Transcriptional and Posttranscriptional Regulation of Neurospora Circadian Clock

描述(申请人提供)：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生物钟的分子和生化机制。丝状真菌粗糙脉孢子菌拥有最被了解的生物钟系统之一，为在分子水平上探索时钟机制提供了一个强大的实验可及系统。像高等真核生物中的昼夜节律振荡器一样，脉孢子虫振荡器由一个自我调节的负反馈环组成。在这个负反馈环中，两个白领蛋白(WC-1和WC-2)是正向元件，形成WC复合体，激活频率(FRQ)基因的转录。负性元件是FRQ和FRH，FRQ是一种与FRQ相互作用的RNA解旋酶，它们形成了抑制WCC活性的复合体。在这项提案中，我们的目标是解决时钟机制的几个基本问题。在特定的目标1中，我们将确定FRQ依赖的WC磷酸化如何调节其定位和活性。本研究将建立脉孢子菌昼夜负反馈过程的生化机制。我们最近发现了一个新的转录后负反馈环，在这个环中，FFC和Exosome通过控制FRQ的RNA降解来调节其昼夜表达。在特定的目标2中，我们将确定调控昼夜节律基因表达的转录后机制。这项研究将确定控制昼夜节律基因表达的重要转录后过程的功能重要性。在具体目标3中，我们将确定一个最近发现的调节昼夜节律周期长度和幅度的时钟基因的昼夜节律功能，并将通过遗传学和深度测序的方法克隆另外两个时钟基因。新的时钟基因的识别及其特征将产生对时钟机制的新的和重要的机制见解。这些相关但独立的目标将有助于我们从遗传学、生化和分子方面阐明脉孢子菌时钟的机制。由于脉孢子虫和动物生物钟之间的相似性，我们的结果将为真核生物生物钟的功能提供重要信息。

DESCRIPTION (provided by applicant): 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 circadian clocks. The filamentous fungus Neurospora crassa, which has one of the best understood circadian clock systems, offers a powerful experimentally-accessible system for exploring the clock mechanism at molecular levels. Like circadian oscillators in the higher eukaryotic organisms, the Neurospora oscillator consists of an autoregulatory negative feedback loop. In this negative feedback loop, two WHITE COLLAR proteins (WC-1 and WC-2) are the positive elements that form a WC complex that activate the transcription of the frequency (frq) gene. The negative elements are FRQ and FRH, a FRQ-interacting RNA helicase, which form a complex that inhibits the WCC activity. In this proposal, we aim to address several fundamental questions of the clock mechanism. In Specific Aim 1, we will determine how the FRQ-dependent WC phosphorylation regulates its localization and activity. This study will establish the biochemical mechanism for the circadian negative feedback process in Neurospora. We recently discovered a novel post-transcriptional negative feedback loop in which FFC and exosome regulate circadian expression of frq by controlling its RNA degradation. In Specific Aim 2, we will determine the post-transcriptional mechanism that regulates circadian gene expression. This study will establish the functional importance of an important post-transcriptional process that controls circadian gene expression. In Specific Aim 3, we will determine the circadian function of a recently identified clock gene that regulates circadian period length and amplitude, and we will clone two additional clock genes by genetics and deep sequencing methods. The identification of new clock genes and their characterization will yield novel and important mechanistic insights into clock mechanisms. These related but independent objectives will help us to elucidate the mechanism of the Neurospora clock in genetic, biochemical, and molecular terms. Because of the similarities between the Neurospora and animal clocks, our results will provide important information on how eukaryotic circadian clocks function.