Mechanisms of Translational Surveillance in C. elegans

线虫的转化监测机制

• 批准号: 10458628
• 负责人: Joshua Arribere
• 金额: $ 31.76万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-18 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10458628
• 关键词: Address; Affect; Animals; Area; Binding; Biochemical; Biochemistry; Biological Assay; Caenorhabditis elegans; Cells; Complex; Data; Dependence; Event; Excision; Freezing; Functional disorder; Gene Abnormality; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Diseases; Genetic Variation; Genomics; Goals; Hereditary Disease; High-Throughput Nucleotide Sequencing; Human; Human Genetics; Inherited; Investigation; Knock-out; Knowledge; Mediating; Messenger RNA; Modeling; Molecular; Monitor; Mus; Mutate; Mutation; Nature; Non-Stop Decay; Nonsense Codon; Nonsense-Mediated Decay; Pathway interactions; Peptides; Postdoctoral Fellow; Process; Production; Property; Proteins; Quality Control; RNA; RNA Helicase; RNA Interference; Reporter; Repression; Ribosomal Interaction; Ribosomal RNA; Ribosomes; Role; SKI gene; Site; Terminator Codon; Testing; Translations; Work; assault; cofactor; design; disease-causing mutation; exosome; field study; fly; genetic approach; human disease; in vivo; insight; mRNA Decay; mRNA Expression; mRNA Transcript Degradation; mutant; novel; nuclease; premature; prevent; recruit; ribosome releasing factor; spatiotemporal

Project Summary/Abstract Cells have evolved quality control mechanisms to identify, remove, and mitigate harm from abnormal gene expression and its intermediates. Among these mechanisms is Nonsense-Mediated mRNA Decay (NMD). During NMD, cells identify and then degrade mRNAs with premature stop codons encoding truncated, deleterious, and potentially dominant-acting proteins. NMD is intimately related with human disease: ~11% of all human inherited diseases are caused by premature stop codons. Despite much effort, an understanding of how NMD target mRNAs are identified and then removed remains elusive. Our ​long-term goal​ is to illuminate how cells recognize and repress mRNAs via NMD in metazoa (animals). The ​key focus​ of this proposal is the second step of this process: the molecular details of how NMD targets are removed from the cell. My lab employs the animal ​C. elegans​, in which the NMD machinery is non-essential, allowing us to exploit powerful genetic approaches not readily available in other animals. Two factors key for NMD target mRNA clearance are the RNA helicase SKI and the ribosome rescue factor PELO. When cells lack SKI and PELO, ribosomes stall on truncated premature stop codons of NMD target mRNAs. We demonstrated that ribosomes stalled on truncated stop codons represent an intermediate of NMD, providing a novel functional landmark in the process of mRNA removal by NMD. Building on this observation, our specific aims are: (Aim 1) We will characterize the nature and timing of RNA cleavage event(s) that give rise to ribosomes stalled on NMD target mRNAs. We will use reporters designed to test distinct models of the spatio-temporal relationship between RNA cleavage and premature translation termination. (Aim 2) We will illuminate the molecular mechanisms by which SKI and PELO remove RNAs and ribosomes during NMD, leveraging our facile ability to mutate SKI and PELO, and to assay their function ​in vivo​. (Aim 3) Finally, we will address a question fundamental to the perceived role of NMD in preventing truncated protein production: How does NMD affect protein production from its target mRNAs? We will determine what protein species are produced during NMD, and determine whether there is repression of nascent protein chains as NMD target mRNAs are degraded. We expect that a clear picture of the cascade of events that comprise target mRNA clearance during NMD will (1) inform models of preceding events, ​i.e.​, how premature stop codons are recognized; (2) illuminate the molecular events of co-translational mRNA decay, important for understanding normal gene expression and regulation; and (3) yield a clear picture of what mRNAs and proteins are expressed from genes harboring premature stop codons, with mechanistic implications for NMD’s roles in human diseases.

项目概要/摘要 细胞已经进化出质量控制机制来识别、消除和减轻异常基因的危害 表达及其中间体。这些机制之一是无义介导的 mRNA 衰变 (NMD)。 在 NMD 期间，细胞识别并用编码截短的过早终止密码子降解 mRNA， 有害且具有潜在主导作用的蛋白质。 NMD 与人类疾病密切相关：~11% 所有人类遗传性疾病都是由过早终止密码子引起的。尽管付出了很多努力，但理解 NMD 靶标 mRNA 是如何被识别然后被移除的仍然是个谜。我们的​长期目标​是照亮 后生动物（动物）中细胞如何通过 NMD 识别和抑制 mRNA。该提案的重点是 该过程的第二步：如何从细胞中去除 NMD 靶标的分子细节。 我的实验室使用动物​C.线虫​，其中 NMD 机械不是必需的，使我们能够 利用其他动物不易获得的强大遗传方法。 NMD目标的两个关键因素 mRNA清除是RNA解旋酶SKI和核糖体救援因子PELO。当细胞缺乏 SKI 且 PELO，核糖体在 NMD 目标 mRNA 的截短提前终止密码子上停滞。我们证明了 停顿在截短终止密码子上的核糖体代表 NMD 的中间体，提供了一种新颖的功能 NMD 去除 mRNA 过程中的里程碑。基于这一观察，我们的具体目标是：（目标 1） 我们将描述导致核糖体停滞的 RNA 裂解事件的性质和时间。 NMD 目标 mRNA。我们将使用旨在测试时空关系的不同模型的记者 RNA 切割和过早翻译终止之间的关系。 （目标 2）我们将阐明分子 SKI 和 PELO 在 NMD 期间去除 RNA 和核糖体的机制，利用我们的简单能力 突变 SKI 和 PELO，并检测它们的体内功能。 （目标3）最后我们要解决一个问题 NMD 在防止截短蛋白质产生方面的作用的基础：NMD 如何影响 从其目标 mRNA 产生蛋白质？我们将确定 NMD 期间产生的蛋白质种类， 并确定当 NMD 靶标 mRNA 降解时是否存在新生蛋白链的抑制。 我们期望能够清楚地了解包括目标 mRNA 清除在内的一系列事件 NMD 将 (1) 告知先前事件的模型，即如何识别过早终止密码子； (2) 照明 共翻译 mRNA 衰变的分子事件，对于理解正常基因表达很重要 和监管； (3) 清晰地了解携带基因的 mRNA 和蛋白质的表达情况 过早终止密码子，对 NMD 在人类疾病中的作用具有机制意义。