Distinct translational profiles reveal elongation bottlenecks and codon-specific stalling during branched-chain amino acid deprivation [Ribo-Seq]
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ABSTRACT:
PROVIDER: PRJNA1234593 | ENA |
REPOSITORIES: ENA
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Project description:Cells regulate protein synthesis in response to fluctuating nutrient availability through highly coordinated mechanisms that affect both translation initiation and elongation. Branched-chain amino acids (BCAAs) - leucine, isoleucine, and valine - are essential nutrients with a significant impact on cellular physiology. However, how their simultaneous depletion affects the translational machinery and dynamics remains largely unclear. Here, we examine the immediate effects of short-term BCAA limitation on translational dynamics in mammalian cells. We performed RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) on NIH3T3 cells subjected to single, double, or triple BCAA deprivation. Our analyses revealed increased ribosome dwell times in the deprived conditions, with especially strong stalling at all valine codons during valine and triple starvation, while leucine and isoleucine depletion triggered more moderate, codon-specific effects. Notably, isoleucine-specific stalling largely diminished under triple deprivation, likely due to early elongation bottlenecks at valine codons. Correlating these stalling events with tRNA charging levels revealed distinct tRNA isoacceptor regulation in each starvation condition. In addition, integrating proteome data showed that many proteins downregulated under BCAA deprivation harbor stalling sites, suggesting that compromised elongation contributes to decreased protein output. Together, these findings suggest that differential ribosome stalling under BCAA limitation reflects a balance between amino acid supply, tRNA charging dynamics, and stress pathway modulation, illustrating that codon usage biases can profoundly influence the global landscape of translation under nutrient stress.
2025-09-02 | GSE291653 | GEO
Project description:Cells regulate protein synthesis in response to fluctuating nutrient availability through highly coordinated mechanisms that affect both translation initiation and elongation. Branched-chain amino acids (BCAAs) - leucine, isoleucine, and valine - are essential nutrients with a significant impact on cellular physiology. However, how their simultaneous depletion affects the translational machinery and dynamics remains largely unclear. Here, we examine the immediate effects of short-term BCAA limitation on translational dynamics in mammalian cells. We performed RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) on NIH3T3 cells subjected to single, double, or triple BCAA deprivation. Our analyses revealed increased ribosome dwell times in the deprived conditions, with especially strong stalling at all valine codons during valine and triple starvation, while leucine and isoleucine depletion triggered more moderate, codon-specific effects. Notably, isoleucine-specific stalling largely diminished under triple deprivation, likely due to early elongation bottlenecks at valine codons. Correlating these stalling events with tRNA charging levels revealed distinct tRNA isoacceptor regulation in each starvation condition. In addition, integrating proteome data showed that many proteins downregulated under BCAA deprivation harbor stalling sites, suggesting that compromised elongation contributes to decreased protein output. Together, these findings suggest that differential ribosome stalling under BCAA limitation reflects a balance between amino acid supply, tRNA charging dynamics, and stress pathway modulation, illustrating that codon usage biases can profoundly influence the global landscape of translation under nutrient stress.
2025-09-02 | GSE291652 | GEO
Project description:Distinct translational profiles reveal elongation bottlenecks and codon-specific stalling during branched-chain amino acid deprivation [RNA-Seq]
| PRJNA1234592 | ENA
Project description:This study investigates the immediate effects of single, double, and triple branched-chain amino acid (BCAA) deprivation on translational dynamics in NIH3T3 cells. Using a multi-omics approach combining RNA-seq, ribosome profiling (Ribo-seq), quantitative proteomics, and tRNA charging assays, we systematically assessed global translational output and codon-specific ribosome dwell times. Our findings reveal that BCAA starvation induces diverse and non-additive translational control patterns, reflecting a complex interplay between mTORC1 and GCN2 pathway activation, tRNA isoacceptor availability, and transcript codon usage. Notably, we identified a positional effect where the enrichment of valine codons at the 5' end of transcripts creates an elongation bottleneck under valine and triple BCAA starvation, impacting downstream ribosome occupancy and overall protein output. This study uncovers a previously underappreciated layer of complexity in BCAA-specific translational regulation.
2025-09-13 | PXD067949 | Pride
Project description:Sense codon-misassociated eRF1 elicits widespread ribosome stalling subjected to quality control
| PRJNA1150324 | ENA
Project description:The ribosome-associated quality control (RQC) is a surveillance system for aberrant translation, sensing ribosome collisions. Although the molecular mechanism has been extensively studied, the endogenous targets of RQC in human cells were poorly understood. Here, starting from the study of codon specificity of eukaryotic termination factor eRF1, we unexpectedly find that misrecognition of UUA sense codon by eRF1 leads to ribosome collisions and provides the source of RQC substrates in humans. eRF1-selective Monosome-Seq and Disome-Seq reveal that eRF1 recruitment to ribosome was not restricted to the stop codons but also the sub-cognate sense codons, including the UUA codon. The UUA misrecognition by eRF1 causes ribosome collision without termination reaction. Remarkably, Disome-Seq with the depletion of ASCC3 and 4EHP, key factors in RQC, showed that ribosome stalled at UUA codons are the predominant sub-populations rescued by RQC. Failure to resolve ribosome collisions by RQC triggers p38 phosphorylation and upregulation of stress response transcription factor ATF3. This study presents the impact of sense codon misrecognition by the termination factor on translation homeostasis in human cells.
