Molecular Genetic Analysis of Fungal Circadian Rhythms

描述（由申请人提供）：生物钟是一种进化上保守的计时机制，通过调节有节奏的基因表达，协调生物体的生理与日常环境周期。由于人类生理和行为的几乎所有方面都与生物钟有关，昼夜节律系统的异常与许多疾病有关，如睡眠障碍、心血管疾病、代谢综合征和癌症。此外，生物钟还控制着药物代谢的时间方面和对细胞毒性药物的易感性。因此，了解哪些基因和蛋白质受生物钟调控，以及这种调控的机制，对于理解生物钟相关疾病和节律性药物代谢是必要的。基因表达昼夜节律控制的研究主要集中在转录水平。然而，有大量证据表明时钟在调节节奏翻译中的作用。尽管如此，基本上我们还不知道翻译是如何由时钟控制的。在之前的资助期内，我们使用神经孢子虫模型系统，发现生物钟控制两个高度保守的mRNA翻译中心调控因子的磷酸化，真核延伸因子2 （eEF2）和真核起始因子2 (eIF2)，两者在晚上的活动达到峰值。此外，我们发现该时钟控制S6激酶的水平，S6激酶是高度保守的起始因子eIF4B的关键调节因子。使用高通量RNA-seq和核糖体分析野生型细胞和翻译因子节律性活动缺陷的细胞，我们将确定时钟是否调节全部或部分蛋白质的翻译。这些研究的结果将使我们能够确定哪些蛋白质大量循环，并使用分子遗传学技术来破译时钟用于控制翻译延伸（Aim 1）和起始（Aim 2）的机制。这项研究将填补我们关于哪些蛋白质随昼夜节律积累以及这种调节如何受到控制的知识的主要空白，从而使我们更深入地了解细胞生理和代谢的哪些方面受到生物钟的调节。

DESCRIPTION (provided by applicant): The circadian clock is an evolutionarily conserved time-keeping mechanism that, through the regulation of rhythmic gene expression, coordinates the physiology of an organism with daily environmental cycles. Because virtually all aspects of human physiology and behavior are linked to the clock, abnormalities in the circadian system are associated with a wide range of diseases such as sleep disorders, cardiovascular disease, metabolic syndrome, and cancer. In addition, the clock controls temporal aspects of drug metabolism and vulnerability to cytotoxic agents. Thus, knowing what genes and proteins are regulated by the clock, and the mechanisms of this regulation, are necessary to understand clock-associated diseases and rhythmic drug metabolism. The primary focus of research on circadian control of gene expression has been at the transcriptional level. However, substantial evidence exists for a role of the clock in regulating rhythmic translation. Despite this, essentialy nothing is known about how translation is controlled by the clock. In the previous funding period, using the model system Neurospora crassa, we discovered that the circadian clock controls the phosphorylation of two highly conserved central regulators of mRNA translation, eukaryotic elongation factor 2 (eEF2), and eukaryotic initiation factor 2� (eIF2�), both peaking in activity a night. In addition, we found that the clock controls the levels of S6 kinase, a key regulator of th highly conserved initiation factor eIF4B. Using high throughput RNA-seq and ribosome profiling in wild type cells, and cells that are defective in rhythmic activity of the translation factors, w will determine if the clock regulates translation of all or just some proteins. Results from these studies will allow us to determine which proteins cycle in abundance, and using molecular genetic techniques, to decipher the mechanism used by the clock to control translation elongation (Aim 1) and initiation (Aim 2). This study will fill major gaps in our knowledge regarding which proteins accumulate with a circadian rhythm and how this regulation is controlled, and thus give us a much deeper understanding of what aspects of cell physiology and metabolism are regulated by the clock.