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Production and purification of endogenously modified tRNA-derived small RNAs.

ABSTRACT: During particular stress conditions, transfer RNAs (tRNAs) become substrates of stress-induced endonucleases, resulting in the production of distinct tRNA-derived small RNAs (tsRNAs). These small RNAs have been implicated in a wide range of biological processes, but how isoacceptor and even isodecoder-specific tsRNAs act at the molecular level is still poorly understood. Importantly, stress-induced tRNA cleavage affects only a few tRNAs of a given isoacceptor or isodecoder, raising the question as to how such limited molecule numbers could exert measurable biological impact. While the molecular function of individual tsRNAs is likely mediated through association with other molecules, addressing the interactome of specific tsRNAs has only been attempted by using synthetic RNA sequences. Since tRNAs carry post-transcriptional modifications, tsRNAs are likely modified but the extent of their modifications remains largely unknown. Here, we developed a biochemical framework for the production and purification of specific tsRNAs using human cells. Preparative scale purification of tsRNAs from biological sources should facilitate experimentally addressing as to how exactly these small RNAs mediate the multitude of reported molecular functions.

SUBMITTER: Drino A

PROVIDER: S-EPMC7549616 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Production and purification of endogenously modified tRNA-derived small RNAs.

Drino Aleksej A Oberbauer Vera V Troger Conor C Janisiw Eva E Anrather Dorothea D Hartl Markus M Kaiser Steffen S Kellner Stefanie S Schaefer Matthias R MR

RNA biology 20200305 8

During particular stress conditions, transfer RNAs (tRNAs) become substrates of stress-induced endonucleases, resulting in the production of distinct tRNA-derived small RNAs (tsRNAs). These small RNAs have been implicated in a wide range of biological processes, but how isoacceptor and even isodecoder-specific tsRNAs act at the molecular level is still poorly understood. Importantly, stress-induced tRNA cleavage affects only a few tRNAs of a given isoacceptor or isodecoder, raising the question ...[more]

PMID: 32138588

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Project description:tRNA‑derived small RNAs (tsRNAs) have been shown to play regulatory roles in many physiological and pathological processes. However, their roles in hypertrophic scars remain unclear. The present study investigated differentially expressed tsRNAs in human hypertrophic scar fibroblasts and normal skin fibroblasts via high‑throughput sequencing. Several dysregulated tsRNAs were validated by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, target prediction, coexpression networks and competing endogenous RNA (ceRNA) networks were evaluated to discover the principal functions of significantly differentially expressed tsRNAs. In total, 67 differentially expressed tsRNAs were detected, of which 27 were upregulated and 40 downregulated in hypertrophic scar fibroblasts. The GO analysis indicated that the dysregulated tsRNAs are associated with numerous biological functions, including 'nervous system development', 'cell adhesion', 'focal adhesion', 'protein binding', 'angiogenesis' and 'actin binding'. KEGG pathway analysis revealed that the most altered pathways include 'Ras signaling pathway', 'Rap1 signaling pathway' and 'cGMP‑PKG signaling pathway'. The target genes of the differentially expressed tsRNAs participate in several signaling pathways important for scar formation. The results of RT‑qPCR were consistent with those of sequencing. MicroRNA (miR)‑29b‑1‑5p was identified as a target of tsRNA‑23678 and was downregulated in hypertrophic scar fibroblasts, constituting a negative regulatory factor for scar formation. Furthermore, tsRNA‑23761 acted as a ceRNA and bound to miR‑3135b to regulate the expression of miR‑3135b targets, including angiotensin‑converting enzyme. Collectively, these findings reveal that tsRNAs are differentially expressed in human hypertrophic scar fibroblasts, and may contribute to the molecular mechanism and treatment of hypertrophic scars.

Project description:Non-coding RNAs (ncRNAs) are a type of RNA that is not translated into proteins. Transfer RNAs (tRNAs), a type of ncRNA, are the second most abundant type of RNA in cells. Recent studies have shown that tRNAs can be cleaved into a heterogeneous population of ncRNAs with lengths of 18-40 nucleotides, known as tRNA-derived small RNAs (tsRNAs). There are two main types of tsRNA, based on their length and the number of cleavage sites that they contain: tRNA-derived fragments and tRNA-derived stress-induced RNAs. These RNA species were first considered to be byproducts of tRNA random cleavage. However, mounting evidence has demonstrated their critical functional roles as regulatory factors in the pathophysiological processes of various diseases, including neurological diseases. However, the underlying mechanisms by which tsRNAs affect specific cellular processes are largely unknown. Therefore, this study comprehensively summarizes the following points: (1) The biogenetics of tsRNA, including their discovery, classification, formation, and the roles of key enzymes. (2) The main biological functions of tsRNA, including its miRNA-like roles in gene expression regulation, protein translation regulation, regulation of various cellular activities, immune mediation, and response to stress. (3) The potential mechanisms of pathophysiological changes in neurological diseases that are regulated by tsRNA, including neurodegeneration and neurotrauma. (4) The identification of the functional diversity of tsRNA may provide valuable information regarding the physiological and pathophysiological mechanisms of neurological disorders, thus providing a new reference for the clinical treatment of neurological diseases. Research into tsRNAs in neurological diseases also has the following challenges: potential function and mechanism studies, how to accurately quantify expression, and the exact relationship between tsRNA and miRNA. These challenges require future research efforts.

Dataset Information

Production and purification of endogenously modified tRNA-derived small RNAs.

Publications

Production and purification of endogenously modified tRNA-derived small RNAs.

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