2024-09-18 | GSE275336 | GEO
Project description:Deregulated mRNA translation in cancer is a source of aberrant protein production when tryptophan shortage is induced by anti-tumor immunity (refs). Tryptophan is decoded by only one codon, and currently the consequences of aberrant protein production in conditions of a shortage of amino acids decoded by multiple codons are unknown. Leucine is decoded by six codons, and its levels influence a variety of key processes in cancer, including the control of mTOR signaling (ref) and epithelial to mesenchymal transition (PMID: 28274951). Here, we show that both leucine deprivation and DNA damage elicit ribosomal stalling and subsequent aberrant protein production predominantly at one codon for leucine, UUA. Mechanistically, we found that the leucine tRNA lowest in abundance is causative for ribosomal stalling and aberrant protein production. In addition, UUA-induced aberrant protein production impacts the landscape of neoepitopes presented at the surface of cancer cells, which can elicit T-cell recognition and killing. Altogether, codon-specific induction of neoepitopes by chemotherapy and leucine diet opens up new avenues for the development of adoptive T cell therapies.
2025-05-06 | PXD044085 | Pride
Project description:Stalling of the ribosomes on the mRNA has been linked to myriad of molecular and biological functions. While a multitude of algorithms predict codon-usage in ribosome profiling data, no tool exists to predict the context-specific stalling of ribosomes on transcripts. Combining these two features can reveal unprecedented insights in the ribosomal function and their usage across cells and conditions. To overcome this lack of tools to comprehensively analyze these features, we created Bumpfinder, a web-server, that provides comprehensive analytical details on differences in total ribosome occupancy on transcripts, ribosomal occupancy across codons, ribosome-stalling and the underlying causal amino-acids/codons, as well as collisions. Employing Bumpfinder on generated and publicly available ribosome-profiling datasets, we studied the response of various cell types to amino-acid starvation conditions. We observed interesting patterns in response to the biological effects, suggesting the impact of quality control mechanisms of protein synthesis. Complimentary analysis of collided ribosomes demonstrated phased ribosome stalling on the identified sites. Thus, by deciphering context-specific ribosome distribution, Bumpfinder provides unprecedented molecular insights in the ribosome function and mRNA translation during stress response conditions.
2023-12-31 | GSE156443 | GEO
Project description:Codon usage is biased between lowly and highly expressed genes in a genome-specific manner. This universal bias has been well assessed in some unicellular species, but remains problematic to assess in more complex species. We propose a new method to compute codon usage bias based on genome wide translational data. A new technique based on sequencing of ribosome protected mRNA fragments (Ribo-seq) allowed us to rank genes and compute codon usage bias with high precision for a great variety of species, including mammals. Genes ranking using Ribo-Seq data confirms the influence of the tRNA pool on codon usage bias and shows a decreasing bias in multicellular species. Ribo-Seq analysis also makes possible to detect preferred codons without information on genes function.
| S-EPMC5499818 | biostudies-literature
Project description:Messenger RNA (mRNA) translation is a tightly controlled process frequently deregulated in cancer. Key to this deregulation are transfer RNAs (tRNAs), whose expression, processing, and post-transcriptional modifications are often altered in cancer to support cellular transformation. In conditions of limiting levels of amino acids, this deregulated control of protein synthesis leads to aberrant protein production in the form of ribosomal frameshifting or misincorporation of non-cognate amino acids. Here, we studied leucine, an essential amino acid coded for in mRNA by six different codons. Surprisingly, we found that leucine deprivation leads to ribosomal stalling and aberrant protein production in various cancer cell types, predominantly at one codon, UUA. Similar effects were observed after treatment with chemotherapeutic agents, implying a shared mechanism controlling the downstream effects on mRNA translation. In both conditions, a limitation in the availability of tRNALeu(UAA) for protein production was shown to be the cause for this dominant effect on UUA codons. The induced aberrant proteins can be processed and immune-presented as neoepitopes, and can direct T-cell killing. Altogether, we uncovered a novel mode of interplay between DNA damage, regulation of tRNA availability for mRNA translation, and aberrant protein production in cancer that could be exploited for anti-cancer therapy.
2024-12-27 | GSE276695 | GEO