Analysing the effect of cellular energy levels on codon-specific ribosome occupancy in vivo
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ABSTRACT:
PROVIDER: PRJNA894167 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR22033369.fastq.gz | Fastqsanger.gz | |||
| SRR22033370.fastq.gz | Fastqsanger.gz | |||
| SRR22033371.fastq.gz | Fastqsanger.gz | |||
| SRR22033372.fastq.gz | Fastqsanger.gz | |||
| SRR22033373.fastq.gz | Fastqsanger.gz |
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Project description:The aim of this study is to evaluate how cellular energy levels shape codon-specific decoding and affect codon-mediated mRNA degradation. To this end we made use of HT5P-seq (Zhang & Pelechano, 2021, PMID:35474692) in S. cerevisiae to footprint the ribosome of co-translationally degraded mRNAs in vivo upon swift changes in the concentration of intracellular ATP and other energy metabolites.
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Project description:Protein translation depends on mRNA-specific initiation, elongation, and termination rates. While the regulation of ribosome elongation is well studied in bacteria and yeast, less is known in higher eukaryotes. Here, we combined ribosome and tRNA profiling to investigate the relations between ribosome elongation rates, (aminoacyl-) tRNA levels and codon usage in mammals. We modeled codon-specific ribosome dwell times and translation fluxes from ribosome profiling, considering pair-interactions between ribosome sites. In mouse liver, the model revealed site and codon specific dwell times, as well as codon pair-interactions clustering by amino acids. While translation fluxes varied significantly across diurnal time and feeding regimen, codon dwell times were highly stable, and conserved in human. Fasting had no effect on codon dwell times in mouse liver. Profiling of total and aminoacylated tRNAs revealed highly heterogeneous levels with specific isoacceptor patterns and a correlation with codon usage. tRNAs for isoleucine, asparagine, aspartate and arginine were lowly loaded and conserved in fasted mice. Finally, codons with low levels of charged tRNAs and high codon usage relative to tRNA abundance exhibited long dwell times. Together, these analyses pave the way towards understanding the complex relation between tRNA loading, codon usage and ribosome dwell times in mammals.
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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.
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Project description:In this study, we examine the impact of poor nutrient conditions on -1 ribosome frameshifting and its effect on mRNA degradation. To explore this process, we utilized Ribosome profiling (Ingolia, et.al 2009, PMID: 19213877), in S.cerevisiae in low nutrients condition. Our goal with Ribosome profiling was to identify gene- and codon-specific features that contribute to genome-wide frameshifting events.
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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.
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Project description:Protein translation depends on mRNA-specific initiation, elongation, and termination rates. While the regulation of ribosome elongation is well studied in bacteria and yeast, less is known in higher eukaryotes. Here, we combined ribosome and tRNA profiling to investigate the relations between ribosome elongation rates, (aminoacyl-) tRNA levels and codon usage in mammals. We modeled codon-specific ribosome dwell times and translation fluxes from ribosome profiling, considering pair-interactions between ribosome sites. In mouse liver, the model revealed site and codon specific dwell times, as well as codon pair-interactions clustering by amino acids. While translation fluxes varied significantly across diurnal time and feeding regimen, codon dwell times were highly stable, and conserved in human. Fasting had no effect on codon dwell times in mouse liver. Profiling of total and aminoacylated tRNAs revealed highly heterogeneous levels with specific isoacceptor patterns and a correlation with codon usage. tRNAs for isoleucine, asparagine, aspartate and arginine were lowly loaded and conserved in fasted mice. Finally, codons with low levels of charged tRNAs and high codon usage relative to tRNA abundance exhibited long dwell times. Together, these analyses pave the way towards understanding the complex relation between tRNA loading, codon usage and ribosome dwell times in mammals.
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Project description:Control of messenger RNA (mRNA) decay rate is intimately connected to translation elongation, but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. We used a combination of cryo–electron microscopy, ribosome profiling, and mRNA stability assays to examine the recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site in Saccharomyces cerevisiae. This interaction occurred when the ribosome lacked accommodated A-site transfer RNA, indicative of low codon optimality. Loss of the interaction resulted in the inability of the mRNA degradation machinery to sense codon optimality. Our findings elucidate a physical link between the Ccr4-Not complex and the ribosome and provide mechanistic insight into the coupling of decoding efficiency with mRNA stability.
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Project description:Analysis of ribosomal occupancy with ribosome profiling.
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Project description:Ribosomes can read through stop codons in a regulated manner, elongating rather than terminating the nascent peptide. Stop codon readthrough is essential to diverse viruses, and phylogenetically predicted to occur in a few hundred genes in Drosophila melanogaster, but the importance of regulated readthrough in eukaryotes remains largely unexplored. Here, we present a ribosome profiling assay (deep sequencing of ribosome-protected mRNA fragments) for Drosophila melanogaster, and provide the first genome-wide experimental analysis of readthrough. Readthrough is far more pervasive than expected: the vast majority of readthrough events evolved within D. melanogaster and were not predicted phylogenetically. The resulting C-terminal protein extensions show evidence of selection, contain functional subcellular localization signals, and their readthrough is regulated, arguing for their importance. We further demonstrate that readthrough occurs in yeast and humans. Readthrough thus provides general mechanisms both to regulate gene expression and function, and to add plasticity to the proteome during evolution. 12 samples of Drosophila ribosome profiling and poly(A)+ mRNA-seq, including technical replicates in S2 cells, and biological replicates of 0-2 hour embryos
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