Ribosome Profiling in Isoleucine tRNA Isoswitch Cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE172354 | GEO | 2023/04/01
REPOSITORIES: GEO
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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.
2020-03-26 | GSE126382 | GEO
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:Polysome Profiling was used to evaluated whether changes in abundance of tRNAIleUAU and tRNAIleGAU would affect codon dependent translation and quantify the abundance of these tRNAs in highly translated fractions.
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Project description:Modeling ribosome dwell times and relationship with tRNA loading and codon usage in mammals (Ribosome Profiling)
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Project description:Modeling ribosome dwell times and relationship with tRNA loading and codon usage in mammals (tRNA Profiling)
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Project description:Techniques for systematically monitoring protein translation have lagged far behind methods for measuring mRNA levels. Here we present a ribosome profiling strategy, based on deep sequencing of ribosome protected mRNA fragments, that enables genome-wide investigation of translation with sub-codon resolution. We used this technique to monitor translation in budding yeast under both rich and starvation conditions. These studies defined the protein sequences being translated and found extensive translational control both for determining absolute protein abundance and for responding to environmental stress. We also observed distinct phases during translation involving a large decrease in ribosome density going from early to late peptide elongation as well as wide-spread, regulated initiation at non-AUG codons. Ribosome profiling is readily adaptable to other organisms, making high-precision investigation of protein translation experimentally accessible. Examine replicates of ribosome footprints and mRNA abundance in biological replicates of log-phase growth and acute amino acid starvation
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Project description:Ribosome profiling data reports on the distribution of translating ribosomes, at steady-state, with codon-level resolution. We present a robust method to extract codon translation rates and protein synthesis rates from these data, and identify causal features associated with elongation and translation efficiency in physiological conditions in yeast. We show that neither elongation rate nor translational efficiency is improved by experimental manipulation of the abundance or body sequence of the rare AGG tRNA. Deletion of three of the four copies of the heavily used ACA tRNA shows a modest efficiency decrease that could be explained by other rate-reducing signals at gene start. This suggests that correlation between codon bias and efficiency arises as selection for codons to utilize translation machinery efficiently in highly translated genes. We also show a correlation between efficiency and RNA structure calculated both computationally and from recent structure probing data, as well as the Kozak initiation motif, which may comprise a mechanism to regulate initiation. We test whether tRNA abundance affects elongation or translation efficiency by changing the tRNA levels through deletion or over expression and measuring the ribosomal dwell time at each codon using a robust statistical method that accounts for flow conservation.
2015-01-05 | E-GEOD-63789 | biostudies-arrayexpress
Project description:The fidelity of start codon recognition by ribosomes is paramount during protein synthesis. The textbook knowledge of eukaryotic translation initiation depicts 5’→3’ unidirectional migration of the pre-initiation complex (PIC) along the 5’UTR. In probing translation initiation from ultra-short 5’UTR, we report that an AUG triplet near the 5’ end can be selected via PIC backsliding. The bi-directional ribosome scanning is supported by competitive selection of closely spaced AUG codons and recognition of two initiation sites flanking an internal ribosome entry site. Transcriptome-wide PIC profiling reveals footprints with an oscillation pattern near the 5’ end and start codons. Depleting the RNA helicase eIF4A leads to reduced PIC oscillations and impaired selection of 5’ end start codons. Enhancing the ATPase activity of eIF4A promotes nonlinear PIC scanning and stimulates upstream translation initiation. The helicase-mediated PIC conformational switch may provide an operational mechanism that unifies ribosome recruitment, scanning, and start codon selection.
2021-11-24 | GSE176058 | GEO
Project description:Proteins begin to fold as they emerge from translating ribosomes. The kinetics of ribosome transit along a given mRNA can influence nascent chain folding, but the extent to which individual codon translation rates impact proteome integrity remains unknown. Here, we show that slower decoding of discrete codons elicits widespread protein aggregation in vivo. Using ribosome profiling, we find that loss of anticodon wobble uridine (U34) modifications in a subset of tRNAs leads to ribosome pausing at their cognate codons in S. cerevisiae and C. elegans. Yeast cells lacking U34 modifications exhibit gene expression hallmarks of proteotoxic stress and accumulate aggregates of endogenous proteins with key cellular functions. Moreover, these cells are severely compromised in clearing stress-induced protein aggregates. Overexpression of hypomodified tRNAs alleviates ribosome pausing, concomitantly restoring protein homeostasis. Our findings demonstrate that modified U34 is an evolutionarily conserved accelerator of decoding and reveal an unanticipated role for tRNA anticodon modifications in maintaining proteome integrity. Ribosome profiling of wild-type and tRNA modification-deficient yeast and nematodes. Yeast samples were generated in various growth conditions (rich medium versus stress induced by treatment with diamide or rapamycin) and paired mRNA-Seq was performed on a subset of samples. Dataset contains three biological replicates for yeast samples and two biological replicates for nematode samples.
2015-06-04 | E-GEOD-67387 | biostudies-arrayexpress
Project description:Transfer RNA (tRNA) modifications enhance the efficiency, specificity and fidelity of translation in all organisms. The anticodon modification mcm5s2U34 is required for normal growth and stress resistance in yeast; mutants lacking this modification have numerous phenotypes. Mutations in the homologous human genes are linked to neurological disease. The yeast phenotypes can be ameliorated by overexpression of specific tRNAs, suggesting that the modifications are necessary for efficient translation of specific codons. We determined the in vivo ribosome distributions at single codon resolution in yeast strains lacking mcm5s2U. We found accumulations at AAA, CAA, and GAA codons, suggesting that translation is slow when these codons are in the ribosomal A site, but these changes appeared too small to affect protein output. Instead, we observed activation of the GCN4-mediated stress response by a non- canonical pathway. Thus, loss of mcm5s2U causes global effects on gene expression due to perturbation of cellular signaling. WT yeast and mutants lacking anticodon tRNA modifications were grown in YPD, and subjected to ribosome footprint profiling (ribo-seq) and RNA-seq of poly-A selected RNA. Dataset contains biological replicates for WT, Δncs6 and Δuba4. Technical replicates were also performed for all RNA-seq datasets (using a different poly-A selection method).
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