleaching, aggregation, and intensity disparities. Using different strengths of germline and somatic drivers, we determined that the availability of the scFv is critical in order to detect translation throughout development. We introduced a new translation imaging method based on a nanobody/tag system named ALFA-array, allowing the sensitive and simultaneous detection of the translation of several distinct mRNA species. Finally, we developed a largely improved RNA imaging system based on an MCP-tdStaygold fusion.

Abstract A078: Functional screen for mediators of onco-mRNA translation specificity in pancreatic cancer

Abstract Oncogenic protein dosage is tightly regulated to enable cancer cells to adapt and survive. In healthy cells, translation initiation of onco-mRNA transcripts encoding oncogenes and growth factors is tightly regulated to maintain accurate protein expression levels. However, this process is broken in cancer cells, which rely on the maintenance of high dosage of oncogenic proteins for their survival and to drive cancer development. Whether this is regulated at the level of translational control and the key factors in cis and trans remain unknown. The Myc oncogene is a central paradigm of an exquisitely regulated oncogene and a major driver of pancreatic ductal adenocarcinoma (PDAC). While there has been a tremendous effort to target Myc in cancer, clinically relevant Myc inhibition remains a challenge. Therefore, identifying selective promoters of MYC translation may present a novel therapeutic opportunity. Employing a genome-wide CRISPRi screen for modulators of MYC mRNA translation initiation in PDAC cells, we identified regulators of selective MYC translation and validated four RNA binding proteins (RBPs), including epitranscriptome modifiers. We focused on a top hit, a little studied RBP, RBM42, which is highly expressed in PDAC and predicts poor patient survival. We found that RBM42 binds and selectively regulates the translation of MYC and a precise, yet vital suite of pro-oncogenic transcripts, including JUN and EGFR. Mechanistically, employing immunoprecipitation-mass spectrometry analysis, we found that RMB42 is a novel ribosome-associated protein (RAP). Through RNA structure determination and mutagenesis analysis, we showed that RBM42 directly binds and remodels the MYC 5’UTR RNA structure, facilitating the formation of the translation pre-initiation complex and increasing MYC translation. Importantly, we demonstrated that RBM42 is necessary for human PDAC cell growth and fitness and PDAC tumorigenesis in xenograft mouse models in a Myc-dependent manner in vivo. In PDAC patient samples, RBM42 expression is correlated with Myc protein levels and transcriptional activity. This work transforms our understanding of the translational code in cancer and offers a new therapeutic opening to target the expression of oncogenes. Citation Format: Joanna R Kovalski, Goksu Sarioglu, Vishvak Subramanyam, Grace Hernandez, Gilles Rademaker, Juan A Oses-Prieto, Macey Slota, Kwun Wah Wen, Grace E Kim, Alma L Burlingame, Hani Goodarzi, Rushika M Perera, Davide Ruggero. Functional screen for mediators of onco-mRNA translation specificity in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A078.

199 Awardee Talk: Insights into translational regulation through tRNA sequencing, RNA sequencing, and ribosome profiling

Abstract Translation acts as an additional layer of regulation that has an important role in gene expression and function. Due to a phenomenon called codon usage bias, highly expressed genes are thought to be codon-biased to support efficient translation. As more than one codon can code for the same amino acid (synonymous codons), organisms may exhibit preferences for specific codons that facilitate increased expression of important genes due to variations in the availability of corresponding tRNAs. Advances in sequencing technologies have recently permitted the study of the translatome, which refers to the entire population of mRNA associated with ribosomes for protein synthesis and can be investigated through ribosome profiling. This cutting-edge technique allows the identification of actively translated regions, but can also reveal translational pausing events that stem from the presence of SNPs. Our previous research has demonstrated variation in tRNA expression across tissues and different states of health, leading us to consider the connection between tRNA abundance and translational stalling due to SNPs. By coupling ribosome profiling with tRNA sequencing and RNA sequencing, we have investigated all elements of translational machinery to predict translational efficiency, estimate proteome composition, and evaluate tRNA abundance as a source of genetic variation. In this work, we utilized ribosome profiling, tRNAseq, and RNAseq and performed an integrative analysis in bovine tissues (kidney, liver and muscle) as well as murine myoblast cell lines (C2C12). By applying these methods to different bovine tissues, we were able to explore the interplay between tRNA availability and translational stalling events. Moreover, we have identified translationally regulated genes underlying tissue-specific biological processes and found that many upregulated and downregulated genes coincided with high and low translational efficiency respectively. We have also successfully defined stalling sites that depict the regulatory information encoded within the coding sequence of transcripts, which could control translation rate and facilitate proper protein folding. Through the implementation of these next generation sequencing (NGS) methods to cell culture, we were able to evaluate the translatome at various time points (0-min, 30-min, 60-min, and 4-h) post-induction of differentiation. Where most studies have focused on molecular changes between differentiating myoblasts and multinucleated myotubes that are present 7 d after differentiation, we focus on the earliest stages of muscle differentiation that initiate muscle development and therefore kickstart the process of meat production. This work offers an atlas of distinctive stalling sites across bovine tissues and various stages of muscle differentiation, which provides an opportunity to predict codon optimality and understand tissue-specific or time-specific mechanisms of regulating protein synthesis.

