Ribosome Profiling Research Articles

RiboSeq.Org: an integrated suite of resources for ribosome profiling data analysis and visualization.

Ribosome profiling (Ribo-Seq) has revolutionisedour understanding of translation, but the increasing complexity and volume of Ribo-Seq data present challenges for its reuse. Here, we formally introduce RiboSeq.Org, an integrated suite of resources designed to facilitate Ribo-Seq data analysis and visualisation within a web browser. RiboSeq.Org comprises several interconnected tools: GWIPS-viz for genome-wide visualisation, Trips-Viz for transcriptome-centric analysis, RiboGalaxy for data processing and the newly developed RiboSeq data portal (RDP) for centralised dataset identification and access. The RDP currently hosts preprocessed datasets corresponding to 14840 sequence libraries (samples) from 969 studies across 96 species, in various file formats along with standardised metadata. RiboSeq.Org addresses key challenges in Ribo-Seq data reuse through standardised sample preprocessing, semi-automated metadata curation and programmatic informationaccess via a REST API and command-line utilities. RiboSeq.Org enhances the accessibility and utility of public Ribo-Seq data, enabling researchers to gain new insights into translational regulation and protein synthesis across diverse organisms and conditions. By providing these integrated, user-friendly resources, RiboSeq.Org aims to lower the barrier to reproducible research in the field of translatomics and promote more efficient utilisation of the wealth of available Ribo-Seq data.

HOT3/eIF5B1 confers Kozak motif-dependent translational control of photosynthesis-associated nuclear genes for chloroplast biogenesis.

Photosynthesis requires chloroplasts, in which most proteins are nucleus-encoded and produced via cytoplasmic translation. The translation initiation factor eIF5B gates the transition from initiation (I) to elongation (E), and the Kozak motif is associated with translation efficiency, but their relationship is previously unknown. Here, with ribosome profiling, we determined the genome-wide I-E transition efficiencies. We discovered that the most prevalent Kozak motif is associated with high I-E transition efficiency in Arabidopsis, rice, and wheat, thus implicating the potential of the Kozak motif in facilitating the I-E transition. Indeed, the effects of Kozak motifs in promoting translation depend on HOT3/eIF5B1 in Arabidopsis. HOT3 preferentially promotes the translation of photosynthesis-associated nuclear genes in a Kozak motif-dependent manner, which explains the chloroplast defects and reduced photosynthesis activity of hot3 mutants. Our study linked the Kozak motif to eIF5B-mediated I-E transition during translation and uncovered the function of HOT3 in the cytoplasmic translational control of chloroplast biogenesis and photosynthesis.

Microprotein-encoding RNA regulation in cells treated with pro-inflammatory and pro-fibrotic stimuli

BackgroundRecent analysis of the human proteome via proteogenomics and ribosome profiling of the transcriptome revealed the existence of thousands of previously unannotated microprotein-coding small open reading frames (smORFs). Most functional microproteins were chosen for characterization because of their evolutionary conservation. However, one example of a non-conserved immunomodulatory microprotein in mice suggests that strict sequence conservation misses some intriguing microproteins.ResultsWe examine the ability of gene regulation to identify human microproteins with potential roles in inflammation or fibrosis of the intestine. To do this, we collected ribosome profiling data of intestinal cell lines and peripheral blood mononuclear cells and used gene expression of microprotein-encoding transcripts to identify strongly regulated microproteins, including several examples of microproteins that are only conserved with primates.ConclusionThis approach reveals a number of new microproteins worthy of additional functional characterization and provides a dataset that can be queried in different ways to find additional gut microproteins of interest.

Absence of SMARCB1 in rhabdoid tumor cells increases sensitivity to translation inhibition and alters translation efficiency of specific mRNAs.

