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

项目总结 核糖体在所有细胞中进行蛋白质合成，将mRNA中包含的信息解释为 产生适量的正确蛋白质。此外，核糖体也参与了信使核糖核酸质量控制。 因此，错误组装的核糖体可以影响蛋白质和mRNAs的序列和丰度，从而 破坏蛋白质的动态平衡。这可能会导致许多疾病，证明了 确保核糖体被准确组装，并以正确的数量生产。 利用生化、遗传、基因组和结构工具的组合，我们将(I)研究机制 促进核糖体蛋白的正确结合和RNA的折叠，(Ii)剖析质量控制 识别并最终降解错误组装的中间体的途径以及(Iii)研究错误组装如何 核糖体促进疾病。 在第一部分中，我们将在已有工作的基础上，对核糖体头部的后期组装进行研究 AS结合了对最新结构的见解和我们的生化工作，以了解Dead-box蛋白质是如何 被用来构建依赖于ATP的调控开关，以控制早期的主要构象转变 40s生物发生学。 在第二部分，我们将扩展我们在质量控制方面的工作，以调查可能的校对 错误组装中间体的机理和确定降解途径，这是该领域的一个新前沿。 在最后一部分，我们将研究包含两个核糖体的亚化学计量水平的核糖体是如何 在缺乏足够组装量的癌细胞中产生的蛋白质Asc1和Rps10 Ltv1因子，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之上 具有不同的组成，并且还考虑了这些蛋白质在mRNA质量中的已知作用 控制力。此外，我们还将研究核糖体数量失调在疾病中的影响。 总之，这项拟议的工作将把机械论的见解与细胞和分子的一个基本问题联系起来 生物学-核糖体是如何组装的，与人类疾病有关。

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.