Mechanisms of Circadian Clock Control of mRNA Translation

项目摘要 生物钟是一种进化上保守的时间保持机制，通过调节生物钟， 节律性基因表达，使生物体的生理与日常环境周期相协调。 因为几乎所有人类生理和行为的方面都与生物钟有关，生物钟功能障碍是 与多种疾病相关，包括睡眠障碍、心血管疾病、代谢紊乱、 综合征和癌症。此外，生物钟控制着许多药物的疗效和毒性。因此，我们认为， 确定哪些基因和蛋白质受生物钟的调节，并确定其机制 调节，是理解生物钟相关疾病和节律性药物代谢的关键。 虽然基因表达的昼夜节律控制的研究主要集中在转录水平， 水平，最近的证据支持时钟在调节转录后机制中的作用。时钟如何 调节mRNA翻译的机制知之甚少。我们发现粗糙脉孢菌的生物钟 控制两个高度保守的mRNA翻译的中央调节器的磷酸化，真核生物 延伸因子2（eEF 2）和真核起始因子2α（eIF 2 α）。使用高通量RNA-seq和 在野生型细胞和翻译因子节律性活性缺陷的细胞中， 发现翻译因子活性的时钟调节影响特定mRNA的翻译，而不是作用于 在全球范围内调节所有mRNA的翻译。在未来5年，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 细胞质核糖体的组成，其中某些核糖体蛋白质在细胞内大量循环， 核糖体这些数据挑战了所有细胞质核糖体都是相同的范式， 表明存在具有不同功能的异源核糖体。我们将利用这些发现， 检验核糖体蛋白质组成、修饰和/或与 辅助蛋白在时间上受生物钟的控制。阐明时钟调节核糖体 修改机制还可以导致对除了时钟之外的信号可能 转录后调节蛋白质表达。

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.