Circadian post-transcriptional regulatory mechanisms

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Circadian clocks are intracellular timekeeping mechanisms that allow cells to coordinate various aspects of their physiology with time of day. These clocks are self-sustained oscillators with periods of about 24 hours and they are known to drive many rhythmic processes ranging from gene expression to complex behaviors. The central feature of this circadian clock is a core transcriptional/translational negative feedback loop in which a set of "clock genes" are transcribed rhythmically and then their protein products feed back to inhibit their own synthesis. This feedback loop takes one circadian cycle (24 hours) to complete. The many rhythmic "outputs" of the clock are thought to be controlled by coordinate regulation of many other genes by the clock genes themselves. The majority of studies on this mechanism have focused on transcriptional and post-translational modifications that are important for proper clock function. However, although there is significant evidence that post-transcriptional mechanisms must also be involved in generating stable circadian rhythmicity (both at the level of the central clock and in control of various output rhythms), almost nothing is known about this level of regulation. The best candidate for mediating circadian posttranscriptional control in vertebrates is the protein nocturnin, a deadenylase (an enzyme that removes the polyA tail from mRNAs) that is expressed in clock-containing cells with high amplitude rhythms. Our central hypothesis is that nocturnin is an important regulatory mechanism of the circadian clock through its control of the half-life (or translatability) of specific rhythmic mRNAs. In this proposal, we examine the biochemical function of nocturnin in detail in four Specific Aims: (1) Characterization of the complex of proteins in which nocturnin resides; (2) Examine nocturnin's intracellular localization; (3) Identify nocturnin's target mRNAs; and (4) Determine how these target mRNAs are recognized. Circadian clocks control many critical aspects of physiology and disruptions of clocks have recently been implicated in many human diseases. The studies proposed here will contribute to the understanding of the molecular underpinnings of these clocks as well as to the general area of post-transcriptional control of gene expression.