Cell-type-specific translational regulation by human DUS enzymes [tRNA NaBH4]
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ABSTRACT:
PROVIDER: PRJNA1034647 | ENA |
REPOSITORIES: ENA
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Project description:Dihydrouridine is an abundant and evolutionary conserved modified nucleoside present on tRNA, but characterization and functional studies of individual modification sites and associated DUS writer enzymes in mammals is lacking. Here we use a chemical probing strategy, RNABPP-PS, to identify 5-chlorouridine as a general activity-based probe for human DUS enzymes. We map D modifications using mechanism-based RNA-protein crosslinking and also through chemical reactivity and mutational profiling to reveal the landscape of D modification sites on human tRNAs. Further, we knockout individual DUS genes in two model human cell lines to investigate their regulation of tRNA expression levels and codon-specific translation elongation. We show that whereas D modifications are present across most tRNA species, loss of D only perturbs the translational function of a subset of tRNAs in a cell-type-specific manner. Our work provides powerful chemical strategies for investigating D and DUS enzymes in diverse biological systems and provides insight into the role of a ubiquitous tRNA modification in translational regulation.
2024-10-31 | GSE246816 | GEO
Project description:Dihydrouridine is an abundant and evolutionary conserved modified nucleoside present on tRNA, but characterization and functional studies of individual modification sites and associated DUS writer enzymes in mammals is lacking. Here we use a chemical probing strategy, RNABPP-PS, to identify 5-chlorouridine as a general activity-based probe for human DUS enzymes. We map D modifications using mechanism-based RNA-protein crosslinking and also through chemical reactivity and mutational profiling to reveal the landscape of D modification sites on human tRNAs. Further, we knockout individual DUS genes in two model human cell lines to investigate their regulation of tRNA expression levels and codon-specific translation elongation. We show that whereas D modifications are present across most tRNA species, loss of D only perturbs the translational function of a subset of tRNAs in a cell-type-specific manner. Our work provides powerful chemical strategies for investigating D and DUS enzymes in diverse biological systems and provides insight into the role of a ubiquitous tRNA modification in translational regulation.
2024-10-31 | GSE246814 | GEO
Project description:Cell-type-specific translational regulation by human DUS enzymes [tRNA DESeq]
| PRJNA1034643 | ENA
Project description:Dihydrouridine is an abundant and evolutionary conserved modified nucleoside present on tRNA, but characterization and functional studies of individual modification sites and associated DUS writer enzymes in mammals is lacking. Here we use a chemical probing strategy, RNABPP-PS, to identify 5-chlorouridine as a general activity-based probe for human DUS enzymes. We map D modifications using mechanism-based RNA-protein crosslinking and also through chemical reactivity and mutational profiling to reveal the landscape of D modification sites on human tRNAs. Further, we knockout individual DUS genes in two model human cell lines to investigate their regulation of tRNA expression levels and codon-specific translation elongation. We show that whereas D modifications are present across most tRNA species, loss of D only perturbs the translational function of a subset of tRNAs in a cell-type-specific manner. Our work provides powerful chemical strategies for investigating D and DUS enzymes in diverse biological systems and provides insight into the role of a ubiquitous tRNA modification in translational regulation.
2024-10-31 | GSE246817 | GEO
Project description:Cell-type-specific translational regulation by human DUS enzymes
| PRJNA1034645 | ENA
Project description:Cell-type-specific translational regulation by human DUS enzymes [iCLIP]
| PRJNA1034648 | ENA
Project description:During mRNA translation, tRNAs are charged by aminoacyl-tRNA synthetases (aaRS) and subsequently used by ribosomes. A multi-enzyme aminoacyl-tRNA synthetase complex (MSC) has long been proposed to increase protein synthesis efficiency by passing charged tRNAs directly to ribosomes. An alternative is that the MSC repurposes specific synthetases for ex-translational functions that are activated by cues that direct specific enzymes to novel targets. To explore this question, we generated mammalian cell clones in which ArgRS and GlnRS were absent from the MSC to give a stable complex lacking the two enzymes (MSCΔRQ). Protein synthesis, under a variety of stress conditions, was unchanged in MSCΔRQ cells. Most strikingly, levels of charged tRNAGln and tRNAArg remained unchanged and no ribosome pausing was observed at codons for Arg and Gln. Thus, increasing or regulating protein synthesis efficiency is not dependent on ArgRS and GlnRS in the MSC. Alternatively, and consistent with previously reported ex-translational roles, we found manipulations that do not affect protein synthesis but instead MSC cellular localization.
2020-11-18 | GSE158212 | GEO
Project description:Quantitative tRNA-sequencing uncovers metazoan tissue-specific tRNA regulation
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Project description:Extensive allele-specific translational regulation in hybrid mice
| PRJEB8233 | ENA
Project description:Small RNAs include tRNA, snRNA, micro-RNA, tRNA fragments and others that constitute >90% of RNA copy numbers in a human cell and perform many essential functions. Popular small RNA-seq strategies limit the insights into coordinated small RNA response to cellular stress. Small RNA-seq also lacks multiplexing capabilities. Here, we report a multiplex small RNA-seq library preparation method (MSR-seq) to investigate cellular small RNA and mRNA response to heat shock, hydrogen peroxide, and arsenite stress. Comparing stress-induced changes of total cellular RNA and polysome-associated RNA, we identify a coordinated tRNA response that involves polysome-specific tRNA abundance and synergistic N3-methylcytosine (m3C) tRNA modification. Combining tRNA and mRNA response to stress we reveal a new mechanism of stress-induced down-regulation in translational elongation. We also find that native tRNA molecules lacking several modifications are biased reservoirs for the biogenesis of tRNA fragments. Our results demonstrate the importance of simultaneous investigation of small RNAs and their modifications in response to varying biological conditions.
2022-03-13 | GSE198441 | GEO