Mechanism and functions of small RNA pathways in Neurospora

描述（由申请人提供）：RNA干扰（RNAi）是一种从真菌到人类的转录后/转录基因沉默机制。在RNAi通路中，大小在20-30个核苷酸（nt）之间的小非编码RNA (sRNAs)，包括microRNAs （miRNAs）和各种小干扰RNA (sirna)，与Argonaute家族蛋白结合并引导其到达信使RNA靶点，从而导致多种生物过程中基因表达的沉默。丝状真菌粗神经孢子菌（Neurospora crassa）是一种广泛使用基因沉默来调控基因表达的生物，它为理解RNAi途径提供了一个独特而强大的系统。我们之前已经建立了dsrna诱导RNAi途径激活的生化机制。通过从神经孢子虫中纯化Argonaute 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 （milrna）和不依赖Dicer的小干扰rna （disiRNAs）。重要的是，我们的研究证明了该生物存在多种sRNA生物发生途径，包括dicer依赖性和dicer非依赖性机制，以及Argonaute蛋白QDE-2在sRNA生物发生中的作用。在特异性目标1中，我们确定了DNA损伤如何诱导qiRNA产生和抑制的机制。在Specific Aim 2中，我们将确定不同的pri- milrna是如何通过不同的途径加工成成熟的milrna的，并将揭示引导不同milrna进入不同途径的“设计”原则。在Specific Aim 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生物发生的几个基本问题，并将扩展我们目前对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.