Genome-wide analysis reveals transcriptional and translational changes during diapause of the Asian corn borer (Ostrinia furnacalis)

BackgroundDiapause, a pivotal phase in the insect life cycle, enables survival during harsh environmental conditions. Unraveling the gene expression profiles of the diapause process helps uncover the molecular mechanisms that underlying diapause, which is crucial for understanding physiological adaptations. In this study, we utilize RNA-seq and Ribo-seq data to examine differentially expressed genes (DEGs) and translational efficiency during diapause of Asian corn borer (Ostrinia furnacalis, ACB).ResultsOur results unveil genes classified as “forwarded”, “exclusive”, “intensified”, or “buffered” during diapause, shedding light on their transcription and translation regulation patterns. Furthermore, we explore the landscape of lncRNAs (long non-coding RNAs) during diapause and identify differentially expressed lncRNAs, suggesting their roles in diapause regulation. Comparative analysis of different types of diapause in insects uncovers shared and unique KEGG pathways. While shared pathways highlight energy balance, exclusive pathways in the ACB larvae indicate insect-specific adaptations related to nutrient utilization and stress response. Interestingly, our study also reveals dynamic changes in the HSP70 gene family and proteasome pathway during diapause. Manipulating HSP protein levels and proteasome pathway by HSP activator or inhibitor and proteasome inhibitor affects diapause, indicating their vital role in the process.ConclusionsIn summary, these findings enhance our knowledge of how insects navigate challenging conditions through intricate molecular mechanisms.

Dog Domestication Strongly Relied on Translation Regulation According to Differential Gene Expression Analysis.

Domestication of dogs from their shared ancestors with wolves occurred more than 15,000 years ago and affected many characteristics of the species. We analyzed the blood RNA sequence data of 12 dogs and 11 wolves from Europe and Asia to shed more light on the domestication history of dogs. We implemented a differential gene expression analysis, a weighted gene correlation network analysis, gene ontology and genetic pathway analyses. We found that both the sample origin (Europe or Asia) and the species had a significant effect on the blood gene expression profiles of the animals. We identified 1567 differentially expressed genes between wolves and dogs and found several significantly overrepresented gene ontology terms, such as RNA polymerase II transcription regulatory region sequence-specific DNA binding or translation. We identified 11 significant gene co-expression networks, hosting a total of 4402 genes, related to DNA replication, metabolism of RNA or metabolism of proteins, for example. Our findings suggest that gene expression regulation played a cardinal role in dog domestication. We recommend further diversifying the analyzed dog and wolf populations in the future by including individuals from different dog breeds and geographical origins, in order to enhance the specificity of detecting significant, true positive genes related to domestication as well as to reduce the false positive rate.

Years of endurance exercise training remodel abdominal subcutaneous adipose tissue in adults with overweight or obesity.

Abnormalities in the structure and metabolic function of abdominal subcutaneous adipose tissue (aSAT) underlie many obesity-related health complications. Endurance exercise improves cardiometabolic health in adults with overweight or obesity, but the effects of endurance training on aSAT are unclear. We included male and female participants who were regular exercisers with overweight or obesity who exercised for >2 years, and cross-sectionally compared them with well-matched non-exercisers with overweight or obesity. Here we show aSAT from exercisers has a higher capillary density, lower Col6a abundance and fewer macrophages compared with non-exercisers. This is accompanied by a greater abundance of angiogenic, ribosomal, mitochondrial and lipogenic proteins. The abundance of phosphoproteins involved in protein translation, lipogenesis and direct regulation of transcripts is also greater in aSAT collected from exercisers. Exploratory ex vivo experiments demonstrate greater angiogenic capacity and higher lipid-storage capacity in samples cultured from aSAT collected from exercisers versus non-exercisers. Regular exercise may play a role in remodelling aSAT structure and proteomic profile in ways that may contribute to preserved cardiometabolic health.

