Circadian clocks are internal timekeeping mechanisms that temporally coordinate many aspects of behavior and physiology with the diurnal cycle. These clocks control thousands of genes that drive many critical processes within the cell, including many metabolic processes. Furthermore, nutrient intake and many metabolic processes also feedback to regulate the central clock mechanism and disruption of the clock results in broad metabolic dysfunction. An important role of the clock may be to temporally coordinate metabolic programs throughout the circadian cycle to increase efficiency and avoid futile reactions. In this proposal, we provide evidence that expands the influence of the clock to yet another critical metabolic component of the cell - the oxidative phosphorylation system of the mitochondrion. We present new evidence demonstrating that the circadian protein Nocturnin is regulating the activity of cytochrome c oxidase (Complex IV) of the electron transport chain. Nocturnin encodes a deadenylase, an enzyme responsible for removing poly A tails from mRNAs, and in this proposal we present data that shows that Nocturnin works in the mitochondria (in addition to its known role in the cytoplasm) to post-transcriptionally control one of the mRNAs that is critical for Complex IV assembly and function. Based on these data, we propose three specific aims designed to test the general hypothesis that the circadian clock regulates oxidative phosphorylation through rhythmic Nocturnin deadenylation of mitochondria-encoded mRNAs. In the first specific aim, we will examine how Nocturnin intracellular localization is regulated and test whether alternative translation initiation regulates whether or not a mitochondrial targeting signal is added to the Nocturnin protein. In the second aim, we will examine the mechanism by which Nocturnin regulates Complex IV activity and mitochondrial oxidative phosphorylation. We will test whether Nocturnin is part of a """"""""machine"""""""" that dynamically regulates mitochondrial mRNA poly A tail length and test whether this results in altered translation of the target mRNA. Finally, in the third aim, we will test whether these processes are directly regulated by the circadian clock and will determine the kinetics of Complex IV assembly at different times of day. Furthermore, we will test whether Nocturnin's rhythmicity is important for proper functioning of the oxidative phosphorylation system using an inducible mouse model that expresses Nocturnin in a non-rhythmic manner. These proposed studies will uncover the mechanism behind this new and exciting role, for the circadian system control of the ATP generating system of the cell. One in 5000 humans have mutations that affect Complex IV assembly and activity resulting in severe disease, including encephalomyopathies, neurodegenerative Leigh syndrome, hypertrophic cardiomyopathies, and fatal lactic acidosis. Therefore, understanding how this system is modulated is of critical importance.
The circadian clock controls many aspects of behavior and physiology and is critical for the maintenance of normal metabolism. We present new evidence that the clock also controls basic cellular respiration. This proposal will determine the molecular mechanism by which the clock controls this critical cellular process.
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