Circadian post-transcriptional regulatory mechanisms

生物钟是细胞内的计时机制，允许细胞协调其生理的各个方面与一天中的时间。这些时钟是自我维持的振荡器，周期约为24小时，已知它们驱动从基因表达到复杂行为的许多有节奏的过程。这种生物钟的中心特征是一个核心的转录/翻译负反馈循环，在这个循环中，一组“时钟基因”被有节奏地转录，然后它们的蛋白质产物反馈到抑制自己的合成。这个反馈循环需要一个昼夜节律周期(24小时)来完成。时钟的许多有节奏的“输出”被认为是由时钟基因本身协调调节许多其他基因所控制的。大多数关于这一机制的研究都集中在转录和翻译后修饰上，这些修饰对正常的时钟功能非常重要。然而，尽管有重要的证据表明转录后机制也必须参与产生稳定的昼夜节律(无论是在中央时钟水平上还是在控制各种输出节律的水平上)，但对于这种水平的调控几乎一无所知。在脊椎动物中，介导昼夜节律转录后调控的最佳候选蛋白是Nocturin蛋白，这是一种死烯基酶(一种从mRNAs中移除Polya尾巴的酶)，它在含有时钟的细胞中以高幅度节律表达。我们的中心假设是，夜转素是生物钟的一个重要调节机制，它通过控制特定节奏mRNAs的半衰期(或可译性)。在本提案中，我们研究了 本文从四个方面详细阐述了夜光蛋白的生物化学功能：(1)研究夜光蛋白所在蛋白质的复合体；(2)研究夜光蛋白在细胞内的定位；(3)确定夜光蛋白的靶向mRNAs；以及(4)确定这些靶向mRNAs的识别方式。生物钟控制着生理学的许多关键方面，而生物钟的紊乱最近被认为与许多人类疾病有关。这里提出的研究将有助于理解这些时钟的分子基础以及转录后基因表达控制的一般领域。

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.