Mechanism and functions of small RNA pathways in Neurospora

描述（由申请人提供）：RNA干扰（RNAi）是从真菌到人类保守的转录后/转录基因沉默机制。在RNAi途径中，大小在20-30个核苷酸（nt）范围内的小的非编码RNA（sRNA），包括microRNA（miRNAs）和各种小干扰RNA（siRNAs），与Argonaute家族蛋白质缔合并将其引导至信使RNA靶标，导致在多种生物过程中基因表达的沉默。丝状真菌粗糙脉孢菌是一种广泛利用基因沉默来调控基因表达的生物体，它为理解RNAi途径提供了一个独特而强大的系统。我们先前已经建立了dsRNA诱导的RNAi途径激活的生化机制。通过从脉孢菌中纯化Argonaute QDE-2相关sRNA，我们最近在该生物体中发现了三种新类型的sRNA，包括DNA损伤诱导的qiRNA、第一种真菌miRNA样sRNA（milRNA）和Dicer非依赖性小干扰RNA（disiRNA）。重要的是，我们的研究证明了在这种生物体中存在多种sRNA生物合成途径，包括Dicer依赖性和Dicer非依赖性机制，以及Argonaute蛋白QDE-2在sRNA生物合成中的作用。在具体目标1中，我们确定了DNA损伤如何诱导qiRNA产生和抑制的机制。在具体目标2中，我们将确定不同的pri-milRNA如何通过不同的途径加工成成熟的milRNA，并将揭示引导不同milRNA进入不同途径的“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生物合成中的几个基本问题，并将扩大我们目前对sRNA生物合成途径和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.