Mechanisms of Translational Surveillance in C. elegans

线虫的转化监测机制

• 批准号: 9932597
• 负责人: Joshua Arribere
• 金额: $ 10.92万
• 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/9932597
• 关键词: Address; Affect; Animals; Area; Binding; Biochemical; Biochemistry; Biological Assay; Caenorhabditis elegans; Cells; Cleaved cell; 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; Structure; 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.

项目摘要/摘要 细胞具有进化的质量控制机制，以识别，去除和减轻异常基因的伤害 表达及其中间体。这些机制包括胡说八道介导的mRNA衰减（NMD）。 在NMD期间，细胞识别并降解mRNA，用编码截短的过早终止密码子降低mRNA， 有害且潜在的作用蛋白质。 NMD与人类疾病密切相关：〜11％ 所有人类遗传疾病都是由过早的停止密码子引起的。尽管付出了很多努力，对 如何识别NMD目标mRNA然后去除，仍然难以捉摸。我们的长期目标是照亮 细胞如何通过Metazoa（动物）中的NMD识别和反映mRNA。该提议的重点是 此过程的第二步：从细胞中去除NMD靶标​​的分子细节。 我的实验室员工动物C.秀丽隐杆线虫，其中NMD机械不必要，使我们能够 在其他动物中利用强大的遗传方法不容易获得。 NMD目标的两个因素 mRNA清除率是RNA解旋酶滑雪和核糖体救援因子Pelo。当细胞缺乏滑雪时 pelo，核糖体在NMD靶标​​mRNA的截短过早终止密码子上插座。我们证明了这一点 停滞在截短的终止密码子上的核糖体代表NMD的中间体，提供了一种新型功能 在NMD删除mRNA的过程中具有里程碑意义。在此观察基础上，我们的具体目标是：（目标1） 我们将表征RNA裂解事件的性质和时机（S）引起核糖体停滞的核糖体 NMD目标mRNA。我们将使用旨在测试空间关系的不同模型的记者 在RNA裂解和过早翻译终止之间。 （目标2）我们将照亮分子 在NMD期间，滑雪和PELO去除RNA和核糖体的机制，利用我们的便利能力 突变滑雪和pelo，并在体内主张它们的功能。 （目标3）最后，我们将解决一个问题 NMD在预防截短蛋白质产生中的感知作用的基础：NMD影响如何 来自其靶标mRNA的蛋白质产生？我们将确定在NMD期间产生哪些蛋白质， 并确定是否存在新生蛋白链作为NMD靶mRNA的表达。 我们期望清晰地了解一系列事件，这些事件在 NMD将（1）告知上述事件模型，即如何确认过早的停止密码子； （2）照明 共同翻译mRNA衰减的分子事件，对于理解正常基因表达很重要 和监管； （3）清楚地了解了从携带的基因表达的mRNA和蛋白质 过早的停止密码子，对NMD在人类疾病中的作用具有机械意义。