Glycosylation of queuosine in tRNAs contributes to optimal translation and post-embryonic growth in vertebrates
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ABSTRACT:
PROVIDER: PRJNA879455 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR21533253.fastq.gz | Fastqsanger.gz | |||
| SRR21533254.fastq.gz | Fastqsanger.gz | |||
| SRR21533255.fastq.gz | Fastqsanger.gz | |||
| SRR21533256.fastq.gz | Fastqsanger.gz | |||
| SRR21533257.fastq.gz | Fastqsanger.gz |
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Project description:Post-transcriptional modification of tRNAs is critical for protein synthesis. Queuosine (Q), a 7-deaza-guanosine derivative, is present at the first position of anticodons of several tRNA species. In vertebrate tRNAs for Tyr and Asp, Q is further glycosylated with galactose and mannose to generate galactosyl-queuosine (galQ) and mannosyl-queuosine (manQ), respectively. However, the biogenesis and physiological relevance of Q-glycosylation remain poorly understood. Here, we biochemically identified two RNA glycosylases, queuosine-tRNA galactosyltransferase (QTGAL) and queuosine-tRNA mannosyltransferase (QTMAN), and successfully reconstituted galQ and manQ formation on the respective tRNAs in the presence of nucleotide diphosphate sugars. Ribosome profiling analyses of human knockout (KO) cells of QTGAL and QTMAN revealed that Q-glycosylation slowed down elongation at the cognate codons, UAC and GAC (GAU), respectively. Furthermore, protein aggregates were significantly increased in both KO cell lines, indicating that Q-glycosylation contributes to proteostasis of nascent proteins. We determined atomic structures of human cytoplasmic tRNAs for Try and Asp bound to the ribosome A-site, providing the molecular basis of codon recognition regulated by galQ and manQ. Furthermore, zebrafish qtgal and qtman KO lines displayed shortened body length, suggesting Q-glycosylation is required for post-embryonic growth in vertebrates. These findings demonstrate that Q-glycosylation modulates codon-specific translation to optimize the decoding speed in protein synthesis, thereby maintaining correct folding of nascent proteins, proteome integrity, and normal growth of vertebrates.
2023-11-21 | GSE213181 | GEO
Project description:Queuosine (Q) is a complex tRNA modification found at position 34 of four tRNAs with a GUN anticodon, and it regulates the translational efficiency and fidelity of the respective codons that differ at the Wobble position. In bacteria, the biosynthesis of Q involves two precursors, preQ0 and preQ1, whereas eukaryotes directly obtain Q from bacterial sources. The study of queuosine has been challenging due to the limited availability of high-throughput methods for its detection and analysis. Here, we have employed direct RNA sequencing using nanopore technology to detect the modification of tRNAs with Q and Q precursors. These modifications were detected with high accuracy on synthetic tRNAs as well as on tRNAs extracted from Schizosaccharomyces pombe and Escherichia coli by comparing unmodified to modified tRNAs using the tool JACUSA2. Furthermore, we present an improved protocol for the alignment of raw sequence reads that gives high specificity and recall for tRNAs ex cellulo that, by nature, carry multiple modifications. Altogether, our results show that such 7-deazaguanine-derivatives are readily detectable using direct RNA sequencing. This advancement opens up new possibilities for investigating these modifications in native tRNAs, furthering our understanding of their biological function.
2023-09-19 | GSE236838 | GEO
Project description:Detection of queuosine and queuosine precursors in tRNAs by direct RNA sequencing
| PRJNA992497 | ENA
Project description:Eukaryotic transfer RNAs (tRNA) contain on average 13 modifications that perform a wide range of roles in translation and in the generation of tRNA fragments that regulate gene expression. Queuosine (Q) modification occurs in the wobble anticodon position of tRNAs for amino acids His, Asn, Tyr, and Asp. In eukaryotes, Q modification is fully dependent on diet or on gut microbiome in multi-cellular organisms. Despite decades of study, cellular roles of Q modification remain to be fully elucidated. Here we show that in human cells, Q modification specifically protects its cognate tRNAHis and tRNAAsn against cleavage by ribonucleases. We generated cell lines that contain completely depleted or fully Q-modified tRNAs. Using these resources, we found that Q modification significantly reduces angiogenin cleavage of its cognate tRNAs in vitro. Q modification does not change the cellular abundance of the cognate full-length tRNAs, but alters the cellular content of their fragments in vivo in the absence and presence of stress. Our results provide a new biological aspect of Q modification and a mechanism of how Q modification alters small RNA pool in human cells.
2018-08-10 | GSE102570 | GEO
Project description:These research areas concentrate on stress induced proteases in recombinant Escherichia coli, glycosylation heterogeneity due to bioprocess conditions produced in mammalian cells, and metabolic engineering of E. coli. The hypothesis of this project is that recombinant protein glycosylation is inefficient under normal bioreactor conditions since the additional glycosylation reactions necessary for the recombinant protein exceed the metabolic capacity of the cells. Normal bioreactor conditions have been optimized for cell growth, and sometimes for protein productivity. Only recently has it been accepted that optimal glycosylation may not occur under optimal growth or protein productivity conditions. Specific Aim: Determine the relationship between bioreactor conditions and glycosylation gene expression in NS0 cells.
