Dissecting the Mechanisms of Regulation and Quality Control in Ribosome Assembly and the Consequences of their Failure

项目摘要 核糖体在所有细胞中进行蛋白质合成，解释mRNA中包含的信息， 产生适量的蛋白质。此外，核糖体还介导mRNA质量控制。 因此，错误装配的核糖体可以影响蛋白质和mRNA的序列和丰度，从而 破坏蛋白质稳态这可能导致许多疾病，这表明 确保核糖体被准确地组装，并以正确的数量产生。 使用生物化学、遗传学、基因组学和结构学工具的组合，我们将（i）研究机制 其促进核糖体蛋白的适当掺入和RNA的折叠，（ii）解剖质量控制 途径，以确定和最终降解错误组装的中间体和（iii）研究如何错误组装 核糖体促进疾病。 在第一部分中，我们将在现有工作的基础上，研究核糖体头部的后期组装，以及 作为联合收割机的见解，从最近的结构和我们的生化工作，以了解如何死亡盒蛋白质 用于构建ATP依赖性调节开关，以控制早期细胞中的主要构象转变。 40 S生物起源。 在第二部分中，我们将扩展我们的质量控制工作，探讨一种可能的校对方法 机制，并确定降解途径的错误组装的中间体，一个新的前沿领域。 在最后一部分中，我们将研究含有两个核糖体亚化学计量水平的核糖体是如何被激活的。 蛋白质Asc 1和Rps 10，它们在缺乏足够数量组装的癌细胞中产生 因子Ltv 1，促进疾病。这项工作建立在我们开发的分离核糖体的遗传系统上 不同的组成，并考虑到已知的作用，这些蛋白质在mRNA的质量 控制此外，我们还将研究疾病中核糖体数量失调的影响。 总之，拟议的工作将联系到细胞和分子的基本问题的机制见解 生物学-核糖体是如何组装的，人类疾病。

PROJECT SUMMARY Ribosomes carry out protein synthesis in all cells, interpreting the information contained in the mRNA to produce the proper amount of the correct protein. In addition, ribosomes also mediate mRNA quality control. Thus, misassembled ribosomes can affect the sequence and abundance of proteins and mRNAs, thereby disrupting protein homeostasis. This can lead to a number of diseases, demonstrating the importance of ensuring ribosomes are accurately assembled, and produced in the correct numbers. Using a combination of biochemical, genetic, genomic and structural tools, we will (i) investigate mechanisms that promote proper incorporation of ribosomal proteins and folding of the RNA, (ii) dissect quality control pathways to identify and ultimately degrade misassembled intermediates and (iii) study how misassembled ribosomes promote disease. In the first part, we will build on our existing work and study the late assembly of the ribosomal head, as well as combine insights from recent structures and our biochemical work to understand how DEAD-box proteins are used to construct ATP-dependent regulatory switches to control major conformational transitions in early 40S biogenesis. In the second part, we will extend our work on quality control to investigate a possible proofreading mechanism and identify degradation pathways for misassembled intermediates, a novel frontier for the field. In the last part, we will investigate how ribosomes containing substoichiometric levels of two ribosomal proteins, Asc1 and Rps10, which are produced in cancer cells that lack sufficient amounts of the assembly factor Ltv1, promote disease. This work builds on a genetic system we have developed to separate ribosomes of distinct composition, and also takes into consideration the known roles of these proteins in mRNA quality control. In addition, we will also investigate the effects from dysregulation of ribosome numbers in disease. Together, the proposed work will link mechanistic insights into a fundamental problem of cell and molecular biology – how ribosomes are assembled, to human disease.