Molecular mechanisms of mammalian circadian clock function

摘要 整个身体的生物钟驱动着数千个基因的有节奏的表达，导致 生物化学、生理和行为的节律。通过基因或基因改变昼夜节律 时差或倒班等环境干扰可能会产生深远的负面后果 并与肥胖、糖尿病、癌症、心血管疾病和精神疾病有关。我们的 工作主要集中在了解哺乳动物的详细分子机制上。 生物钟机制及其控制节律基因的机制 表情。根据目前的模型，这种时钟机构的核心部分是负极 转录因子异二聚体CLOCK/BMAL1驱动转录的反馈环 与之相互作用的“时钟”蛋白周期(PER)1、PER 2、隐花色素(CRY)1和CRY 2 相互抑制Clock/BMAL1的活性，从而抑制自身的合成。我们已经解决了 CLOCK/BMAL1和CRY2/PER2络合物的晶体结构和这些数据允许 鉴定了蛋白质中进化保守的功能结构域，揭示了 对这些蛋白质的运作机制和设定昼夜节律的更多见解。 在接下来的五年里，我们将对这些信息进行扩展，以确定如何 这只钟走时。这些核心生物钟转录因子在驱动节律中的作用 转录是有充分证据的，但最近的数据表明，转录后控制， 尽管人们对此知之甚少，但它对正常有节奏的蛋白质表达谱也是至关重要的。 一种类型的转录后控制是调节mRNA聚(A)尾长，它影响 信使核糖核酸的稳定性和翻译调控。我们已经鉴定了数百个小鼠肝脏的mRNAs 在它们的Poly(A)尾巴的长度上表现出强烈的昼夜节律。在许多这样的案例中， 有节律性的尾巴长度是细胞质有节律性的多聚腺苷和死烯基化的结果 细胞质多聚腺苷和死烯基化机制的节律和许多成分是 在昼夜节律的控制下。此外，有节奏的聚(A)尾巴是密切相关的 有节律性的蛋白质表达。因此，生物钟调节动态 许多可驱动节律性蛋白表达的mRNAs的多聚腺苷化状态独立于 消息的稳定状态级别。夜转素是一种强健的有节奏的蛋白质，可以去除聚(A)尾巴 来自mRNAs。我们已经证明，在小鼠中丢失该基因会导致对饮食诱导的抵抗力 肥胖和胆固醇和甘油三酯代谢节律的改变，这意味着它是一个重要的 昼夜节律转录后调节因子。在未来五年，我们将专注于确定 胞浆和线粒体中的Nocturin的mRNA底物。

Abstract Circadian clocks throughout the body drive rhythmic expression of thousands of genes, resulting in rhythms in biochemistry, physiology and behavior. Disruption of circadian clocks through genetics or environmental perturbations such as jet lag or shift-work, can have profound negative consequences and has been linked to obesity, diabetes, cancer, cardiovascular disease and mental illness. Our work is focused generally on understanding the detailed molecular mechanisms of the mammalian circadian clock machinery and the mechanisms by which these clocks control rhythmic gene expression. According to the current model, the core part of this clock mechanism is a negative feedback loop whereby the transcription factor heterodimer CLOCK/BMAL1 drives transcription of the “clock” proteins PERIOD (PER) 1, PER 2, CRYPTOCHROME (CRY) 1 and CRY 2 which interact with each other to repress the activity of CLOCK/BMAL1, and thus their own synthesis. We have solved crystal structures for the CLOCK/BMAL1 and CRY2/PER2 complexes and these data have allowed the identification of evolutionarily conserved functional domains throughout the proteins and revealed additional insights into the mechanisms by which these proteins operate and set the circadian period. Over the next five years, we will expand on this information to determine the atomic details of how this clock keeps time. The roles of these core circadian clock transcription factors in driving rhythmic transcription is well-documented, but recent data have demonstrated that post-transcriptional control, although much less well understood, is also critical for normal rhythmic protein expression profiles. One type of post-transcriptional control is regulation of mRNA poly(A) tail length, which impacts the stability and translational regulation of mRNA. We have identified hundreds of mouse liver mRNAs that exhibit robust circadian rhythms in the length of their poly(A) tails. In many of these cases, the rhythmic tail lengths are the result of rhythmic cytoplasmic polyadenylation and deadenylation rhythms and many components of the cytoplasmic polyadenylation and deadenylation machinery are themselves under circadian control. Furthermore, the rhythmic poly(A) tails are closely correlated with the rhythmic protein expression. Therefore, the circadian clock regulates dynamic polyadenylation status of many mRNAs that can drive rhythmic protein expression independent of the steady-state levels of the message. Nocturnin is a robustly rhythmic protein that removes poly(A) tails from mRNAs. We have shown that loss of this gene in mice causes resistance to diet-induced obesity and altered rhythms in cholesterol and triglyceride metabolism, implicating it as an important circadian post-transcriptional mediator. Over the next five years, we will focus on identifying the mRNA substrates of Nocturnin both in the cytosol and in the mitochondria.