The proteomic landscape and temporal dynamics of mammalian gastruloid development.

Gastrulation is the highly coordinated process by which the early embryo breaks symmetry, establishes germ layers and a body plan, and sets the stage for organogenesis. As early mammalian development is challenging to study in vivo, stem cell-derived models have emerged as powerful surrogates, e.g. human and mouse gastruloids. However, although single cell RNA-seq (scRNA-seq) and high-resolution imaging have been extensively applied to characterize such in vitro embryo models, a paucity of measurements of protein dynamics and regulation leaves a major gap in our understanding. Here, we sought to address this by applying quantitative proteomics to human and mouse gastruloids at four key stages of their differentiation (naïve ESCs, primed ESCs, early gastruloids, late gastruloids). To the resulting data, we perform network analysis to map the dynamics of expression of macromolecular protein complexes and biochemical pathways, including identifying cooperative proteins that associate with them. With matched RNA-seq and phosphosite data from these same stages, we investigate pathway-, stage- and species-specific aspects of translational and post-translational regulation, e.g. finding peri-gastrulation stages of human and mice to be discordant with respect to the mitochondrial transcriptome vs. proteome, and nominating novel kinase-substrate relationships based on phosphosite dynamics. Finally, we leverage correlated dynamics to identify conserved protein networks centered around congenital disease genes. Altogether, our data (https://gastruloid.brotmanbaty.org/) and analyses showcase the potential of intersecting in vitro embryo models and proteomics to advance our understanding of early mammalian development in ways not possible through transcriptomics alone.

Proteomic Profiling of Unannotated Microproteins in Human Placenta Reveals XRCC6P1 as a Potential Negative Regulator of Translation.

Ribosome profiling and mass spectrometry have revealed thousands of previously unannotated small and alternative open reading frames (sm/alt-ORFs) that are translated into micro/alt-proteins in mammalian cells. However, their prevalence across human tissues and biological roles remains largely undefined. The placenta is an ideal model for identifying unannotated microproteins and alt-proteins due to its considerable protein diversity that is required to sustain fetal development during pregnancy. Here, we profiled unannotated microproteins and alt-proteins in human placental tissues from preeclampsia patients or healthy individuals by proteomics, identified 52 unannotated microproteins or alt-proteins, and demonstrated that five microproteins can be translated from overexpression constructs in a heterologous cell line, although several are unstable. We further demonstrated that one microprotein, XRCC6P1, associates with translation initiation factor eIF3 and negatively regulates translation when exogenously overexpressed. Thus, we revealed a hidden sm/alt-ORF-encoded proteome in the human placenta, which may advance the mechanism studies for placenta development as well as placental disorders such as preeclampsia.

A PARP14/TARG1-Regulated RACK1 MARylation Cycle Drives Stress Granule Dynamics in Ovarian Cancer Cells.

Mono(ADP-ribosyl)ation (MARylation) is emerging as a critical regulator of ribosome function and translation. Herein, we demonstrate that RACK1, an integral component of the ribosome, is MARylated on three acidic residues by the mono(ADP-ribosyl) transferase (MART) PARP14 in ovarian cancer cells. MARylation of RACK1 is required for stress granule formation and promotes the colocalization of RACK1 in stress granules with G3BP1, eIF3η, and 40S ribosomal proteins. In parallel, we observed reduced translation of a subset of mRNAs, including those encoding key cancer regulators (e.g., AKT). Treatment with a PARP14 inhibitor or mutation of the sites of MARylation on RACK1 blocks these outcomes, as well as the growth of ovarian cancer cells in culture and in vivo. To re-set the system after prolonged stress and recovery, the ADP-ribosyl hydrolase TARG1 deMARylates RACK1, leading to the dissociation of the stress granules and the restoration of translation. Collectively, our results demonstrate a therapeutically targetable pathway that controls stress granule assembly and disassembly in ovarian cancer cells.

Phenotypic Plasticity in Locusts: Trade-Off Between Migration and Reproduction.