Rhabdoid tumors, characterized and driven by the loss of the mSWI/SNF (mammalian SWItch/Sucrose Non-Fermentable) subunit SMARCB1, are very aggressive childhood cancers that can arise in the brain, the kidney, or soft tissues. Cell lines derived from these tumors are specifically sensitivity to the translation inhibitor homoharringtonin (HHT). Having recently demonstrated mSWI/SNF roles in translation, we assessed SMARCB1 potential roles in translation in rhabdoid tumor cells. We first revealed by cell viability assays that rhabdoid tumor cells' sensitivity to HHT were dependent on the absence of SMARCB1. Polysome profiling and immunoprecipitation experiments demonstrated the interaction of SMARCB1 with translation machinery. Global translation assays and ribosome profiling experiments further revealed that SMARCB1 re-expression increased global translation and altered translation efficiency of specific mRNAs. Most regulated mRNAs presented an increased translation efficiency and were involved in differentiation. In comparison with the entire transcriptome, these mRNAs presented a longer coding sequence and were enriched in GC. Finally, we demonstrated that SMARCB1 re-expression increased cytoplasmic localization of these mRNAs and that gene encoding these transcripts were bound by SMARCA4 and SMARCC1. In conclusion, this study reveals that the loss of SMARCB1 in rhabdoid tumors has specific consequences on mRNAs translation with potential to unveil new dependencies.

Translation profiling of stress-induced small proteins reveals a novel link among signaling systems.

Signaling networks allow adaptation to stressful environments by activating genes that counteract stressors. Small proteins (≤ 50 amino acids long) are a rising class of stress response regulators. Escherichia coli encodes over 150 small proteins, most of which lack phenotypes and their biological roles remain elusive. Using magnesium limitation as a stressor, we identify stress-induced small proteins using ribosome profiling, RNA sequencing, and transcriptional reporter assays. We uncover 17 small proteins with increased translation initiation, several of them transcriptionally upregulated by the PhoQ-PhoP two-component signaling system, crucial for magnesium homeostasis. Next, we describe small protein-specific deletion and overexpression phenotypes, underscoring their physiological significance in low magnesium stress. Most remarkably, we elucidate an unusual connection via a small membrane protein YoaI, between major signaling networks - PhoR-PhoB and EnvZ-OmpR in E. coli, advancing our understanding of small protein regulators in cellular signaling.

MK-801-exposure induces increased translation efficiency and mRNA hyperacetylation of Grin2a in the mouse prefrontal cortex

ABSTRACT Acute exposure to MK-801, the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, induces schizophrenia-like behavioural changes in juvenile male mice. However, the effects of acute MK-801 exposure on brain gene expression at the translation level remain unclear. Here, we conducted ribosome profiling analysis on the prefrontal cortex (PFC) of acute MK-801-exposed juvenile male mice. We found 357 differentially translated genes, with the N 4-acetylcytidine (ac4C) consensus motif enriched in the transcripts with increased translation efficiency. Acetylated RNA immunoprecipitation sequencing revealed 148 differentially acetylated peaks, of which 121 were hyperacetylated, and 27 were hypoacetylated. Genes harbouring these peaks were enriched in pathways related to axon guidance, Hedgehog signalling pathway, neuron differentiation, and memory. Grin2a encodes an NMDA receptor subunit NMDAR2A, and its human orthologue is a strong susceptibility gene for schizophrenia. Grin2a mRNA was hyperacetylated and exhibited significantly increased translation efficiency. NMDAR2A protein level was increased in MK-801-exposed PFC. Pretreatment of Remodelin, an inhibitor of N-acetyltransferase 10, returned the NMDAR2A protein levels to normal and partially reversed schizophrenia-like behaviours of MK-801-exposed mice, shedding light on the possible role of mRNA acetylation in the aetiology of schizophrenia.

Ribosome binding of phasiRNA precursors accelerates the 24-nt phasiRNA burst in meiotic maize anthers.

