Investigating Circadian Post-Transcriptional Regulation.

项目概要/摘要： 昼夜节律是高度保守的，大约24小时，生理周期，调整无数 影响着从细菌发光到人类睡眠的一切。通过理想的编程 人们相信，这些节律通过确保许多生物体功能被维持， 与昼夜节律的适当阶段最佳地同步。扰乱正常的昼夜节律 对人类的长期医学前景产生负面影响，因此了解其机制至关重要 在细胞生理学上的昼夜节律调节。昼夜节律是通过一个高度调节的 基于转录-翻译的负反馈回路或时钟。目前的时钟调节模式， 细胞生理学是转录-翻译负臂的正臂的转录活性 反馈环驱动调节生物体行为的基因启动子的表达。然而，在这方面， 越来越多的证据表明，昼夜节律调节被赋予细胞生理学， 转录和负臂可能在这种调节中发挥作用。我们工作的长期目标是 确定这种转录后调节对细胞生理学的影响程度，并确定其机制。 昼夜节律转录后调节的基础。 作为一种保持时间的机制，转录-翻译负反馈环是高度保守的 我们对分子钟的了解，大部分来自对模型系统的研究。 因此，我们将利用模型系统的简单性和可重复性来经济有效地解决我们的问题。 假设为了确定昼夜转录后调节的程度，我们将分析 转录组和蛋白质组的小鼠巨噬细胞在昼夜节律的时间。由于小鼠是一种常见的模型， 人类免疫系统，我们的研究将获得深入了解昼夜转录后的程度 调节以及研究免疫系统的时钟调节。为了解决机械基础问题， 我们将利用粗糙脉孢菌（Neurospora crassa），一种面包霉菌， 基因操纵在其他真核生物的时钟模型系统中是无与伦比的。我们假设 负臂可以通过瞬时蛋白质-蛋白质相互作用来控制昼夜节律输出， 由负臂固有的灵活的生化性质所实现的定时构象变化。我们 将创建一个昼夜节律负臂蛋白的构象/时间相互作用组（CTI）图谱，以验证我们的 假说.由于时钟结构的保守性，这项工作的结果有可能定义 在细胞生理学的时钟调节中的几个新的和未被认识的范例。

Project Summary/Abstract: Circadian rhythms are highly conserved, roughly 24-hour, physiological cycles that adjust innumerable actions, affecting everything from luminescence in bacteria to sleep in humans. Through the ideal programming of behavior, it is believed that these rhythms enhance fitness by ensuring that many organismal functions are optimally synchronized with the appropriate phase of the circadian day. Disruption of proper circadian timing negatively impacts the human long-term medical outlook, making it critical to understand the mechanism underlying circadian regulation over cellular physiology. Circadian rhythms are controlled via a highly-regulated transcription-translation based negative feedback loop, or clock. The current paradigm for clock regulation over cellular physiology is that transcriptional activity from the positive arm of the transcription–translation negative feedback loop drives the expression of a host of gene promoters that modulate organismal behavior. However, mounting evidence suggests that circadian regulation is imparted on cellular physiology beyond the level of transcription and that the negative arm may play a role in this regulation. The long-term goal of our work is to determine the extent of this post-transcriptional regulation on cellular physiology and to identify the mechanistic underpinnings of circadian post-transcriptional regulation. As a mechanism for keeping time, transcription–translation negative feedback loops are highly conserved and much of what is understood about the molecular clock comes from the investigation of model systems. Therefore, we will exploit the simplicity and reproducibility of model systems to cost-effectively address our hypotheses. To determine the extent of circadian post-transcriptional regulation, we will analyze the transcriptome and proteome of murine macrophages over circadian time. As mice are a common model for the human immune system, our study will garner insights into both the extent of circadian post-transcriptional regulation as well as investigate clock regulation on the immune system. To tackle the mechanistic underpinnings of post-transcriptional regulation, we will utilize Neurospora crassa, a bread mold whose ease of biochemical and genetic manipulation is unparalleled in any other eukaryotic clock model system. We hypothesize that the negative arm may control circadian output via transient protein-protein interactions, which are synchronized by timed conformational changes that are enabled by the negative arm’s inherently flexible biochemical nature. We will create a Conformational/Temporal Interactome (CTI) map of circadian negative arm proteins to validate our hypothesis. Due to the conservation of clock architecture, the results of this work have the potential to define several novel and unrecognized paradigms in clock regulation over cellular physiology.