Investigating Circadian Post-Transcriptional Regulation.

项目摘要/摘要： 昼夜节律是高度保守的，大约24小时的生理周期，可以进行无数次的调整 行动，影响一切，从细菌的发光到人类的睡眠。通过理想的编程 在行为方面，人们认为这些节奏通过确保许多器官功能是 与昼夜节律的适当阶段保持最佳同步。打乱正常的昼夜节律 对人类长期的医学前景产生负面影响，这使得理解这种机制变得至关重要 对细胞生理的潜在的昼夜节律调节。昼夜节律是通过高度调节的 基于转录翻译的负反馈环或时钟。当前时钟调节的范例超过 细胞生理学是指转录活动从正臂到负臂的转录-翻译 反馈环驱动了一系列基因启动子的表达，这些基因启动子调节着生物的行为。然而， 越来越多的证据表明，昼夜节律对细胞生理学的影响超出了 转录，并认为负臂可能在这一调控中发挥作用。我们工作的长期目标是 确定这种转录后调控对细胞生理的影响程度，并确定其机制 昼夜节律转录后调控的基础。 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 人们对分子钟的大部分了解都来自于对模型系统的研究。 因此，我们将利用模型系统的简单性和重复性来经济高效地解决我们的 假设。为了确定昼夜节律转录后调控的程度，我们将分析 小鼠巨噬细胞的转录组和蛋白质组的昼夜变化。因为小鼠是一种常见的 人类免疫系统，我们的研究将深入了解昼夜节律转录后的程度 以及研究对免疫系统的生物钟调节。解决机械基础问题 在转录后调控方面，我们将利用粗糙脉孢菌，一种易于生化的面包霉菌 基因操作在任何其他真核生物时钟模型系统中都是无与伦比的。我们假设 负臂可能通过瞬时的蛋白质-蛋白质相互作用来控制昼夜节律输出，这种相互作用是由 由负臂固有的灵活的生化性质实现的定时构象变化。我们 将创建昼夜负臂蛋白的构象/时间相互作用组(CTI)图来验证我们的 假设。由于时钟结构的守恒，这项工作的结果有可能定义 细胞生理学中时钟调节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.