Reproductive phasiRNAs (phased, small interfering RNAs), produced from numerous PHAS loci, are essential for plant anther development. PHAS transcripts are enriched on endoplasmic reticulum-bound ribosomes in maize (Zea mays), but the impact of ribosome binding on phasiRNA biogenesis remains elusive. Through ribosome profiling of maize anthers at 10 developmental stages, we demonstrated that 24-PHAS transcripts are bound by ribosomes, with patterns corresponding to the timing and abundance of 24-PHAS transcripts. Ribosome binding to 24-PHAS transcripts is conserved among different maize inbred lines, with ribosomes enriched upstream of miR2275 target sites. We detected short open reading frames (sORFs) in the ribosome-binding regions of some 24-PHAS transcripts and observed a 3-nt periodicity in most sORFs, but mass spectrometry failed to detect peptides corresponding to the sORFs. Deletion of the entire ribosome-binding region of 24PHAS_NO296 locus eliminated ribosome binding and decreased 24-nt phasiRNA production, without affecting 24PHAS_NO296 transcript levels. In contrast, disrupting only the sORFs in 24PHAS_NO296 did not substantially affect the generation of 24-nt phasiRNAs. A newly formed sORF in these mutants may have re-directed ribosome binding to its transcripts. Overall, these findings demonstrate that sORFs facilitate ribosome binding to 24-PHAS transcripts, thereby promoting phasiRNA biogenesis in meiotic anthers.

Deep learning prediction of ribosome profiling with Translatomer reveals translational regulation and interprets disease variants

Deep learning prediction of ribosome profiling with Translatomer reveals translational regulation and interprets disease variants

PneumoBrowse 2: an integrated visual platform for curated genome annotation and multiomics data analysis of Streptococcus pneumoniae.

Streptococcus pneumoniae is an opportunistic human pathogen responsible for high morbidity and mortality rates. Extensive genome sequencing revealed its large pangenome, serotype diversity, and provided insight into genome dynamics. However, functional genome analysis has lagged behind, as that requires detailed and time-consuming manual curation of genome annotations and integration of genomic and phenotypic data. To remedy this, PneumoBrowse was presented in 2018, a user-friendly interactive online platform, which provided the detailed annotation of the S. pneumoniae D39V genome, alongside transcriptomic data. Since 2018, many new studies on S. pneumoniae genome biology and protein functioning have been performed. Here, we present PneumoBrowse 2 (https://veeninglab.com/pneumobrowse), fully rebuilt in JBrowse 2. We updated annotations for transcribed and transcriptional regulatory features in the D39V genome. We added genome-wide data tracks for high-resolution chromosome conformation capture (Hi-C) data, chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq), ribosome profiling, CRISPRi-seq gene essentiality data and more. Additionally, we included 18 phylogenetically diverse S. pneumoniae genomes and their annotations. By providing easy access to diverse high-quality genome annotations and links to other databases (including UniProt and AlphaFold), PneumoBrowse 2 will further accelerate research and development into preventive and treatment strategies, through increased understanding of the pneumococcal genome.

Versatile Dual Reporter to Identify Ribosome Pausing Motifs Alleviated by Translation Elongation Factor P.

Protein synthesis is influenced by the chemical and structural properties of the amino acids incorporated into the polypeptide chain. Motifs containing consecutive prolines can slow the translation speed and cause ribosome stalling. Translation elongation factor P (EF-P) facilitates peptide bond formation in these motifs, thereby alleviating stalled ribosomes and restoring the regular translational speed. Ribosome pausing at various polyproline motifs has been intensively studied using a range of sophisticated techniques, including ribosome profiling, proteomics, and in vivo screening, with reporters incorporated into the chromosome. However, the full spectrum of motifs that cause translational pausing in Escherichia coli has not yet been identified. Here, we describe a plasmid-based dual reporter for rapid assessment of pausing motifs. This reporter contains two coupled genes encoding mScarlet-I and chloramphenicol acetyltransferase to screen motif libraries based on both bacterial fluorescence and survival. In combination with a diprolyl motif library, we used this reporter to reveal motifs of different pausing strengths in an E. coli strain lacking efp. Subsequently, we used the reporter for a high-throughput screen of four motif libraries, with and without prolines at different positions, sorted by fluorescence-associated cell sorting (FACS) and identify new motifs that influence the translational efficiency of the fluorophore. Our study provides an in vivo platform for rapid screening of amino acid motifs that affect translational efficiencies.

