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，并且已知它们驱动从基因表达到复杂行为的许多节律过程。这种生物钟的中心特征是一个核心的转录/翻译负反馈环，其中一组“时钟基因”有节奏地转录，然后它们的蛋白质产物反馈以抑制它们自己的合成。这个反馈循环需要一个昼夜循环（24小时）才能完成。生物钟的许多有节奏的“输出”被认为是由生物钟基因自身对许多其他基因的协调调节控制的。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，这些修饰对正确的时钟功能很重要。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（在中央时钟水平和控制各种输出节律），几乎没有什么是已知的这种水平的调节。在脊椎动物中，介导昼夜节律转录后控制的最佳候选者是蛋白质β-turnin，这是一种去腺苷酸酶（一种从mRNA中去除polyA尾的酶），在具有高振幅节律的含时钟细胞中表达。我们的中心假设是，β-turnin是一个重要的调节机制的生物钟，通过其控制的半衰期（或翻译）的特定节奏的mRNA。在本建议书中，我们研究了 在四个具体目标中详细介绍了转铁蛋白的生化功能：（1）转铁蛋白所在蛋白复合物的表征;（2）检查转铁蛋白的细胞内定位;（3）鉴定转铁蛋白的靶mRNA;（4）确定这些靶mRNA是如何识别的。生物钟控制着生理学的许多关键方面，生物钟的中断最近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，以及基因表达的转录后控制的一般领域。

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.