Posttranscriptional Regulation of the Neurospora Circadian Clock

描述（由申请人提供）：生物钟调节几乎所有真核生物中的各种细胞、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其对广泛的细胞和生理过程的普遍影响中显而易见。已知生物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振荡器类似，脉孢菌昼夜振荡器具有自动调节负反馈回路。在这个负反馈回路中，白色-1（WC-1）和WC-2是形成WC复合物（WCC）的正元件，其激活频率（frq）基因的转录。我们最近确定了一个FRQ相互作用的RNA解旋酶，FRH，作为另一个核心组成部分的脉孢菌昼夜节律钟。我们发现FRQ-FRH复合物（FFC）作为昼夜负反馈回路中的负元件，通过促进CK-1a和CKII介导的WC磷酸化来抑制WCC活性。我们还发现，WCC磷酸化负调控其DNA结合活性。此外，我们还发现蛋白磷酸酶2A（PP 2A）在昼夜负反馈环的功能中起重要作用。在具体目标1中，我们将确定FFC如何通过促进WC磷酸化来抑制WCC活性。我们将确定FFC依赖的WCC磷酸化事件，并检查其功能的重要性。我们将在体外重建FFC依赖的WCC抑制。在具体目标2中，我们将通过遗传和生物化学方法确定PP 2A和其他磷酸酶在昼夜负反馈回路中的作用。在具体目标3中，我们将确定FRH在frq mRNA转录后调控中的作用。我们将确定外泌体和FRH在调节frq mRNA降解中的作用。我们还将确定FRQ在此过程中的作用。由于脉孢菌的生物钟与高等真核生物的生物钟之间的相似性，我们在脉孢菌中的研究结果将对理解真核生物的生物钟机制具有重要意义。

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.