2011-04-29 | GSE28920 | GEO
Project description:These research areas concentrate on stress induced proteases in recombinant Escherichia coli, glycosylation heterogeneity due to bioprocess conditions produced in mammalian cells, and metabolic engineering of E. coli. The hypothesis of this project is that recombinant protein glycosylation is inefficient under normal bioreactor conditions since the additional glycosylation reactions necessary for the recombinant protein exceed the metabolic capacity of the cells. Normal bioreactor conditions have been optimized for cell growth, and sometimes for protein productivity. Only recently has it been accepted that optimal glycosylation may not occur under optimal growth or protein productivity conditions. Specific Aim: Determine the relationship between bioreactor conditions and glycosylation gene expression in NS0 cells. EXPERIMENT: Mouse NS0 myeloma cells were grown in culture, stressed with 5 mM NaCl, 10 mM proline plus 5 mM ammonia, or 5 mM ammonia, along with an unstressed control group. The growth of the cultures were followed until the late exponential phase (90 hours), at which time two 50 mLs of cells were harvested and RNA extracted. Samples were prepared in triplicate, for a total of 12 samples. The RNA was amplified and labeled by Microarray Core (E) and hybridized to the GLYCOv3 microarrays.
2011-04-29 | E-GEOD-28920 | biostudies-arrayexpress
Project description:Queuosine (Q) is a conserved tRNA modification at the wobble anticodon position of tRNAs that read the codons of amino acids Tyr, His, Asn, and Asp. Q-modification in tRNA plays important roles in the regulation of translation efficiency and fidelity. Queuosine tRNA modification is synthesized de novo in bacteria, whereas the substrate for Q-modification in tRNA in mammals is queuine, the catabolic product of the Q-base of gut bacteria. This gut microbiome dependent tRNA modification may play pivotal roles in translational regulation in different cellular contexts, but extensive studies of Q-modification biology are hindered by the lack of high throughput sequencing methods for its detection and quantitation. Here, we describe a periodate-treatment method of biological RNA samples that enables single base resolution profiling of Q-modification in tRNAs by Nextgen sequencing. Periodate oxidizes the Q-base, which results in specific deletion signatures in the RNA-seq data. Unexpectedly, we found that periodate-treatment also enables the detection of several 2-thio-modifications including τm5s2U, mcm5s2U, cmnm5s2U, and s2C by sequencing in human and E. coli tRNA. We term this method Periodate-dependent analysis of queuosine and thio modification sequencing (PAQS-seq). We assess Q- and 2-thio-modifications at the tRNA isodecoder level, and 2-thio modification changes in stress response. PAQS-seq should be widely applicable in the biological studies of Q- and 2-thio-modifications in mammalian and microbial tRNAs.
2022-06-22 | GSE196016 | GEO
Project description:Queuosine (Q) is a complex tRNA modification found in bacteria and eukaryotes at position 34 of four tRNAs with a GUN anticodon, and it regulates the translational efficiency and fidelity of the respective codons that differ at the Wobble position. In bacteria, the biosynthesis of Q involves two precursors, preQ0 and preQ1, whereas eukaryotes directly obtain Q from bacterial sources. The study of queuosine has been challenging due to the limited availability of high-throughput methods for its detection and analysis. Here, we have employed direct RNA sequencing using nanopore technology to detect the modification of tRNAs with Q and Q precursors. These modifications were detected with high accuracy on synthetic tRNAs as well as on tRNAs extracted from Schizosaccharomyces pombe and Escherichia coli by comparing unmodified to modified tRNAs using the tool JACUSA2. Furthermore, we present an improved protocol for the alignment of raw sequence reads that gives high specificity and recall for tRNAs ex cellulo that, by nature, carry multiple modifications. Altogether, our results show that 7-deazaguanine-derivatives such as queuosine are readily detectable using direct RNA sequencing. This advancement opens up new possibilities for investigating these modifications in native tRNAs, furthering our understanding of their biological function.
| S-EPMC10639084 | biostudies-literature
Project description:In most eukaryotes and bacteria, queuosine (Q) replaces the guanosine at the wobble position of tRNAs harboring a GUN anticodon. To investigate whether other RNAs are also Q-or preQ1-modified in Schizosaccharomyces pombe, Q-RIP-Seq was established and applied to RNAs from WT (AEP1) and qtr2∆ cells (AEP288). Metabolic labeling of RNAs with azido-propyl-preQ1 (preQ1-L1), chemical clicking of a biotin-alkyne followed by immunoprecipitation on streptavidin beads and sequencing of enriched RNAs in WT compared to qtr2∆ allowed identification of Q-modified RNAs.
2022-09-30 | GSE210404 | GEO
Project description:Tgt is the enzyme modifying the guanine (G) in tRNAs with GUN anticodon to queuosine (Q). tgt is required for optimal growth of Vibrio cholerae in the presence of sub-lethal aminoglycoside concentrations. We further explored here the role of the Q in the efficiency of codon decoding upon tobramycin exposure. We characterized its impact on the overall bacterial proteome, and elucidated the molecular mechanisms underlying the effects of Q modification in antibiotic translational stress response. Using molecular reporters, we showed that Q impacts the efficiency of decoding at tyrosine TAT and TAC codons. Proteomics analyses revealed that the anti-SoxR factor RsxA is better translated in the absence of tgt. RsxA displays a codon bias towards tyrosine TAT and overabundance of RsxA leads to decreased expression of genes belonging to SoxR oxidative stress regulon. We also identified conditions that regulate tgt expression. We propose that regulation of Q modification in response to environmental cues leads to translational reprogramming of genes bearing a biased tyrosine codon usage. In silico analysis further identified candidate genes possibly subject to such translational regulation, among which DNA repair factors. Such transcripts, fitting the definition of modification tunable transcripts, are plausibly central in the bacterial response to antibiotics.
2025-01-06 | GSE231087 | GEO