Mechanisms of Circadian Clock Control of mRNA Translation

总结 生物钟对人体健康和药物代谢至关重要，它调节蛋白质的节律性产生 从而影响细胞功能和新陈代谢。许多蛋白质的水平显示出强大的昼夜节律， 从没有节奏的mRNA中分离出来。使用模式真核生物粗糙脉孢菌，我们发现多达一半的 这种蛋白质水平的昼夜节律调节是通过生物钟控制一种保守的调节因子的活性， 翻译起始（eIF 2 α）和翻译核糖体的蛋白质组成。我们还做出了令人惊讶的 生物钟控制核糖体通过正常停止阅读的可能性的观察 密码子来产生具有羧基末端延伸和潜在新功能的蛋白质。此外，我们还发现， 生物钟调节tRNA合成酶的水平，这些合成酶为tRNA提供适当的氨基酸， 翻译，因此对准确的蛋白质合成至关重要。在接下来的5年里，我们将利用这些 研究结果测试了令人兴奋的假设，即生物钟控制着翻译保真度的日常变化， 因此蛋白质的多样性超出了基因组的编码范围。我们将确定核糖体的时钟控制 组成是必要的，以及需要哪些特定的核糖体蛋白，节奏终止密码子 通读。此外，我们将测试是否生物钟控制的结合辅伴侣佐丁核糖体 调节蛋白质折叠的日常节奏。我们将确定昼夜节律对甲硫氨酰-tRNA的影响， 在三个不同的细胞上，研究了磷酸化MetRS的激酶的活性，MetRS水平和磷酸化MetRS的激酶活性的节律。 MetRS调控途径。这些包括通过引发剂甲硫氨酰tRNA的充电的翻译起始， 翻译延伸通过加载延伸子甲硫氨酰tRNA，和错误掺入的甲硫氨酸， 蛋白质合成通过非同源tRNA的错误充电。这项工作将对我们的 了解细胞在一天中的不同时间是如何不同的，以及蛋白质组如何能够更好地 比我们从基因组序列中预测的要多样。

Summary The circadian clock, critical to human health and drug metabolism, regulates rhythmic protein production and thus cell function and metabolism. Many proteins whose levels show robust circadian rhythms are produced from mRNAs that are not rhythmic. Using the model eukaryote Neurospora crassa, we found that up to half of this circadian regulation of protein levels is through clock control of the activities of a conserved regulator of translation initiation (eIF2α), and the protein composition of translating ribosomes. We also made the surprising observation that the circadian clock controls the probability that ribosomes will read through the normal stop codon to produce proteins with carboxy-terminal extensions and potentially new functions. In addition, we found that the clock regulates the levels of tRNA synthetases that charge tRNAs with the appropriate amino acids for translation, and thus are critical for accurate protein synthesis. Over the next 5 years, we will capitalize on these findings to test the exciting hypothesis that the circadian clock controls daily changes in translation fidelity and thus protein diversity beyond what is encoded for in the genome. We will determine if clock control of ribosome composition is necessary, and which specific ribosomal proteins are required, for rhythmic stop codon readthrough. In addition, we will test if circadian clock control of binding of the co-chaperone Zuotin to ribosomes regulates daily rhythms in protein folding. We will determine the impact of circadian rhythms in methionyl-tRNA synthetase (MetRS) levels, and rhythms in the activities of kinases that phosphorylate MetRS, on three different MetRS regulatory pathways. These include translation initiation through charging of the initiator methionyl tRNA, translation elongation through charging of elongator methionyl tRNA, and misincorporation of methionine during protein synthesis through the mischarging of non-cognate tRNA. This work will significantly impact our understanding of both how a cell is different at different times of the day, and how the proteome can be more diverse than what one would predict from the genome sequence.