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

 描述(由申请人提供)：最近在小非编码RNA(SncRNAs)方面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SNcRNAs作为基因调节因子的基本角色的确定。TRNA衍生的RNA片段(TRFs)是最近发现的一个SNcRNA家族，迅速被认为在从原核生物到人类的各种生物中普遍表达；然而，TRFs的生物学功能和机制(S)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最常见的单一原因。我们最近的研究表明，受RSV诱导最强的SncRNAs属于TRF家族。值得注意的是，至少有两种最丰富的RSV诱导的TRFs具有与miRNAs不同的基因反式沉默功能，其中一种被称为tRF5-GluCTC，它促进RSV的复制并在转录后水平上调节基因。这种TRF的生物发生也是特异的，由特定的内切酶(血管生成素，Ang)介导，而不是由其他核酸酶介导。然而，tRF5-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tRF5-GluCTC不是tRNA降解的随机副产物，而是一个对宿主-RSV相互作用重要的功能分子。本项目将重点探索tRF5-GluCTC对RSV复制的调控作用的分子机制，以及宿主对其的反应。我们的实验将确定呼吸道合胞病毒感染的呼吸道上皮细胞中tRF5-GluCTC的靶点(目标1)，确定控制tRF5-GluCTC反式沉默活性的病毒因子(S)(目标2)，并确定参与Ang介导的TRFs生物发生的病毒成分(S)(目标3)。初步数据表明，RSV蛋白N和P将分别成为AIMS 2和3的重点。该项目的总体目标是结合分子病毒学、蛋白质和RNA生物化学以及细胞和结构生物学技术来阐明TRF介导的RSV复制及其引起的宿主反应的调节机制。这一新的调控机制将为研究RSV与宿主的相互作用提供一个新的视角，并无疑有助于发现新的基因调控机制来应对RSV感染。我们在抗病毒靶标识别、靶向机制和TRF生物发生方面的研究结果 这也将为设计RSV感染的预防和治疗策略提供重要的新见解。鉴于对TRFs的研究还处于早期阶段，本研究中获得的关于tRF5-GluCTC的知识和技术将对探索其他TRFs在各种生物系统中的功能非常重要，因此将使研究界广泛受益。

 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.