Mechanisms of Translational Surveillance

项目摘要/摘要 相当一部分人类遗传性致病突变引入了一个早期终止密码子， 在一种被称为无意义介导的信使核糖核酸衰变的过程中，截断蛋白质的产生并引发信使核糖核酸的衰变 (NMD)。尽管做了很多工作，但人们仍然不清楚早期终止密码子是如何被识别的，以及它们是如何产生的 信使核糖核酸衰变。这项工作的长期目标是阐明细胞是如何识别和抑制mRNA的 动物体内的早期终止密码子。过去几十年的研究突显了其中的许多因素 以及它们的一些生化能力，但所涉及的分子的步骤和结构/功能 目前仍不清楚。在这项提案中，PI和他的实验室将剖析蛋白质合成和 含早期终止密码子的mRNAs在体内的降解。拟议工作的具体目标是：[目标 1]：表征mRNA切割反应、其产物及其对蛋白质因子的依赖。结果 从这一目的将提供有关NMD背后的RNA衰变反应的生物化学信息。[瞄准 2]：研究UPF1因子在许可mRNAs用于衰退方面的作用。这一目标的结果将照亮 影响mRNAs衰变的时机、招募和承诺的因素。[目标3]：确定角色的特征 核糖体参与了NMD途径。这一目标的结果将展示核糖体如何向细胞 在国家导弹防御系统中导致核糖核酸衰变的机制。实验将：(A)分析NMD突变体的表型 秀丽隐杆线虫菌株，(B)概述在NMD期间产生的RNA物种，特别是通过 短(Illumina)和长读(牛津纳米孔)测序，以及(C)对纯化的NMD进行生化分析 复合体。这项工作的结果将阐明细胞识别途径的分子细节 并抑制与许多人类致病等位基因相关的早期终止密码子突变。

Project Summary/Abstract A substantial fraction of human inherited disease-causing mutations introduce an early stop codon that truncates protein production and elicits mRNA decay in a process called Nonsense-Mediated mRNA Decay (NMD). Despite much work, it is still unclear how early stop codons are recognized and how they bring about mRNA decay. The long-term goal of the work is to illuminate how cells recognize and repress mRNAs with early stop codons in animals. Work over the last several decades has highlighted many of the factors involved as well as some of their biochemical capabilities, but the steps and structure/function of the molecules involved remains unclear. In this proposal, the PI and his lab will dissect the pathway of protein synthesis and degradation of early stop codon-containing mRNAs in vivo. The specific aims of the proposed work are to: [Aim 1]: characterize the mRNA cleavage reaction, its products, and its dependencies on protein factors. Results from this aim will provide information about the biochemistry of the RNA decay reaction underlying NMD. [Aim 2]: study the role the factor UPF1 has in licensing mRNAs for decay. Results from this aim will illuminate the factors underlying the timing, recruitment, and commitment of mRNAs to decay. [Aim 3]: characterize the role ribosomes have in the NMD pathway. Results from this aim will showcase how ribosomes signal to cellular machinery to bring about RNA decay during NMD. Experiments will: (a) analyze the phenotype of NMD mutant C. elegans strains, (b) profile the RNA species produced during NMD and in particular mutant backgrounds via both short (Illumina) and long-read (Oxford Nanopore) sequencing, and (c) biochemically analyze purified NMD complexes. Results from this work will illuminate the molecular details of the pathway by which cells recognize and repress early stop codon mutations, relevant to many human disease-causing alleles.