Circadian clocks regulate a wide variety of cellular, physiological, and behavioral activities in almost all eukaryotic organisms. The importance of the circadian clock in human physiology and mental health is evident from its ubiquitous influence on a wide range of cellular and physiological processes. The malfunction of the clock is known to be associated with several forms of human sleep disorders and psychiatric illness. Similar to the circadian oscillators in the higher eukaryotic organisms, the Neurospora circadian oscillator has an autoregulatory negative feedback loop. In this negative feedback loop, WHITE COLLAR-1 (WC-1) and WC-2 are the positive elements that form a WC complex (WCC) that activate the transcription of the frequency (frq) gene. We recently identified a FRQ-interacting RNA helicase, FRH, as another core component of the Neurospora circadian clock. We showed that the FRQ-FRH complex (FFC) acts as the negative element in the circadian negative feedback loop that inhibits WCC activity by promoting CK-1a- and CKII-mediated WC phosphorylation. We also showed that WCC phosphorylation negatively regulates its DNA binding activity. In addition, we found that protein phosphatase 2A (PP2A) is important for the function of circadian negative feedback loop.
In Specific Aim 1, we will determine how FFC inhibits the WCC activity by promoting WC phosphorylation. We will determine the FFC-dependent WCC phosphorylation events and examine their functional importance. We will reconstitute the FFC-dependent WCC inhibition in vitro.
In Specific Aim 2, we will determine the roles of PP2A and other phosphatases in the circadian negative feedback loop by genetic and biochemical approaches.
In Specific Aim 3, we will determine the role of FRH in the posttranscriptional regulation of frq mRNA. We will determine the role of exosome and FRH in the regulation of frq mRNA degradation. We will also determine the role of FRQ in this process. Because of the similarities between the Neurospora circadian clock and those of higher eukaryotes, our results in Neurospora will have important implications for the understanding of eukaryotic circadian clock mechanisms. ? ? ?
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