Deep sequencing of yeast and mouse tRNAs and tRNA fragments using OTTR
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus Saccharomyces cerevisiae
PROVIDER: GSE197651 | GEO | 2022/03/06
REPOSITORIES: GEO
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Project description:Deep sequencing of yeast and mouse tRNAs and tRNA fragments using OTTR
| PRJNA811502 | ENA
Project description:Transfer RNAs (tRNAs) and their derived small RNAs (tDRs) vary across mammalian tissues, but the . We applied Ordered Two-Template Relay sequencing (OTTR-seq) to profile small RNAs in 19 tissues from adult mice, quantifying mature tRNAs and tDRs at isodecoder resolution and inferring select base‑modification signatures from reverse‑transcriptase misincorporations. We observe tissue‑biased expression of many isodecoders and identify tDRs with greater tissue specificity than their parent tRNAs. Modification‑associated mismatch profiles are largely consistent across tissues, with notable isodecoder‑specific differences at canonical sites (e.g., A34→I, G37→m1G/wybutosine, C47d→m3C). Because RT‑based signatures do not capture all modifications without chemical pretreatments, our modification inferences represent a subset of the tRNA epitranscriptome. These data provide an isodecoder‑resolved reference for tissue‑biased tRNA and tDR regulation in mice and a framework for exploring disease‑associated remodeling of the tRNA pool. Recent studies have begun to illustrate that transfer RNA (tRNA) expression, processing, and modification can vary among different mammalian tissues and cell types. The full scope of these differences and the regulatory mechanisms that modulate production of a wide variety of tRNAs and tDRs (tRNA-derived small RNAs) across tissues, cell types, and changing cellular conditions is poorly understood. Dysregulation of these transcripts has increasingly been implicated as a critical factor in disease progression; thus, a deeper understanding of the homeostatic variance across somatic tissues is vital. Here, we systematically profile the relative changes in abundance and key sites of base modification within tRNAs, tDRs, and other small RNAs using a powerful small RNA sequencing method, Ordered Two-Template Relay sequencing (OTTR-seq), across a panel of 19 mouse tissues. By analyzing a diverse range of healthy tissues, we can better recognize tissue-biased regulation of tRNAs and identify tDRs that exhibit significantly greater tissue specificity than their corresponding source tRNAs. Furthermore, we leveraged reverse transcriptase-associated base misincorporations to construct an isodecoder-specific map of select nucleotide modifications across all mouse tRNAs. Differentially modified isodecoders can alter their translation functionality, stability, and basis for production of unique tDR profiles. This dataset provides the most comprehensive view to date of the dynamic relationships between tRNAs, their modifications, and tDRs in a mammalian model, offering an important knowledgebase for understanding tissue-specific regulation and the role of these molecules in disease.
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Project description:Here, we apply tRNA-seq and YAMAT-seq to profile the expressions of tRFs and tRNAs in plants. We provide a high-quality expression atlas of tRFs and tRNAs in Arabidopsis and rice, and uncover complex tRF population and the dynamic expressions of tRNA genes in plants.
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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.
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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. 121 samples analyzed in total, representing time courses of 10 different yeast strains; Biological replicates for each time point are included
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Project description:Specific environmental insults cause the limited fragmentation of transfer RNAs (tRNAs) into tRNA-derived small RNAs (tsRNAs), which have been implicated in a wide range of biological processes. tRNA fragmentation results from endonucleolytic activities targeting single-stranded tRNA regions. However, how a tRNA with a single hydrolyzed phosphodiester bond in the anticodon loop (‘nicked’ tRNA) gives rise to distinct tsRNAs remains poorly understood. By utilizing biochemical fractionation of tsRNA-containing ribonucleoprotein complexes (RNPs) coupled with LC-MS/MS, we identified several RNA helicase enzymes that represent putative tRNA/tsRNA processing enzymes. Furthermore, a combination of biochemical and computational approaches revealed that specific RNA helicases indeed bind specific tRNAs with high affinity, as well as process nicked tRNAs into individual tsRNAs. In summary, these findings reveal that tRNA-derived duplexes can be substrates of well-known RNA helicases, thereby expanding their potential for cellular function to the creation of individual tsRNAs during the cellular stress response.
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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:Specific environmental insults cause the limited fragmentation of transfer RNAs (tRNAs) into tRNA-derived small RNAs (tsRNAs), which have been implicated in a wide range of biological processes. tRNA fragmentation results from endonucleolytic activities targeting single-stranded tRNA regions. However, how a tRNA with a single hydrolyzed phosphodiester bond in the anticodon loop (‘nicked’ tRNA) gives rise to distinct tsRNAs remains poorly understood. After identifying RNA helicases that were able to unwind ‘nicked’ tRNAs in vitro, the specificity of one of those enzymes, DDX3X, was determined by RNA helicase assays on tRNAs, which had been subjected to recombinant Angiogenin. Both in vivo ‘nicked’ tRNAs, DDX3X-unwound ‘nicked’ tRNAs as well as heat-denatured ‘nicked’ tRNAs were subjected to small RNA sequencing.
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