Characterization of tRNA-derived RNA Fragments (tRFs) in Respiratory Syncytial Vi

描述(申请人提供)：具有调节功能的小非编码RNA(SncRNAs)的发现是生物学上最近的一项突破。在SncRNAs中，microRNAs(MiRNAs)和病毒来源的SncRNAs已成为控制病毒复制和宿主对病毒感染反应的关键元件。然而，其他类型的SncRNAs在病毒感染后的表达模式和功能方面完全没有被探索。为了确定SncRNAs的表达模式，以及发现应对病毒感染的新的调控SncRNAs，我们对感染了人类呼吸道合胞病毒(RSV)的细胞进行了下一代测序(NGS)，RSV是导致婴幼儿肺部和呼吸道感染的最常见的呼吸道粘膜病毒，与未感染的细胞相比。我们发现，RSV感染导致大量tRNA衍生的RNA片段(TRFs)的产生，这些片段长约30个核苷酸(NTs)，相当于成熟tRNAs的5‘-一半(TRF5)。TRF5的诱导是病毒特异性和RSV复制依赖的。这些tRF5中至少有一个成员来自tRNA-Glu-CTC(tRF5-GluCTC)，它降低了细胞质中靶mRNAs的水平。它在RSV感染中也起着关键作用，因为RSV的复制通过它的抑制而减弱，但通过它的异位表达而促进。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。该项目的中心假设是，TRFs不是tRNA降解的随机副产品，而是功能分子。这一中心假说将通过确定呼吸道合胞病毒感染的呼吸道上皮细胞中tRF5-GluCTC的靶标(目标1)、确定控制TRFs反式沉默活性的病毒因子(S)(目标2)以及确定促进Ang介导的TRFs的生物发生的病毒成分(S)来解决(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。值得注意的是，尽管RSV对公共卫生具有重要意义，但目前还没有有效的治疗干预措施或疫苗可用。因此，本项目的总体目标是结合分子病毒学、蛋白质和RNA生物化学、细胞和结构生物学技术，确定这种新型TRF对RSV复制的调控作用的分子机制。本项目的研究结果将为研究宿主和RSV之间的相互作用提供重要的新视角和新的调控机制，并为基于TRFs与其靶标(宿主和病毒来源的相互作用)和TRFs的生物发生机制的生物标志物、治疗干预和疫苗设计的未来发展奠定基础。

DESCRIPTION (provided by applicant): The discovery of small non-coding RNAs (sncRNAs) with regulatory functions is a recent breakthrough in biology. Among sncRNAs, microRNAs (miRNAs) and virus-derived sncRNAs have emerged as elements of critical importance in controlling viral replication and host responses to viral infection. However, the expression patterns and functional aspects of other types of sncRNAs after viral infection are completely unexplored. To define the expression patterns of sncRNAs, as well as to discover novel regulatory sncRNAs in response to viral infection, we applied next-generation sequencing (NGS) to cells infected with human respiratory syncytial virus (RSV), the most common respiratory mucosal virus that causes lung and airway infections in infants and young children, in comparison to uninfected cells. We found that RSV infection leads to abundant production of tRNA-derived RNA fragments (tRFs) that are approximately 30 nucleotides (nts) long and correspond to the 5'- half of mature tRNAs (tRF5). The induction of tRF5 is virus-specific and RSV replication-dependent. At least one member of these tRF5s, which is derived from tRNA-Glu-CTC (tRF5-GluCTC), decreases the level of target mRNAs in the cytoplasm. It also plays a critical role in RSV infection, as RSV replication is attenuated by its suppression, but promoted by its ectopic expression. The biogenesis of this tRF is also specific, and mediated by a particular endonuclease (angiogenin, ANG), and not by other nucleases. The central hypothesis of this project is that tRFs are not a random by-product of tRNA degradation, but rather are functional molecules. This central hypothesis will be addressed by identifying the targets of tRF5-GluCTC in RSV-infected airway epithelial cells (Aim 1), determining the viral factor(s) controlling the trans-silencing activity of tRFs (Aim 2), and defining 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. Of note, despite the public health importance of RSV, no effective therapeutic interventions or vaccines are available. Therefore, the overall goal of this project is to use a combination of molecular virology, protein and RNA biochemistry, cellular and structural biology techniques, to identify the molecular mechanisms underlying the regulatory effects of this novel tRF on RSV replication. The results of this project will provide an important new perspective and novel regulatory mechanisms to study the interactions between the host and RSV, and lay the foundation for the future development of biomarkers, therapeutic interventions, and vaccine designs based on the interaction of tRFs with their targets, both host and virus-derived, and the biogenic mechanisms of tRFs.