Locusts exhibit phenotypic plasticity in response to population density changes, with distinct phenotypes in the solitary and gregarious phases. In the past decade, many studies have revealed the molecular mechanisms underlying phase changes, which include the change of body coloration, pheromones, behavior, flight, fecundity, immunity, and aging. Our understanding of the molecular mechanisms related to these phenotypic differences has expanded in breadth and depth with the decoding of the locust genome, involving transcriptional, post-transcriptional, translational, and epigenetic regulation. Large-scale regulation networks composed of genes and noncoding RNAs reflect the systematic modifications of the locust phase transition in response to environmental changes. Gene manipulation techniques have verified the functions of specific genes and related pathways in phase changes. This review highlights the latest advances in studies of locust phase changes and suggests that the divergence of energy and metabolism allocation in gregarious and solitary locusts is an adaptive strategy for long-distance migration and local reproduction, respectively. Finally, we propose future research directions and discuss emerging questions in the area of phenotypic plasticity of locusts.

Crosstalk between ubiquitination and translation in neurodevelopmental disorders.

Ubiquitination is one of the most conserved post-translational modifications and together with mRNA translation contributes to cellular protein homeostasis (proteostasis). Temporal and spatial regulation of proteostasis is particularly important during synaptic plasticity, when translation of specific mRNAs requires tight regulation. Mutations in genes encoding regulators of mRNA translation and in ubiquitin ligases have been associated with several neurodevelopmental disorders. RNA metabolism and translation are regulated by RNA-binding proteins, critical for the spatial and temporal control of translation in neurons. Several ubiquitin ligases also regulate RNA-dependent mechanisms in neurons, with numerous ubiquitination events described in splicing factors and ribosomal proteins. Here we will explore how ubiquitination regulates translation in neurons, from RNA biogenesis to alternative splicing and how dysregulation of ubiquitin signaling can be the underlying cause of pathology in neurodevelopmental disorders, such as Fragile X syndrome. Finally we propose that targeting ubiquitin signaling is an attractive novel therapeutic strategy for neurodevelopmental disorders where mRNA translation and ubiquitin signaling are disrupted.

The RNA binding protein Arid5a drives IL-17-dependent autoantibody-induced glomerulonephritis.

Autoantibody-mediated glomerulonephritis (AGN) arises from dysregulated renal inflammation, with urgent need for improved treatments. IL-17 is implicated in AGN and drives pathology in a kidney-intrinsic manner via renal tubular epithelial cells (RTECs). Nonetheless, downstream signaling mechanisms provoking kidney pathology are poorly understood. A noncanonical RNA binding protein (RBP), Arid5a, was upregulated in human and mouse AGN. Arid5a-/- mice were refractory to AGN, with attenuated myeloid infiltration and impaired expression of IL-17-dependent cytokines and transcription factors (C/EBPβ, C/EBPδ). Transcriptome-wide RIP-Seq revealed that Arid5a inducibly interacts with conventional IL-17 target mRNAs, including CEBPB and CEBPD. Unexpectedly, many Arid5a RNA targets corresponded to translational regulation and RNA processing pathways, including rRNAs. Indeed, global protein synthesis was repressed in Arid5a-deficient cells, and C/EBPs were controlled at the level of protein rather than RNA accumulation. IL-17 prompted Arid5a nuclear export and association with 18S rRNA, a 40S ribosome constituent. Accordingly, IL-17-dependent renal autoimmunity is driven by Arid5a at the level of ribosome interactions and translation.

Unveiling the Network regulatory mechanism of ncRNAs on the Ferroptosis Pathway: Implications for Preeclampsia.

Non-coding RNAs (ncRNAs) are transcripts originating from the genome that do not serve as templates for protein synthesis. They function as epigenetic and translational regulators in various pathophysiological mechanisms, including cell proliferation and apoptosis. The ferroptosis signaling pathway, a novel mode of cell death, participates in numerous pathophysiological processes. Its signaling transmission is both complex and precise, featuring interconnected and interdependent pathways. Recent studies suggest that ncRNAs can finely regulate key genes in the ferroptosis pathway, thus modulating cellular functions, reducing oxidative stress, and maintaining maternal-fetal interface homeostasis. Future strategies targeting the ncRNA/ferroptosis axis may provide new perspectives and potential intervention points for treating preeclampsia. This article clarifies how the ncRNA/ferroptosis axis impacts preeclampsia, revealing how ncRNAs interact with ferroptosis, and pinpointing new molecular targets for the treatment of preeclampsia, thereby providing theoretical support for clinical strategies.

