Mechanisms of Circadian Clock and Gene Sliencing in Neurospora

 描述（由申请人提供）：这个最大化的研究者研究奖将集中在如何基本生物现象的机制的理解：昼夜节律钟和RNA干扰。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。允许测量时间的分子机制被称为“昼夜节律钟”，其输出为昼夜节律。我们的长期目标是了解真核生物钟的分子和生化机制。RNA干扰（RNA interference，RNAi）是一种转录后/转录后基因沉默机制，从真菌到人类都存在。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为生物钟和RNAi机制提供了一个强大的实验系统。我们以前的研究对昼夜节律和RNAi领域都做出了重要贡献。对于我们未来的生物钟研究，我们建议专注于生物钟振荡器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及其结构构象。这项研究将帮助 建立了脉孢菌昼夜负反馈过程的关键生化机制。在具体目标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。在具体目标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，这些目标利用了一个完善的模型系统，以解决三个基本问题，这是我们理解真核生物昼夜节律钟的关键，并将阐明脉孢菌时钟的遗传，生化和分子机制。由于脉孢菌和动物时钟之间的保守性，我们的研究结果将提供重要的见解真核生物的时钟机制。对于我们未来的RNAi和小RNA研究，我们将重点关注quelling，milRNA和dicer非依赖性小RNA途径的机制。在具体目标4中，我们确定了DNA损伤如何诱导qiRNA产生以及qiRNA如何促进同源重组的机制。在Specific Aim 5中，我们将确定milRNA的产生途径并破译milRNA的设计原理。在具体目标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生物合成中的几个基本问题，并将显着扩展我们目前对sRNA生物合成途径和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.