The translatome of glioblastoma.

Glioblastoma (GB), the most common and aggressive brain tumor, demonstrates intrinsic resistance to current therapies, resulting in poor clinical outcomes. Cancer progression can be partially attributed to the deregulation of protein translation mechanisms that drive cancer cell growth. In this study, we present the translatome landscape of GB as a valuable data resource. Eight patient-derived GB sphere cultures (GSCs) were analyzed using ribosome profiling and messenger RNA (mRNA) sequencing. We investigated inter-cell-line differences through differential expression analysis at both the translatome and transcriptome levels. Translational changes post-radiotherapy were assessed at 30 and 60 min. The translation of non-coding RNAs (ncRNAs) was validated using in-house and public mass spectrometry (MS) data, whereas RNA expression was confirmed by quantitative PCR (qPCR). Our findings demonstrate that ribosome sequencing provides more detailed information than MS or transcriptional analyses. Transcriptional similarities among GSCs correlate with translational similarities, aligning with previously defined subtypes such as proneural and mesenchymal. Additionally, we identified a broad spectrum of open reading frame types in both coding and non-coding mRNA regions, including long non-coding RNAs (lncRNAs) and pseudogenes undergoing active translation. Translation of ncRNAs into peptides was independently confirmed by in-house data and external MS data. We also observed that translational regulation of histones (downregulated) and splicing factors (upregulated) occurs in response to radiotherapy. These data offer new insights into genome-wide protein synthesis, identifying translationally regulated genes and alternative translation initiation sites in GB under normal and radiotherapeutic conditions, providing a rich resource for GB research. Further functional validation of differentially expressed genes after radiotherapy is needed. Understanding translational control in GB can reveal mechanistic insights and identify currently unknown biomarkers, ultimately enhancing the diagnosis and treatment of this aggressive brain cancer.

Transient Inhibition of Translation Improves Cardiac Function After Ischemia/Reperfusion by Attenuating the Inflammatory Response.

The myocardium adapts to ischemia/reperfusion (I/R) by changes in gene expression, determining the cardiac response to reperfusion. mRNA translation is a key component of gene expression. It is largely unknown how regulation of mRNA translation contributes to cardiac gene expression and inflammation in response to reperfusion and whether it can be targeted to mitigate I/R injury. To examine translation and its impact on gene expression in response to I/R, we measured protein synthesis after reperfusion in vitro and in vivo. Underlying mechanisms of translational control were examined by pharmacological and genetic targeting of translation initiation in mice. Cell type-specific ribosome profiling was performed in mice that had been subjected to I/R to determine the impact of mRNA translation on the regulation of gene expression in cardiomyocytes. Translational regulation of inflammation was studied by quantification of immune cell infiltration, inflammatory gene expression, and cardiac function after short-term inhibition of translation initiation. Reperfusion induced a rapid recovery of translational activity that exceeds baseline levels in the infarct and border zone and is mediated by translation initiation through the mTORC1 (mechanistic target of rapamycin complex 1)-4EBP1 (eIF4E-binding protein 1)-eIF (eukaryotic initiation factor) 4F axis. Cardiomyocyte-specific ribosome profiling identified that I/R increased translation of mRNA networks associated with cardiac inflammation and cell infiltration. Short-term inhibition of the mTORC1-4EBP1-eIF4F axis decreased the expression of proinflammatory cytokines such as Ccl2 (C-C motif chemokine ligand 2) of border zone cardiomyocytes, thereby attenuating Ly6Chi monocyte infiltration and myocardial inflammation. In addition, we identified a systemic immunosuppressive effect of eIF4F translation inhibitors on circulating monocytes, directly inhibiting monocyte infiltration. Short-term pharmacological inhibition of eIF4F complex formation by 4EGI-1 or rapamycin attenuated translation, reduced infarct size, and improved cardiac function after myocardial infarction. Global protein synthesis is inhibited during ischemia and shortly after reperfusion, followed by a recovery of protein synthesis that exceeds baseline levels in the border and infarct zones. Activation of mRNA translation after reperfusion is driven by mTORC1/eIF4F-mediated regulation of initiation and mediates an mRNA network that controls inflammation and monocyte infiltration to the myocardium. Transient inhibition of the mTORC1-/eIF4F axis inhibits translation and attenuates Ly6Chi monocyte infiltration by inhibiting a proinflammatory response at the site of injury and of circulating monocytes.