MiR-31-mediated local translation at the mitotic spindle is important for early development.

miR-31 is a highly conserved microRNA that plays crucial roles in cell proliferation, migration and differentiation. We discovered that miR-31 and some of its validated targets are enriched on the mitotic spindle of the dividing sea urchin embryo and mammalian cells. Using the sea urchin embryo, we found that miR-31 inhibition led to developmental delay correlated with increased cytoskeletal and chromosomal defects. We identified miR-31 to directly suppress several actin remodeling transcripts, including β-actin, Gelsolin, Rab35 and Fascin. De novo translation of Fascin occurs at the mitotic spindle of sea urchin embryos and mammalian cells. Importantly, miR-31 inhibition leads to a significant a increase of newly translated Fascin at the spindle of dividing sea urchin embryos. Forced ectopic localization of Fascin transcripts to the cell membrane and translation led to significant developmental and chromosomal segregation defects, highlighting the importance of the regulation of local translation by miR-31 at the mitotic spindle to ensure proper cell division. Furthermore, miR-31-mediated post-transcriptional regulation at the mitotic spindle may be an evolutionarily conserved regulatory paradigm of mitosis.

Viral Factors in Modulation of Host Immune Response: A Route to Novel Antiviral Agents and New Therapeutic Approaches.

Viruses utilize host cells at all stages of their life cycle, from the transcription of genes and translation of viral proteins to the release of viral copies. The human immune system counteracts viruses through a variety of complex mechanisms, including both innate and adaptive components. Viruses have an ability to evade different components of the immune system and affect them, leading to disruption. This review covers contemporary knowledge about the virus-induced complex interplay of molecular interactions, including regulation of transcription and translation in host cells resulting in the modulation of immune system functions. Thorough investigation of molecular mechanisms and signaling pathways that are involved in modulating of host immune response to viral infections can help to develop novel approaches for antiviral therapy. In this review, we consider new therapeutic approaches for antiviral treatment. Modern therapeutic strategies for the treatment and cure of human immunodeficiency virus (HIV) are considered in detail because HIV is a unique example of a virus that leads to host T lymphocyte deregulation and significant modulation of the host immune response. Furthermore, peculiarities of some promising novel agents for the treatment of various viral infections are described.

Translational Regulation of Duplicated Gene Expression Evolution in Allopolyploid Cotton.

Polyploidy, a prevalent event in plant evolution, drives phenotypic diversification and speciation. While transcriptional changes and regulation in polyploids have been extensively studied, the translational level impact remains largely unexplored. To address this gap, we conducted a comparative transcriptomic and translatomic analysis of cotton leaves from allopolyploid species G. hirsutum (AD1) and G. barbadense (AD2) relative to their model A-genome and D-genome diploid progenitors. Our data revealed that while allopolyploidization significantly affects the transcriptional landscape, its impact on translation was relatively modest, evidenced by a narrower expression range and fewer expression changes in ribosome-protected fragments than in mRNA levels. Allopolyploid-specific changes commonly identified in both AD1 and AD2 were observed in 7393 genes at either transcriptional or translational levels. Interestingly, the majority of translational changes exhibited concordant down-regulation in both ribosome-protected fragments and mRNA, particularly associated with terpenoid synthesis and metabolism (352 genes). Regarding translational efficiency (TE), at least one-fifth of cotton genes exhibit translational level regulation, with a general trend of more down-regulation (13.9-15.1%) than up-regulation (7.3-11.2%) of TE. The magnitude of translational regulation was slightly reduced in allopolyploids compared with diploids, and allopolyploidy tends to have a more profound impact on genes and functional associations with ultra-low TE. Moreover, we demonstrated a reduced extent of homeolog expression biases during translation compared with transcription. Our study provides insights into the regulatory consequences of allopolyploidy post-transcription, contributing to a comprehensive understanding of regulatory mechanisms of duplicated gene expression evolution.

LncRNA BC200 is processed into a stable Alu monomer.

