Mechanisms of Translational Surveillance

项目总结/摘要 很大一部分人类遗传性致病突变都会引入早期终止密码子， 在一个称为无义介导的mRNA衰变的过程中， （NMD）。尽管做了大量的工作，但仍不清楚早期终止密码子是如何被识别的，以及它们是如何导致 mRNA衰变。这项工作的长期目标是阐明细胞如何识别和抑制mRNA， 动物中的早期终止密码子过去几十年的工作已经强调了许多相关的因素 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 仍不清楚在这个提议中，PI和他的实验室将剖析蛋白质合成的途径， 体内含早期终止密码子的mRNA的降解。拟议工作的具体目标是：[目的 1]：表征mRNA切割反应、其产物及其对蛋白质因子的依赖性。结果 从这个目标出发，将提供有关NMD背后的RNA衰变反应的生物化学信息。[目的 2]：研究UPF 1因子在许可mRNA衰变中的作用。这一目标的结果将阐明 这些因素是mRNA衰变的时间、募集和承诺的基础。[Aim 3]：描述角色 核糖体在NMD通路中的作用。这一目标的结果将展示核糖体如何向细胞发出信号， 在NMD过程中导致RNA衰变的机制。实验将：（a）分析NMD突变体的表型 C.线虫菌株，（B）分析NMD期间产生的RNA种类，特别是突变体背景， 短（Illumina）和长读（Oxford Nanopore）测序，以及（c）生物化学分析纯化的NMD 配合物这项工作的结果将阐明细胞识别 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.