The ribosome profiling landscape of yeast reveals a high diversity in pervasive translation

BackgroundPervasive translation is a widespread phenomenon that plays a critical role in the emergence of novel microproteins, but the diversity of translation patterns contributing to their generation remains unclear. Based on 54 ribosome profiling (Ribo-Seq) datasets, we investigated the yeast Ribo-Seq landscape using a representation framework that allows the comprehensive inventory and classification of the entire diversity of Ribo-Seq signals, including non-canonical ones.ResultsWe show that if coding regions occupy specific areas of the Ribo-Seq landscape, noncoding regions encompass a wide diversity of Ribo-Seq signals and, conversely, populate the entire landscape. Our results show that pervasive translation can, nevertheless, be associated with high specificity, with 1055 noncoding ORFs exhibiting canonical Ribo-Seq signals. Using mass spectrometry under standard conditions or proteasome inhibition with an in-house analysis protocol, we report 239 microproteins originating from noncoding ORFs that display canonical but also non-canonical Ribo-Seq signals. Each condition yields dozens of additional microprotein candidates with comparable translation properties, suggesting a larger population of volatile microproteins that are challenging to detect. Our findings suggest that non-canonical translation signals may harbor valuable information and underscore the significance of considering them in proteogenomic studies. Finally, we show that the translation outcome of a noncoding ORF is primarily determined by the initiating codon and the codon distribution in its two alternative frames, rather than features indicative of functionality.ConclusionOur results enable us to propose a topology of a species’ Ribo-Seq landscape, opening the way to comparative analyses of this translation landscape under different conditions.

Zika viruses encode 5′ upstream open reading frames affecting infection of human brain cells

Zika virus (ZIKV), an emerging mosquito-borne flavivirus, is associated with congenital neurological complications. Here, we investigate potential pathological correlates of virus gene expression in representative ZIKV strains through RNA sequencing and ribosome profiling. In addition to the single long polyprotein found in all flaviviruses, we identify the translation of unrecognised upstream open reading frames (uORFs) in the genomic 5′ region. In Asian/American strains, ribosomes translate uORF1 and uORF2, whereas in African strains, the two uORFs are fused into one (African uORF). We use reverse genetics to examine the impact on ZIKV fitness of different uORFs mutant viruses. We find that expression of the African uORF and the Asian/American uORF1 modulates virus growth and tropism in human cortical neurons and cerebral organoids, suggesting a potential role in neurotropism. Although the uORFs are expressed in mosquito cells, we do not see a measurable effect on transmission by the mosquito vector in vivo. The discovery of ZIKV uORFs sheds new light on the infection of the human brain cells by this virus and raises the question of their existence in other neurotropic flaviviruses.

Mechanisms of Electroacupuncture in Alleviating Visceral Hypersensitivity in Post-Infectious Irritable Bowel Syndrome Mice: The Role of GDNF Signaling Pathway and Gut Microbiota

Introduction: Post-infectious irritable bowel syndrome (PI-IBS) is a functional bowel disease that develops following an acute gastrointestinal infection. Electroacupuncture (EA) can regulate the gut microbiota and alleviate visceral hypersensitivity. Glial cell-derived neurotrophic factor (GDNF) is a potential factor in visceral hypersensitivity reactions. The aim of this study was to explore whether EA could alleviate visceral hypersensitivity in PI-IBS by regulating gut microbiota through GDNF signaling. Methods: 2,4,6-trinitrobenzene sulfonic acid was used to induce visceral hypersensitivity in PI-IBS mice. Intestinal visceral sensitivity was assessed by using the abdominal withdrawal reflex (colorectal distention). 16S ribosomal RNA sequencing profiles the gut microbiome community. Results: GDNF can exacerbate the imbalances of the gut microbiota and increase visceral hypersensitivity compared with the model group. Whereas EA treatment increases the richness and diversity of the gut microbiota, decreases differences among species and alleviates visceral sensitivity. Conclusion: EA can alleviate visceral hypersensitivity in PI-IBS by regulating the gut microbiota via GDNF signaling, providing new insights for mechanistic research on EA in PI-IBS treatment.

