Posttranscriptional Regulation of the Neurospora Circadian Clock

描述(申请人提供)：生物钟在几乎所有真核生物中调节各种各样的细胞、生理和行为活动。生物钟在人类生理和心理健康中的重要性从它对广泛的细胞和生理过程的普遍影响中可见一斑。众所周知，时钟的故障与几种形式的人类睡眠障碍和精神疾病有关。与高等真核生物中的昼夜节律振荡器类似，脉孢子虫昼夜节律振荡器具有自我调节的负反馈环。在这个负反馈环中，白领-1(WC-1)和WC-2是正向元件，形成WC复合体(WCC)，激活频率(FRQ)基因的转录。我们最近发现FRQ相互作用的RNA解旋酶FRH是脉孢子虫生物钟的另一个核心组成部分。我们发现FRQ-FRH复合体(FFC)在昼夜负反馈环中扮演负元件的角色，通过促进CK-1a和CKII介导的WC磷酸化来抑制WCC的活性。我们还发现WCC的磷酸化负向调节其DNA结合活性。此外，我们还发现蛋白磷酸酶2A(PP2A)对昼夜节律负反馈环的功能起着重要作用。在特定的目标1中，我们将确定FFC如何通过促进WC的磷酸化来抑制WCC的活性。我们将确定FFC依赖的WCC磷酸化事件，并检测它们的功能重要性。我们将在体外重建FFC依赖的WCC抑制。在特定的目标2中，我们将通过遗传和生化方法来确定PP2A和其他磷酸酶在昼夜负反馈环路中的作用。在特定的目标3中，我们将确定FRH在FRQ mRNA转录后调控中的作用。我们将确定外切体和FRH在调节FRQ mRNA降解中的作用。我们还将确定FRQ在这一进程中的作用。由于脉孢子虫的生物钟与高等真核生物的生物钟相似，我们在脉孢子虫中的研究结果将对理解真核生物的生物钟机制具有重要的意义。

DESCRIPTION (provided by applicant): Circadian clocks regulate a wide variety of cellular, physiological, and behavioral activities in almost all eukaryotic organisms. The importance of the circadian clock in human physiology and mental health is evident from its ubiquitous influence on a wide range of cellular and physiological processes. The malfunction of the clock is known to be associated with several forms of human sleep disorders and psychiatric illness. Similar to the circadian oscillators in the higher eukaryotic organisms, the Neurospora circadian oscillator has an autoregulatory negative feedback loop. In this negative feedback loop, WHITE COLLAR-1 (WC-1) and WC-2 are the positive elements that form a WC complex (WCC) that activate the transcription of the frequency (frq) gene. We recently identified a FRQ-interacting RNA helicase, FRH, as another core component of the Neurospora circadian clock. We showed that the FRQ-FRH complex (FFC) acts as the negative element in the circadian negative feedback loop that inhibits WCC activity by promoting CK-1a- and CKII-mediated WC phosphorylation. We also showed that WCC phosphorylation negatively regulates its DNA binding activity. In addition, we found that protein phosphatase 2A (PP2A) is important for the function of circadian negative feedback loop. In Specific Aim 1, we will determine how FFC inhibits the WCC activity by promoting WC phosphorylation. We will determine the FFC-dependent WCC phosphorylation events and examine their functional importance. We will reconstitute the FFC-dependent WCC inhibition in vitro. In Specific Aim 2, we will determine the roles of PP2A and other phosphatases in the circadian negative feedback loop by genetic and biochemical approaches. In Specific Aim 3, we will determine the role of FRH in the posttranscriptional regulation of frq mRNA. We will determine the role of exosome and FRH in the regulation of frq mRNA degradation. We will also determine the role of FRQ in this process. Because of the similarities between the Neurospora circadian clock and those of higher eukaryotes, our results in Neurospora will have important implications for the understanding of eukaryotic circadian clock mechanisms.