Functional Portraits of tRNA-derived Small Non-coding RNAs

 描述（由申请人提供）：最近关于小的非编码RNA（sncRNA）的发现显著地推进了人类遗传学和分子生物学，这主要是由于鉴定了microRNA（miRNAs）作为基因调节剂的基本作用，miRNAs是最好表征的sncRNA家族. tRNA衍生的RNA片段（tRFs）是最近发现的sncRNA家族，其在从原核生物到人类的生物体中广泛表达，但其生物学功能和这些功能的机制在很大程度上是未知的。我们小组的研究表明，呼吸道合胞病毒（RSV）诱导的sncRNA最高属于tRF家族，而miRNA表达的变化很小。这些诱导的tRF是功能性的，并且参与RSV复制的调节。值得注意的是，至少三种测试的tRF具有与miRNA的基因反式沉默功能在机制上不同的基因反式沉默功能。然而，需要更多的实验证据来确定哺乳动物tRFs对基因表达的调控是否有广泛的影响，更重要的是，tRFs使用什么机制来实现其基因调控功能。在本项目中，我们提出了一个新的假设，即RSV诱导的功能性tRFs具有共同的基因反式沉默功能。由于RSV诱导的tRF定位于细胞质中（因此不太可能在调节基因转录中发挥作用，通常是核事件），我们还假设tRF的基因抑制作用发生在转录后步骤，例如，mRNA稳定性或翻译。在这个探索性项目中，这些假设将在两个特定目标中进行测试。在目标1中，将鉴定对RSV复制、病毒基因表达和相关趋化因子/细胞因子诱导重要的tRF。然后我们将确定这些功能性tRF是否具有基因反式沉默活性。目的2将检验我们的第二个假设，即tRFs的基因抑制作用发生在转录后步骤。高通量的方法，包括微阵列或蛋白质组学分析，然后将被用来确定一个代表性的tRF的目标候选人。将通过qRT-PCR和Western印迹分析对靶候选物进行实验确认。还将进行诱变研究，以确认tRF对其靶标的影响是直接和特异性的。该项目可能具有重要的翻译意义，通过提出新的治疗机会来调节病毒复制，为建立功能性tRF数据库提供实验证据，更重要的是鉴定tRF* 靶标相互作用的共有特征以开发tRF靶标预测算法，这无疑将有助于发现新的基因调控网络，从而使研究界广泛受益。这些目标将在未来的R 01应用程序中根据该探索性项目的结果进行探索。

 DESCRIPTION (provided by applicant): Recent discoveries about small non-coding RNAs (sncRNAs) have significantly advanced human genetics and molecular biology, largely due to the identification of a fundamental role of microRNAs (miRNAs), the best- characterized sncRNA family, as gene regulators. tRNA-derived RNA Fragments (tRFs) are a recently discovered sncRNA family that is ubiquitously expressed in organisms ranging from prokaryotes to humans, yet their biological functions and the mechanism(s) underlying those functions are largely unknown. Studies by our group have shown that the sncRNAs most highly induced by respiratory syncytial virus (RSV) belong to the tRF family, while the change in miRNA expression is minimal. These induced tRFs are functional and involved in the regulation of RSV replication. Notably, at least three tested tRFs have a gene trans- silencing function that is mechanistically distinct from that of miRNAs. However, more experimental evidence is needed to determine whether there is a widespread impact of mammalian tRFs on the regulation of gene expression, and more importantly, what mechanism(s) tRFs use for their gene regulatory function. In this project we propose the novel hypothesis that functional tRFs induced by RSV have a common gene trans-silencing function. Since RSV-induced tRFs are localized in the cytoplasm (and so are unlikely to play a role in regulating gene transcription, usually a nuclear event), we also hypothesize that the gene-suppression effect of tRFs occurs at post-transcriptional steps, e.g., mRNA stability or translation. In this exploratory project, these hypotheses will be tested in two Specific Aims. In Aim 1, tRFs important for RSV replication, viral gene expression and associated chemokine/cytokine induction will be identified. We will then determine whether these functional tRFs have gene trans-silencing activity. Aim 2 will test our second hypothesis that the gene- suppression effect of tRFs occurs at post-transcriptional steps. High-throughput methods, including microarray or proteomics analyses, will then be used to identify target candidates for a representative tRF. Target candidates will be experimentally confirmed by qRT-PCR and Western blot analysis. A mutagenesis study will also be performed to confirm that the effect of the tRF on its target(s) is direct and specific. This project may hav important translational implications by suggesting new therapeutic opportunities to modulate viral replication, providing experimental evidence to build a database of functional tRFs, and of more importance to identify the consensus features of tRF*target interaction for the development of a tRF target- prediction algorithm, which will undoubtedly facilitate the discovery of new gene regulatory networks and so will broadly benefit the research community. These goals will be explored in a future R01 application based on the results of this exploratory project.