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.

项目总结/摘要 细胞已经进化出质量控制机制，以识别、去除和减轻异常基因的伤害。 表达及其中间体。无义介导的mRNA衰变（NMD）。 在NMD过程中，细胞识别并降解具有提前终止密码子的mRNA， 有害的，潜在的显性作用蛋白质。NMD与人类疾病密切相关：约11%的 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， 如何鉴定NMD靶向mRNA并随后将其去除仍然是难以捉摸的。我们的长期目标是 在后生动物（动物）中，细胞如何通过NMD识别和抑制mRNA。该提案的重点是 这个过程的第二步：NMD目标如何从细胞中移除的分子细节。 我的实验室使用动物C。在这种情况下，国家导弹防御系统不是必不可少的， 利用在其他动物中不容易获得的强大的遗传方法。NMD目标的两个关键因素 mRNA清除是RNA解旋酶SKI和核糖体拯救因子PELO。当细胞缺乏SKI， PELO，核糖体在NMD靶mRNA的截短的提前终止密码子上停滞。我们证明了 截短的终止密码子上的核糖体代表NMD的中间体，提供了一种新的功能 NMD去除mRNA过程中的里程碑。基于这一观点，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切割事件的性质和时间。 NMD靶向mRNA。我们将使用旨在测试时空关系的不同模型的报告器 RNA切割和过早翻译终止之间的关系。(Aim 2）我们将阐明分子 SKI和PELO在NMD过程中去除RNA和核糖体的机制，利用我们轻松的能力， 突变SKI和PELO，并检测其体内功能。(Aim（3）最后，我们将讨论一个问题。 NMD在防止截短蛋白质产生方面的作用的基本认识：NMD如何影响 从其目标mRNA中产生蛋白质？我们将确定在NMD期间产生哪些蛋白质种类， 并确定当NMD靶mRNA降解时是否存在新生蛋白链的抑制。 我们期望，一个清晰的图片级联的事件，包括目标mRNA清除过程中， NMD将（1）告知先前事件的模型，即如何识别过早的终止密码子;（2）阐明 共翻译mRNA衰变的分子事件，对于理解正常基因表达很重要 和调控;和（3）产生一个清晰的图片，什么mRNA和蛋白质表达的基因含有 过早的终止密码子，与NMD在人类疾病中的作用机制的影响。