tRNA-derived RNA Fragments (tRFs) and their Functions in Respiratory Syncytial Virus (RSV) Infection

 描述（由申请人提供）：最近对小的非编码RNA（sncRNA）的发现极大地促进了人类遗传学和分子生物学的发展，这主要是由于sncRNA作为基因调控因子的基本作用的鉴定。tRNA衍生的RNA片段（tRFs）代表最近发现的sncRNA家族，其被迅速认为在从原核生物到人类的生物体中普遍表达;然而，tRFs的生物学功能及其潜在的机制在很大程度上是未知的。呼吸道合胞病毒（RSV）是婴儿下呼吸道疾病的单一最常见原因。我们最近的出版物表明，最高度诱导RSV的sncRNA属于tRF家族。值得注意的是，至少两种最丰富的RSV诱导型tRF具有与miRNA不同的基因转沉默功能，并且这两种中的一种称为tRF 5-GluCTC，促进RSV复制并在转录后水平调节基因。这种tRF的生物发生也是特异性的，并且由特定的核酸内切酶（血管生成素，ANG）介导，而不是由其他核酸酶介导。然而，tRF 5-GluCTC的生物学功能和诱导的分子机制尚不清楚。我们在这个项目中的中心假设是，RSV诱导的tRF 5-GluCTC不是tRNA降解的随机副产物，而是宿主-RSV相互作用的重要功能分子。本项目将重点探讨tRF 5-GluCTC对RSV复制的调控作用及其宿主应答的分子机制。我们的实验将鉴定RSV感染的气道上皮细胞中tRF 5-GluCTC的靶标（目的1），确定控制tRF 5- GluCTC的反式沉默活性的病毒因子（目的2），并确定有助于ANG介导的tRF生物发生的病毒组分（目的3）。初步数据表明，RSV蛋白N和P将分别成为目标2和3的重点。该项目的总体目标是使用分子病毒学、蛋白质和RNA生物化学以及细胞和结构生物学技术的组合来阐明tRF介导的RSV复制和其引起的宿主反应的调节机制。这种新的调控应该提供一个关键的新的视角RSV-宿主相互作用，无疑有利于发现新的基因调控机制，在RSV感染。我们在抗病毒靶点鉴定、靶向机制和tRF生物发生方面的研究结果 还将为RSV感染的预防和治疗策略的设计提供重要的新见解。由于tRFs的研究尚处于早期阶段，本研究中获得的tRF 5-GluCTC的知识和技术对于探索其他tRFs在各种生物系统中的功能非常重要，因此将广泛受益于研究界。

 DESCRIPTION (provided by applicant): Recent discoveries on small non-coding RNAs (sncRNAs) have significantly advanced human genetics and molecular biology, largely due to the identification of a fundamental role of sncRNAs as gene regulators. tRNA- derived RNA Fragments (tRFs) represent a recently discovered sncRNA family that is quickly being recognized as ubiquitously expressed in organisms ranging from prokaryotes to humans; however, tRFs' biological functions and the mechanism(s) underlying them are largely unknown. Respiratory syncytial virus (RSV) is the single most common cause of lower respiratory tract illness in infants. Our recent publication has shown that the sncRNAs most highly induced by RSV belong to the tRF family. Notably, at least the two most abundant RSV-inducible tRFs have a gene trans-silencing function that is mechanistically distinct from that of miRNAs, and one of these two, called tRF5-GluCTC, promotes RSV replication and regulates genes at a post-transcriptional level. The biogenesis of this tRF is also specific, and mediated by a particular endonuclease (angiogenin, ANG), and not by other nucleases. However, the molecular mechanisms underlying the biological functions and induction of tRF5-GluCTC are not known. Our central hypothesis in this project is that the RSV-induced tRF5-GluCTC is not a random by-product of tRNA degradation, but rather a functional molecule important for host-RSV interactions. This project will focus on exploring the molecular mechanisms underlying the regulatory effects of tRF5-GluCTC on RSV replication, and the host responses thereto. Our experiments will identify the targets of tRF5-GluCTC in RSV- infected airway epithelial cells (Aim 1), determine the viral factor(s) controlling the trans-silencing activity of tRF5- GluCTC (Aim 2), and define the viral component(s) contributing to ANG-mediated biogenesis of tRFs (Aim 3). Preliminary data suggest that the RSV proteins N and P will be a focus of Aims 2 and 3, respectively. The overall goal of this project is to use a combination of molecular virology, protein and RNA biochemistry, and cellular and structural biology techniques to elucidate tRF-mediated regulatory mechanisms for both RSV replication and the host responses it elicits. This novel regulation should provide a key new perspective on RSV-host interactions and undoubtedly facilitate the discovery of new gene regulatory mechanisms in response to RSV infection. Our results on antiviral target identification, targeting mechanisms, and tRF biogenesis will also provide new insights important for the design of preventive and therapeutic strategies for RSV infection. Given the early stage of studies on tRFs, the knowledge and techniques obtained in this study on tRF5-GluCTC will be important for exploring the functions of other tRFs in various biological systems, and therefore will broadly benefit the research community.