Mechanism and functions of small RNA pathways in Neurospora

描述(申请人提供)：RNA干扰(RNAi)是一种转录后/转录后基因沉默机制，从真菌到人类都是如此。在RNAi途径中，大小从20-30个核苷酸(NT)的小非编码RNAs(SRNAs)，包括microRNAs(MiRNAs)和各种小干扰RNAs(SiRNAs)，结合并引导ArgAerte家族蛋白信使RNA靶标，导致多种生物过程中基因表达的沉默。丝状真菌粗糙脉孢霉是一种广泛使用基因沉默来调节基因表达的生物，它为理解RNAi途径提供了一个独特而强大的系统。我们以前已经建立了dsRNA诱导RNAi途径激活的生化机制。通过从脉孢菌中分离纯化与ArgAerte QDE-2相关的sRNAs，我们最近在该生物体中发现了三种新的sRNAs，包括DNA损伤诱导的qRNA、第一真菌miRNA样sRNAs(MilRNAs)和Diller非依赖性小干扰RNAs(DisiRNAs)。重要的是，我们的研究证实了该生物体中存在不同的sRNA生物发生途径，包括依赖和非依赖的机制，以及ArgAerte蛋白QDE-2在sRNA发生中的作用。在特定的目标1中，我们确定了DNA损伤如何诱导qRNA的产生和抑制的机制。在具体目标2中，我们将确定不同的pri-milRNAs是如何通过不同的途径被加工成成熟的milRNAs的，并将揭示将不同的milRNAs引导到不同的途径的“设计”原则。在具体目标3中，我们将确定disiRNAs的生物发生途径和功能。总之，这些研究解决了小RNA生物发生中的几个基本问题，并将扩大我们目前对sRNA生物发生途径和sRNA功能的了解。

DESCRIPTION (provided by applicant): RNA interference (RNAi) is a post-transcriptional/transcriptional gene silencing mechanism conserved from fungi to humans. In RNAi pathways, small non-coding RNAs (sRNAs) with sizes ranging from 20-30 nucleotides (nt), including microRNAs (miRNAs) and various small interfering RNAs (siRNAs), associate with and guide Argonaute family proteins to messenger RNA targets, resulting in the silencing of gene expression in diverse biological processes. The filamentous fungus Neurospora crassa, an organism that broadly employs gene silencing in regulation of gene expression, offers a unique and powerful system for understanding of RNAi pathways. We have previously established the biochemical mechanism for the dsRNA-induced activation of the RNAi pathway. By purifying the Argonaute QDE-2- associated sRNAs from Neurospora, we recently discovered three new types of sRNAs in this organism, including the DNA damage-induced qiRNA, first fungal miRNA-like sRNAs (milRNAs) and Dicer- independent small interfering RNAs (disiRNAs). Importantly, our studies demonstrated the existence of diverse sRNA biogenesis pathways in this organism, including Dicer-dependent and Dicer-independent mechanisms, and the roles of the Argonaute protein QDE-2 in sRNA biogenesis. In Specific Aim 1, we determine the mechanism of how DNA damage induces of qiRNA production and quelling. In Specific Aim 2, we will determine how different pri-milRNAs are processed into mature milRNAs by different pathways and will uncover the "design" principles that steer different milRNAs into different pathways. In Specific Aim 3, we will determine the biogenesis pathway and function of disiRNAs. Together, these studies address several fundamental questions in small RNA biogenesis and will expand our current knowledge of sRNA biogenesis pathways and sRNA function.