Mechanisms of Translational Surveillance in C. elegans

项目摘要/摘要 细胞已经进化出质量控制机制，以识别、移除和减轻异常基因的危害 表达及其中间体。在这些机制中，无意义介导的mRNA衰退(NMD)就是其中之一。 在NMD期间，细胞识别并降解编码截短的过早终止密码子的mRNAs， 有害的，可能起主导作用的蛋白质。NMD与人类疾病密切相关：约11% 所有人类遗传性疾病都是由过早终止密码子引起的。尽管付出了很大的努力，但对 NMD靶基因的mRNAs是如何被识别和移除的，目前仍不清楚。我们的​长期目标​是照亮 后生动物(动物)细胞如何通过NMD识别和抑制mRNAs。本提案的​Key Focus​是 这一过程的第二步：NMD目标如何从细胞中移除的分子细节。 我的实验室使用了动物​C.elegans​，其中国家导弹防御系统的机器是非必要的，允许我们 利用其他动物不容易获得的强大的遗传方法。NMD目标的两个关键因素 MRNA的清除是RNA解旋酶SKI和核糖体拯救因子PELO。当细胞缺乏滑雪板和 Pelo，核糖体在NMD靶向mRNAs的截短提前终止密码子上停滞。我们证明了这一点 停滞在截短终止密码子上的核糖体是NMD的中间体，提供了一种新的功能 在NMD去除mRNA的过程中具有里程碑意义。在此基础上，我们的具体目标是：(目标1) 我们将描述导致核糖体停滞的核糖体裂解事件(S)的性质和时间 NMD靶向mRNAs。我们将使用旨在测试时空关系的不同模型的记者 RNA裂解和提前翻译终止之间的关系。(目标2)我们将阐明分子 SKI和PELO在NMD期间移除RNA和核糖体的机制，利用我们灵活的能力 突变SKI和PELO，并在活体​中检测它们的功能​。(目标3)最后，我们将回答一个问题 NMD在防止截短蛋白生产中所起作用的基础：NMD如何影响 从其目标mRNAs中产生蛋白质？我们将确定在NMD期间产生哪些蛋白质物种， 并确定随着NMD靶向mRNAs的降解，是否存在对新生蛋白链的抑制。 我们预计，包括靶向mRNA清除的一系列事件的清晰图景 国家气象局将(1)告知先前事件的模型，​，即​，如何识别提前停止密码子；(2)阐明 共翻译信使核糖核酸衰变的分子事件，对理解正常基因表达很重要 和调控；以及(3)提供了一个清晰的图像，说明了从含有 过早终止密码子，对NMD在人类疾病中的作用具有机械学意义。

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.