Conserved 5-methyluridine tRNA modification modulates ribosome translocation
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ABSTRACT:
PROVIDER: PRJNA1142031 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR30048378_1.fastq.gz | Fastqsanger.gz | |||
| SRR30048378_2.fastq.gz | Fastqsanger.gz | |||
| SRR30048379_1.fastq.gz | Fastqsanger.gz | |||
| SRR30048379_2.fastq.gz | Fastqsanger.gz | |||
| SRR30048380_1.fastq.gz | Fastqsanger.gz |
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Project description:While the centrality of post-transcriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m5U54). Here, we uncover contributions of m5U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m5U in the T-loop (TrmA inE. coli, Trm2 inS. cerevisiae) exhibit altered tRNA modifications patterns. Furthermore, m5U54 deficient tRNAs are desensitized to small molecules that prevent translocationin vitro.This finding is consistent with our observations that, relative to wild-type cells,trm2Δ cell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m5U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.
2024-08-08 | GSE273506 | GEO
Project description:In addition to the conserved translation elongation factors eEF1A and eEF2, fungi require a third essential elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the A and E sites, its exact mechanism of action is unclear. Here we show that eEF3 acts at the mRNA–tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl-tRNA recruitment to the A site. Depletion of eEF3 in vivo leads to a general slow-down in translation elongation due to accumulation of ribosomes with an occupied A site. Cryo-EM analysis of native eEF3-ribosome complexes shows that eEF3 facilitates late steps of translocation by favoring non-rotated ribosomal states as well as by opening the L1 stalk to release the E-site tRNA. Additionally, our analysis provides structural insights into novel translation elongation states, enabling presentation of a revised yeast translation elongation cycle.
2020-10-28 | GSE160206 | GEO
Project description:eEF3 promotes late stages of tRNA translocation on the ribosome
| PRJNA672335 | ENA
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).
2013-08-01 | E-GEOD-45366 | biostudies-arrayexpress
Project description:In the ribosome complex, tRNA is a critical element of mRNA translation. We reported a new technology for profiling ribosome-embedded tRNAs and their modifications. With the method, we generated a comprehensive survey of the quanity and quality of intra-ribosomal tRNAs (Ribo-tRNA-seq). Ribo-tRNA-seq can provide new insights on translation control mechanism in diverse biological contexts.
2018-03-19 | E-MTAB-6576 | biostudies-arrayexpress
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.
2020-03-26 | GSE126383 | GEO
Project description:In higher eukaryotes, the large numbers of nuclear-encoded tRNA genes partially ensure the robustness of cytoplasmic protein translation. Here we discover that a loss-of-function in n-Tr20, a member of the nuclear-encoded tRNA Arg UCU family that is expressed specifically in the central nervous systems leads to low but detectable levels of ribosome stalling. In the absence of GTPBP2, a novel binding partner of the ribosome recycling protein Pelota, ribosome stalling increases, leading to widespread neurodegeneration. Our results not only define GTPBP2 as a ribosome rescue factor, but also unmask the disease potential of mutations in nuclear-encoded tRNA genes. In this submission we provide ribosome footprinting data from the cerebella of four strains derived from the C57BL/6J strain with combinations of n-Tr20 and GTPBP2 mutations. Examination of ribosome stalling in cerebella from 4 mouse strains derived from the: C57BL/6J (B6J) strain. The nmf205-/- strain has a homozygous mutation in the gene GTPBP2 while the B6J strain has normal GTPBP2. The n-Tr20 J/J strain has a defect in the n-Tr20 tRNA while the n-Tr20 N/N strain has a functional n-Tr20 tRNA. The 4 strains are the 2x2 combinations of these defects and correctly functioning sequences. 2 replicates for each strain. Please note that only BAM files are included in the records since they form the basis of the study's conclusions. The raw data ribosomal RNA have been filtered and then unique reads mapping to mm10 were computed using tophat and igenome annotations.
2014-07-28 | E-GEOD-56127 | biostudies-arrayexpress
Project description:tRNA modifications play important roles in maintaining translation accuracy in all domains of life. Disruptions in the tRNA modification machinery, especially of the anticodon stem loop, can be lethal for many bacteria and lead to a broad range of phenotypes in baker’s yeast. Very little is known about the function of tRNA modifications in host-pathogen interactions, where rapidly changing environments and stresses require fast adaptations. We found that two closely related fungal pathogens of humans, the highly pathogenic Candida albicans and its much less pathogenic sister species, Candida dubliniensis, differ in the function of a tRNA-modifying enzyme. This enzyme, Hma1, exhibits speciesspecific effects on the ability of the two fungi to grow in the hypha morphology, which is central to their virulence potential. We show that Hma1 has tRNA-threonylcarbamoyladenosine dehydratase activity, and its deletion alters ribosome occupancy, especially at 37°C—the body temperature of the human host. A C. albicans HMA1 deletion mutant also shows defects in adhesion to and invasion into human epithelial cells and shows reduced virulence in a fungal infection model. This links tRNA modifications to host-induced filamentation and virulence of one of the most important fungal pathogens of humans.
2024-04-04 | GSE199421 | GEO
Project description:Post-transcriptional modifications are important for transfer RNAs (tRNAs) to be efficient and accurate in translation on the ribosome. The m1G37 modification on a subset of tRNAs in bacteria are generated by a conserved methyltransferase TrmD and is essential for bacterial growth. Previous studies showed that m1G37 has an important role in preventing translational frameshifting and also that this modification is coupled with aminoacylation of tRNAs for proline. Here we performed suppressor screening to isolate a mutant E. coli cell that lacks TrmD but is viable, and the whole-genome sequencing revealed several mutations on prolyl-tRNA synthetase (ProRS) gene conferring cell viability in the absence of TrmD. Biochemical assays confirmed uncoupling of m1G37 modification and aminoacylation, and cell-based assays uncovered the critical role of m1G37 in supporting Wobble decoding.
2022-09-24 | E-MTAB-12178 | biostudies-arrayexpress