AlkAnilineSeq analysis of human tRNAs
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB56781 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR10849213.fastq.gz | Fastqsanger.gz | |||
| ERR10849214.fastq.gz | Fastqsanger.gz | |||
| ERR10849215.fastq.gz | Fastqsanger.gz | |||
| ERR10849216.fastq.gz | Fastqsanger.gz | |||
| ERR10849217.fastq.gz | Fastqsanger.gz |
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Project description:RNA modifications have a substantial impact on tRNA function. While modifications in the anticodon loop play an important role in translational fidelity, modifications in the tRNA core influence tRNA structural stability. In bacteria, tRNA modifications play important roles in the stress response and expression of virulence factors. While tRNA modifications are well characterized in a few model organisms, our knowledge of tRNA modifications in human pathogens, such as Pseudomonas aeruginosa is lacking. Here we leveraged two orthogonal approaches to build a reference landscape of tRNA modifications in E. coli, which we used to identify tRNA modifications in P. aeruginosa. We determined conservation for many modifications between the two organisms. We also identified potential sites of tRNA modification in P. aeruginosa tRNA that are not present in E. coli. One of these sites is found at the same positions as acacp3U, a modification previously identified in Vibrio cholerae. Identifying which modifications are present on different tRNAs will uncover the pathways impacted by the different tRNA modifying enzymes, some of which may play roles in determining virulence and pathogenicity.
2024-04-03 | GSE236676 | GEO
Project description:The participation of transfer RNAs (tRNAs) in fundamental aspects of biology and disease necessitates an accurate, experimentally confirmed annotation of tRNA genes, and curation of precursor and mature tRNA sequences. This has been challenging, mainly because RNA secondary structure and nucleotide modifications, together with tRNA gene multiplicity, complicate sequencing and sequencing read mapping efforts. To address these issues, we developed hydro-tRNAseq, a method based on partial alkaline RNA hydrolysis that generates fragments amenable for sequencing. To identify transcribed tRNA genes, we further complemented this approach with Photoactivatable Crosslinking and Immunoprecipitation (PAR-CLIP) of SSB/La, a conserved protein involved in pre-tRNA processing. Our results show that approximately half of all predicted tRNA genes are transcribed in human cells. We also report predominant nucleotide modification sites, their order of introduction, and identify tRNA leader, trailer and intron sequences. By using complementary sequencing-based methodologies, we present a human tRNA atlas, and determine expression levels of mature and processing intermediates of tRNAs in human cells.
2017-07-17 | GSE95683 | GEO
Project description:<p>During translation, transfer RNAs (tRNAs) deliver specific amino acids to the ribosome in a coordinated manner with the sequence encoded by the mRNA. Despite their central role in protein synthesis, the precise contribution of tRNAs to the modulation of translation remains poorly understood, primarily due to lack of methods to characterise tRNA abundances and their modifications from actively translating ribosomes. Here we develop RiboNano-tRNAseq, a novel method to quantify native ribosome-associated tRNAs (ribo-tRNAs) from actively translating ribosomes, achieved by coupling tag-free pulldown of active ribosomes with native tRNA nanopore sequencing. Applying RiboNano-tRNAseq, we investigate how the ribosome-associated tRNAome varies under diverse stress conditions. Our work reveals highly tailored tRNA responses depending on the stress type. For example, under leucine starvation, we observe enrichment of a subset of tRNALeu isoacceptors in ribo-tRNAs. By contrast, methionine starvation leads to a decrease in methyl-based tRNA modifications, such as m1A or m2,2G, reflecting the decreased availability of the methyl donor S-adenosylmethionine (SAM). Strikingly, arsenite starvation does not affect tRNA modification profiles, but instead causes a drastic fragmentation of tRNAs, predominantly within ribosome-associated tRNAs. Altogether, our work findings demonstrate that RiboNano-tRNAseq enables comprehensive characterization of the tRNAome and its dynamics by simultaneously capturing tRNA abundance, modification and fragmentation patterns, both in total and actively translating tRNA pools.</p>
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Project description:Transfer RNAs (tRNAs) carry abundant chemical modifications, and growing evidence shows dysregulation of chemical modifications on human tRNAs causes an expanding number of diseases including cancers, diabetes, neurological diseases, and mitochondrial disorders. Sensitive and robust detection and quantification of chemical modifications are critical to discovering functionally relevant and regulatory tRNA chemical modifications. Here we report a method “MapID-tRNA-seq” deploying a recently evolved processive reverse transcriptase to map modifications in human tRNAs and MapID-based data processing pipeline for de novo modification identification and tRNA expression quantification. MapIDs refer to consolidated sequences of human tRNA genes with reduced redundancy and annotations of genetic variation sites. MapID-based data processing largely improved the accuracy of modification detection and expression quantification by tRNA-seq, which otherwise got compromised from reads mis- and multi-alignment to the highly redundant human tRNA genome. “MapID-tRNA-seq” robustly detected the occurrence and stoichiometries of N1-methyladenosines, providing insights into the function and biosynthesis of N1-methyladenosine in human tRNAs. Our data revealed unique signatures of the evolved reverse transcriptase against N1-methyladenosine and four other types of chemical modifications. “MapID-tRNA-seq” provides a generalizable platform for de novo identification and quantitation of chemical modifications in human tRNAs to elucidate their functions in biology and diseases.
