Production and purification of endogenously modified tRNA-derived small RNAs

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.

在特定应激条件下，转运RNA（transfer RNAs, tRNAs）会成为应激诱导核酸内切酶的底物，进而产生不同的tRNA衍生小RNA（tRNA-derived small RNAs, tsRNAs）。这类小RNA已被证实参与诸多生物学过程，但针对同工受体tRNA（isoacceptor）乃至同功能异构体tRNA（isodecoder）特异性tsRNAs的分子作用机制，目前仍不甚明晰。值得注意的是，应激诱导的tRNA剪切仅会影响特定同工受体或同功能异构体tRNA中的少数分子，这引发了一个关键科学问题：如此有限的分子数量如何能产生可观测的生物学效应？尽管单个tsRNA的分子功能可能通过与其他分子结合来介导，但此前仅通过合成RNA序列来尝试解析特定tsRNA的相互作用组。由于tRNA携带有转录后修饰，tsRNAs大概率也带有此类修饰，但其修饰程度在很大程度上仍未明确。本研究建立了一套基于人类细胞生产和纯化特定tsRNAs的生化实验框架。从生物样本中规模化制备纯化tsRNAs，将助力通过实验明确这类小RNA究竟如何介导诸多已被报道的分子功能。