Transcriptional and Posttranscriptional Regulation of Neurospora Circadian Clock

描述（由申请人提供）：生物钟控制真核生物体中各种基本的细胞、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生物钟的分子和生化机制。丝状真菌粗糙脉孢菌（Neurospora crassa）拥有最好的生物钟系统之一，为在分子水平上探索生物钟机制提供了一个强大的实验可用系统。与高等真核生物中的昼夜节律振荡器一样，脉孢菌振荡器由一个自动调节负反馈回路组成。在这个负反馈回路中，两个白色蛋白（WC-1和WC-2）是形成WC复合物的正元件，该复合物激活频率（frq）基因的转录。负元件是FRQ和FRH，一种FRQ相互作用的RNA解旋酶，它们形成抑制WCC活性的复合物。在本提案中，我们旨在解决时钟机制的几个基本问题。 在具体目标1中，我们将确定FRQ依赖性WC磷酸化如何调节其定位和活性。本研究将为脉孢菌昼夜负反馈过程的生化机制奠定基础。我们最近发现了一个新的转录后负反馈环，其中FFC和外泌体通过控制其RNA降解来调节frq的昼夜表达。在具体目标2中，我们将确定调节昼夜节律基因表达的转录后机制。这项研究将建立一个重要的转录后过程，控制昼夜基因表达的功能重要性。在具体目标3中，我们将确定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，我们将通过遗传学和深度测序方法克隆另外两个时钟基因。新的时钟基因的识别和表征将产生新的和重要的机械见解时钟机制。这些相关但独立的目标将帮助我们从遗传、生化和分子方面阐明脉孢菌生物钟的机制。由于脉孢菌和动物生物钟之间的相似性，我们的研究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.