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2011-11-17 | E-GEOD-33756 | biostudies-arrayexpress
Project description:Decoding human tRNA modifications and crosstalk by enhanced single-read analysis
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Project description:tRNA modifications regulate gene expression and protein synthesis. Human tRNAome contains ~40 chemical modification types distributed on average at 13 sites in cytosolic tRNA and 6 sites in mitochondrial tRNA. tRNA modifications display dynamic patterns dependent on cell type and cell state, underscoring the need for advancing methodologies to assess their variations and crosstalks transcriptome-wide. Here, we develop enhanced single-read analysis of tRNA crosstalks (eSLAC), an integrative platform that combines the NGS sequencing method of multiplex small RNA sequencing (MSR-seq), expanded capability to measure pseudouridine (Ψ), 5-formylcytidine (f5C), and N4-acetylcytidine (ac4C), and a single-read analysis pipeline that examines the modification-modification and modification-charging crosstalks. We achieve assessments of over 60% of all human tRNA modification sites and assign Ψ sites to three Ψ writer enzymes. Both Ψ-Ψ and Ψ-charging have strong positive crosstalks. Applying polysome tRNA profiling, we identify differential tRNA isodecoder utilization and tRNA Ψ variations in the E-site versus A and P sites on the polysome. Our approach establishes a foundation for revealing the interconnectedness and the functional complexity of the tRNA modome.
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Project description:A subset of eukaryotic tRNAs is methylated in the anticodon loop to form the 3-methylcytosine (m3C) modification. In mammals, the number of tRNAs containing m3C has expanded to include mitochondrial (mt) tRNA-Ser-UGA and mt-tRNA-Thr-UGU. Whereas the enzymes catalyzing m3C formation in nuclear-encoded cytoplasmic tRNAs have been identified, the proteins responsible for m3C modification in mt-tRNAs are unknown. Here, we show that m3C formation in human mt-tRNAs is dependent upon the Methyltransferase-Like 8 (METTL8) enzyme. We find that METTL8 is a mitochondria-associated protein that interacts with mitochondrial seryl-tRNA synthetase along with mt-tRNAs containing m3C. Human cells deficient in METTL8 exhibit loss of m3C modification in mt-tRNAs but not nuclear-encoded tRNAs. Consistent with the mitochondrial import of METTL8, the formation of m3C in METTL8-deficient cells can be rescued by re-expression of wildtype METTL8 but not by a METTL8 variant lacking the N-terminal mitochondrial localization signal. Notably, METTL8-deficiency in human cells causes alterations in the native migration pattern of mt-tRNA-Ser-UGA suggesting a role for m3C in tRNA folding. Altogether, these findings demonstrate that METTL8 is required for m3C formation in mitochondrial tRNAs and uncover a potential role for m3C modification in mitochondrial tRNA structure.
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Project description:The CCA-adding enzyme adds CCA to the 3' ends of transfer RNAs (tRNAs), a critical step in tRNA biogenesis that generates the amino acid attachment site. We found that the CCA-adding enzyme plays a key role in tRNA quality control by selectively marking unstable tRNAs and tRNA-like small RNAs for degradation. Instead of adding CCA to the 3' ends of these transcripts, CCA-adding enzymes from all three kingdoms of life add CCACCA. Here, we report deep sequencing analysis of the 3' ends of tRNA-Ser-CGA and tRNA-Ser-UGA from S. cerevisiae strains and show that hypomodified mature tRNAs are subjected to CCACCA (or poly(A) addition) as part of a rapid tRNA decay pathway in vivo. We conjecture that CCACCA addtion is a universal mechanism for controlling tRNA levels and preventing errors in translation.
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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.
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