8681 Preoteogenomic Identification Of Novel Translated And Regulatory Open Reading Frames In Human Beta-Cells

Abstract Disclosure: K. Walters: None. R.C. Guiterrez: None. S. Sakhar: None. A. Baldwin: None. E.S. Nakayasu: None. H. Russ: None. N. Mukherjee: None. Preoteogenomic Identification of Novel Translated and Regulatory Open Reading Frames in Human Beta-cells The discovery of novel autoreactive T cells that target β cells during type 1 diabetes (T1D) has been challenging due to our limited knowledge in autoreactive β cell antigens. Most of these antigens are derived from canonical open reading frames (ORFs) of protein coding genes, such as preproinsulin and chromogranin A. However, a defective ribosomal product (DRiP) was recently identified from the insulin mRNA (INS-DRiP). This antigen is the result of translation from a novel or unannotated open reading frames (nuORFs). nuORFs have a range of functions from regulating translation to encoding novel small bioactive peptides to producing neoantigens. In this study, we use a transcriptome-wide approach to identify nuORFs in human β cells in an unbiased manner. We performed ribosome profiling (ribo-seq) in stem cell derived β cells (sBCs). Human pluripotent stem cells were differentiated into sBCs expressing a flag-tagged ribosomal protein (RPL22-Flag) driven by the rat insulin promoter. We performed IP-ribo-seq on sBCs enriching for RPL22-Flag containing ribosomes. These data exhibited high-resolution 3-nucleotide periodicity consistent with translating codons and identified sequencing reads consistent with translation of the INS-DRiP. We performed de novo ORF-calling using two software using complementary statistical approaches (RiboCode and ORFQuant). We identified over 12,000 canonical annotated ORFs of which ∼85% were had protein level support from matched proteomics confirming the high specificity of our approach. Of nearly 1000 nuORFs identified in sBCs, over half had protein level support in sBCs and cadaveric human islets. We found that 149 nuORFs were associated with Type-1 Diabetes (T1D) genes, including a novel upstream ORF (uORF) in TYK2, which had the strongest association with T1D and is a promising therapeutic target to halt T1D progression. Using reporter assay, we show that the novel uORF in TYK2 is a translational activator and warrants further investigation. Finally, our data represents a valuable database for the search of novel autoreactive antigens in β cells and accelerate the discovery of novel autoreactive T cells implicated in T1D pathogenesis. Presentation: 6/2/2024

CEAM is a mitochondrial-localized, amyloid-like motif-containing microprotein expressed in human cardiomyocytes

Microproteins synthesized through non-canonical translation pathways are frequently found within mitochondria. However, the functional significance of these mitochondria-localized microproteins in energy-intensive organs such as the heart remains largely unexplored. In this study, we demonstrate that the long non-coding RNA CD63-AS1 encodes a mitochondrial microprotein. Notably, in ribosome profiling data of human hearts, there is a positive correlation between the expression of CD63-AS1 and genes associated with cardiomyopathy. We have termed this microprotein CEAM (CD63-AS1 encoded amyloid-like motif containing microprotein), reflecting its sequence characteristics. Our biochemical assays show that CEAM forms protease-resistant aggregates within mitochondria, whereas deletion of the amyloid-like motif transforms CEAM into a soluble cytosolic protein. Overexpression of CEAM triggers mitochondrial stress responses and adversely affect mitochondrial bioenergetics in cultured cardiomyocytes. In turn, the expression of CEAM is reciprocally inhibited by the activation of mitochondrial stresses induced by oligomycin. When expressed in mouse hearts via adeno-associated virus, CEAM impairs cardiac function. However, under conditions of pressure overload-induced cardiac hypertrophy, CEAM expression appears to offer a protective benefit and mitigates the expression of genes associated with cardiac remodeling, presumably through a mechanism that suppresses stress-induced translation reprogramming. Collectively, our study uncovers a hitherto unexplored amyloid-like microprotein expressed in the human cardiomyocytes, offering novel insights into myocardial hypertrophy pathophysiology.

