Investigating Circadian Post-Transcriptional Regulation.

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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.