The noncoding RNA BC200 is elevated in human cancers and is implicated in translation regulation as well as cell survival and proliferation. Upon BC200 overexpression, we observed correlated expression of a second, smaller RNA species. This RNA is expressed endogenously and exhibits cell-type-dependent variability relative to BC200. Aptamer-tagged expression constructs confirmed that the RNA is a truncated form of BC200, and sequencing revealed a modal length of 120 nt; thus, we refer to the RNA fragment as BC120. We present a methodology for accurate and specific detection of BC120 and establish that BC120 is expressed in several normal human tissues and is also elevated in ovarian cancer. BC120 exhibits remarkable stability relative to BC200 and is resistant to knockdown strategies that target the 3' unique sequence of BC200. Combined knockdown of BC200 and BC120 exhibits greater phenotypic impacts than knockdown of BC200 alone, and overexpression of BC120 negatively impacts translation of a GFP reporter, providing insight into a potential translational regulatory role for this RNA. The presence of a novel, truncated, and stable form of BC200 adds complexity to the investigation of this noncoding RNA that must be considered in future studies of BC200 and other related Alu RNAs.

Local protein synthesis at neuromuscular synapses is required for motor functions

Motor neurons are highly polarized, and their axons extend over great distances to form connections with myofibers via neuromuscular junctions (NMJs). Local translation at the NMJs in vivo has not been identified. Here, we utilized motor neuron-labeled RiboTag mice and the TRAP (translating ribosome affinity purification) technique to spatiotemporally profile the translatome at NMJs. We found that mRNAs associated with glucose catabolism, synaptic connection, and protein homeostasis are enriched at presynapses. Local translation at the synapse shifts from the assembly of cytoskeletal components during early developmental stages to energy production in adulthood. The mRNA of neuronal Agrin (Agrn), the key molecule for NMJ assembly, is present at motor axon terminals and locally translated. Disrupting the axonal location of Agrn mRNA causes impairment of synaptic transmission and motor functions in adult mice. Our findings indicate that spatiotemporal regulation of mRNA local translation at NMJs plays critical roles in synaptic transmission and motor functions invivo.

Editing and genome-wide analysis upstream open reading frames contributes to enhancing salt tolerance in tomato.

The salinization of soil constitutes a substantial hindrance to the advancement of sustainable agriculture. Our research seeks to elucidate the role of a Rab GTPase-activating protein (RabGAP) family member, SlRabGAP22, in salt tolerance and its translational regulation under salt stress in tomatoes, employing gene-editing techniques and ribosome profiling methodologies. Findings demonstrate that SlRabGAP22 acts as a positive regulator of tomato salt tolerance, with four predicted upstream open reading frames (uORFs) classified into three categories. Functional uORFs were found to be negative regulation. Editing these uORFs along with altering their classifications and characteristics mitigated the inhibitory effects on primary ORFs and fine-tuned gene expression. Enhanced tomato salt tolerance was attributed to improved scavenging of reactive oxygen species, reduced toxicity Na+, and diminished osmotic stress effects. Furthermore, we conducted genome-wide analysis of ORFs to lay the foundation for further research on uORFs in tomatoes. In summary, our findings offer novel perspectives and important data for the enhancement of genetic traits via uORF-based strategies and translational regulation against the backdrop of salt stress.

Cellular RNA-binding proteins LARP4 and PABPC1 synergistically facilitate viral translation of coronavirus PEDV

Coronaviruses are causing epizootic diseases and thus are a substantial threat for both domestic and wild animals. These viruses depend on the host translation machinery to complete their life cycle. The current paper identified cellular RNA-binding proteins (RBPs), La-related protein 4 (LARP4) and polyadenylate-binding protein cytoplasmic 1 (PABPC1), as critical regulators of efficient translation of the coronavirus porcine epidemic diarrhea virus (PEDV) mRNA. In Vero cells, PEDV infection caused LARP4 to migrate from the nucleus to the cytoplasm in a chromosome region maintenance1 (CRM1)-independent pathway. In the absence of the nuclear export signal of LARP4, viral translation was not promoted by LARP4. A further study unveiled that the cytoplasmic LARP4 binds to the 3′-terminal untranslated region (3′UTR) of PEDV mRNA with the assistance of PABPC1 to facilitate viral translation. LARP4 knockdown reduced the promotion of the PABPC1-induced 3′UTR translation activity. Moreover, the rabbit reticulocyte lysate (RRL) system revealed that the prokaryotic expressed protein LARP4 and PABPC1 enhance PEDV mRNA translation. To our knowledge, this is the first study demonstrating that PEDV induces nucleo-cytoplasmic shuttling of LARP4 to enhance its own replication, which broadens our insights into how viruses use host's RBPs for the efficient translation of viral mRNA.