Mechanisms of Circadian Clock Control of mRNA Translation

项目摘要 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，使生物体的生理与日常环境周期相协调。 因为人类生理和行为的几乎所有方面都与时钟有关，所以时钟功能障碍 与多种疾病有关，包括睡眠障碍、心血管疾病、新陈代谢 综合症和癌症。此外，时钟还控制着许多药物的疗效和毒性。因此， 确定哪些基因和蛋白质受时钟调控，并确定其机制 调节，是理解时钟相关疾病和药物节律代谢的关键。 虽然对基因表达的昼夜节律控制的研究主要集中在转录水平 在水平上，最近的证据支持时钟在调节转录后机制中的作用。时钟是如何 调节信使核糖核酸的翻译目前还知之甚少。我们发现粗糙脉孢子虫的昼夜生物钟 控制两个高度保守的信使核糖核酸翻译中心调节因子的磷酸化。 延伸因子2(EEF2)和真核细胞起始因子2α(eIF2α)。使用高吞吐量的RNA-seq和 野生型细胞和翻译因子节律性活动缺陷的细胞的核糖体图谱，我们 发现翻译因子活动的时钟调节影响特定mRNAs的翻译，而不是起作用 在全球范围内规范所有mRNAs的翻译。在接下来的5年里，我们将利用我们的专业知识和工具 以确定这种特异性的机制。在一个令人兴奋的突破中，我们发现时钟控制着 核糖体细胞质核糖体的组成，某些核糖体蛋白在细胞内大量循环 核糖体。这些数据挑战了所有细胞质核糖体都相同的范式，而不是 提示存在具有不同功能的异源核糖体。我们将利用这些发现来 测试核糖体蛋白组成、修饰和/或相互作用发生变化的假设 附属蛋白在时钟的控制下暂时出现。阐明时钟调控的核糖体 修改机制还可能导致对时钟以外的信号可能出现的意外方式的洞察 转录后调控蛋白质表达。

Project Summary 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 nearly all aspects of human physiology and behavior are linked to the clock, clock dysfunction is associated with a wide range of diseases, including sleep disorders, cardiovascular disease, metabolic syndrome, and cancer. In addition, the clock controls the efficacy and toxicity of many drugs. Therefore, identifying what genes and proteins are regulated by the clock, and determining the mechanisms for this regulation, are key to understanding clock-associated diseases and rhythmic drug metabolism. While the primary focus of research on circadian control of gene expression has been at the transcriptional level, recent evidence supports a role for the clock in regulating posttranscriptional mechanisms. How the clock regulates mRNA translation is poorly understood. We found that the Neurospora crassa circadian clock controls the phosphorylation of two highly conserved central regulators of mRNA translation, eukaryotic elongation factor 2 (eEF2), and eukaryotic initiation factor 2α (eIF2α). Using high throughput RNA-seq and ribosome profiling in wild type cells, and cells that are defective in rhythmic activity of the translation factors, we found that clock regulation of translation factor activity affects translation of specific mRNAs, rather than acting globally to regulate translation of all mRNAs. During the next 5 years, we will leverage our expertise and tools to determine the mechanisms for this specificity. In an exciting breakthrough, we found that the clock controls the composition of cytoplasmic ribosomes, whereby certain ribosomal proteins cycle in abundance in ribosomes. These data challenge the paradigm that all cytoplasmic ribosomes are the same, and instead suggest the existence of heterologous ribosomes with distinct functions. We will capitalize on these findings to test the hypothesis that changes in ribosome protein composition, modification, and/or interactions with accessory proteins occur temporally under control of the clock. Elucidating the clock-regulated ribosome modification mechanism may also lead to insights into unexpected ways that signals other than the clock might postranscriptionally regulate protein expression.