Mechanisms of Circadian Clock and Gene Sliencing in Neurospora

 描述(由申请者提供)：这一最大化研究者研究奖将集中于对基本生物现象：生物钟和RNA干扰的机制的理解。生物钟控制着真核生物中各种基本的细胞、生理和行为过程。能够测量时间的分子机器被称为“生物钟”，它的输出是昼夜节律。我们的长期目标是了解真核生物生物钟的分子和生化机制。RNA干扰(RNAi)是一种从真菌到人类的转录后/转录后基因沉默机制。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。丝状真菌粗糙脉孢菌为生物钟和RNAi机制提供了一个强大的实验可及系统。我们以前的研究在昼夜节律和RNAi领域都做出了根本性的贡献。对于我们未来的生物钟研究，我们建议集中在生物钟机制的三个不同的关键方面。在具体目标1中，我们将确定FRQ的磷酸化如何调节其活性和结构构象。这项研究将会有所帮助 建立对脉孢子菌昼夜负反馈过程至关重要的生化机制。在特定的目标2中，我们将确定CatP如何通过调节染色质结构来调节FRQ转录的机制。在特定的目标3中，我们将确定QRF转录的反义转录如何通过转录干扰调节FRQ表达的机制。总之，这些目标利用一个成熟的模型系统来解决三个基本问题，这三个问题对于我们理解真核生物的生物钟至关重要，并将阐明脉孢子菌生物钟的遗传、生化和分子机制。由于脉孢子虫和动物时钟之间的保守性，我们的结果将为真核生物的时钟机制提供重要的见解。在未来的RNAi和小RNA研究中，我们将重点研究抑制、milRNA和非依赖于DICER的小RNA途径的机制。在特定的目标4中，我们确定了DNA损伤如何诱导qRNA产生以及qRNAs如何促进同源重组的机制。在具体目标5中，我们将确定milRNA的生产途径，并破译milRNAs的设计原则。在特定的目标6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，并将极大地扩展我们目前对sRNA生物发生途径和sRNA功能的了解。

 DESCRIPTION (provided by applicant): This Maximizing Investigators' Research Award will be focused on the understanding of mechanisms of how fundamental biological phenomena: circadian clock and RNA interference. Circadian clocks control a wide variety of fundamental cellular, physiological, and behavioral processes in eukaryotic organisms. The molecular machinery that permits the measurement of time is referred to as the "circadian clock" and its output as circadian rhythms. Our long-term goal is to understand the molecular and biochemical mechanisms of eukaryotic circadian clocks. 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 offers a powerful experimentally-accessible system for both circadian clock and RNAi mechanisms. Our previous studies have made fundamental contributions to both circadian and RNAi fields. For our future circadian clock research, we propose to focus on three different key aspects of the circadian oscillator mechanism. In Specific Aim 1, we will determine how phosphorylation of FRQ regulates its activity and its structural conformation. This study will help establish a biochemical mechanism critical for the circadian negative feedback process in Neurospora. In Specific Aim 2, we will determine the mechanism for how CATP regulates frq transcription by regulating the chromatin structure. In Specific Aim 3, we will determine the mechanism for how antisense transcription of qrf transcription regulates frq expression by transcriptional interference. Together, these objectives take advantage of a well-established model system to address three fundamental questions that are critical for our understanding of eukaryotic circadian clocks and will elucidate the genetic, biochemical, and molecular mechanism of the Neurospora clock. Because of the conservation between the Neurospora and animal clocks, our results will provide important insights into eukaryotic clock mechanisms. For our future RNAi and small RNA research, we will focus on the mechanisms of quelling, milRNA and dicer-independent small RNA pathways. In Specific Aim 4, we determine the mechanism of how DNA damage induces of qiRNA production and how qiRNAs promote homologous recombination. In Specific Aim 5, we will determine the milRNA production pathways and decipher the design principles of milRNAs. In Specific Aim 6, we will determine the biogenesis pathway and function of disiRNAs. Together, these studies address several fundamental questions in small RNA biogenesis and will significantly expand our current knowledge of sRNA biogenesis pathways and sRNA function.