G3BP isoforms differentially affect stress granule assembly and gene expression during cellular stress.

Stress granules (SGs) are macromolecular assemblies that form under cellular stress. Formation of these membrane-less organelles is driven by the condensation of RNA and RNA-binding proteins such as G3BPs. G3BPs form SGs following stress-induced translational arrest. Three G3BP paralogs (G3BP1, G3BP2A, and G3BP2B) have been identified in vertebrates. However, the contribution of different G3BP paralogs to stress granule formation and gene expression changes is incompletely understood. Here, we probed the functions of G3BPs by identifying important residues for stress granule assembly at their N-terminal domain such as V11. This conserved amino acid is required for formation of the G3BP-Caprin-1 complex, hence promoting SG assembly. Total RNA sequencing and ribosome profiling revealed that a G3BPV11A mutant leads to changes in mRNA levels and ribosome engagement during the integrated stress response (ISR). Moreover, we found that G3BP2B preferentially forms stress granules and promotes changes in mRNA expression under endoplasmic reticulum (ER) stress. Furthermore, our work is a resource for researchers to study gene expression changes under cellular stress. Together, this work suggests that perturbing protein-protein interactions mediated by G3BPs affect stress granule assembly and gene expression during the ISR, and such functions are differentially regulated by G3BP paralogs under ER stress.

Genome-wide impact of codon usage bias on translation optimization in Drosophila melanogaster

Accuracy and efficiency are fundamental to mRNA translation. Codon usage bias is widespread across species. Despite the long-standing association between optimized codon usage and improved translation, our understanding of its evolutionary basis and functional effects remains limited. Drosophila is widely used to study codon usage bias, but genome-scale experimental data are scarce. Using high-resolution mass spectrometry data from Drosophila melanogaster, we show that optimal codons have lower translation errors than nonoptimal codons after accounting for these biases. Genomic-scale analysis of ribosome profiling data shows that optimal codons are translated more rapidly than nonoptimal codons. Although we find no long-term selection favoring synonymous mutations in D. melanogaster after diverging from D. simulans, we identify signatures of positive selection driving codon optimization in the D. melanogaster population. These findings expand our understanding of the functional consequences of codon optimization and serve as a foundation for future investigations.

RPFdb v3.0: an enhanced repository for ribosome profiling data and related content.

RPFdb (http://www.rpfdb.org or http://sysbio.gzzoc.com/rpfdb/) is a comprehensive repository dedicated to hosting ribosome profiling (Ribo-seq) data and related content. Herein, we present RPFdb v3.0, a significant update featuring expanded data content and improved functionality. Key enhancements include (i) increased data coverage, now encompassing 5018 Ribo-seq datasets and 2343 matched RNA-seq datasets from 496 studies across 34 species; (ii) implementation of translation efficiency, combining Ribo-seq and RNA-seq data to provide gene-specific translation efficiency; (iii) addition of pausing score, facilitating the identification of condition-specific triplet amino acid motifs with enhanced ribosome enrichment; (iv) refinement of open reading frame (ORF) annotation, leveraging RibORF v2.0 for more sensitive detection of actively translated ORFs; (v) introduction of a resource hub, curating advances in translatome sequencing techniques and data analytics tools to support a panoramic overview of the field; and (vi) redesigned web interface, providing intuitive navigation with dedicated pages for streamlined data retrieval, comparison and visualization. These enhancements make RPFdb a more powerful and user-friendly resource for researchers in the field of translatomics. The database is freely accessible and regularly updated to ensure its continued relevance to the scientific community.