Mechanism and targets of non-stop decay in eukaryotic translation

描述（由申请人提供）：基因表达在转录、RNA加工、翻译和RNA降解等多个水平上受到调控。信使RNA （mRNA）在翻译过程中受到至少三种质量控制机制的监视，这些机制致力于消除有缺陷的mRNA；无义介导衰变（NMD）、不去衰变（NGD）和不间断衰变（NSD）。NSD针对缺乏停止密码子的转录本，这些转录本不仅会产生异常蛋白，还会使核糖体停滞，导致翻译效率整体下降。真核生物NSD的发生机制尚不清楚。NSD可能是由核糖体在不间断mRNA转录物上的停顿触发的。在葡萄球菌中，当外泌体相关因子Ski7与停滞的核糖体相互作用触发3‘到5’的衰变时，不间断的mRNA被降解，或者通过第二种途径，mRNA被核酸酶以5‘到3’的方式分解和降解。当真核核糖体不间断地翻译mRNA时，翻译可能通过聚(A)尾进行，产生具有聚赖氨酸c末端的蛋白质产物，从而破坏功能。这些异常的多赖氨酸蛋白被认为是特定机制的目标。最近对？酿酒葡萄球菌的ltn敲除表明，不间断蛋白可能被核糖体相关的泛素连接酶Ltn1标记为降解。然而，Ltn1对NSD的确切作用尚不清楚。本文的目的是通过研究酿酒酵母不间断衰变的机制和靶点，进一步阐明真核质量控制途径mRNA不间断衰变的机制。具体来说，这些研究将利用格林实验室开发的方法来测量体外翻译过程中肽延伸率、肽释放率和核糖体释放率，研究真核生物中NSD的靶点和基本机制。除了标准的生化分析外，还将采用基于质谱的泛素化分析和新的深度测序技术（核糖体分析）。这些研究的目的是解决不间断mNRA的翻译如何影响核糖体，调查Ltn1对NSD的精确贡献，并确定NSD的靶向mrna。本研究还将探讨NSD与NMD一样，不仅针对有缺陷的mRNA，而且通过调节mRNA转录子子集来控制基因表达水平，在细胞中发挥更广泛的功能的可能性。这项工作将揭示缺乏停止密码子的mrna在真核生物中被NSD衰变的基本机制，并在真核生物中鉴定NSD靶点，为人类健康和疾病提供相关信息。

DESCRIPTION (provided by applicant): Gene expression is regulated on numerous levels including transcription, RNA processing, translation, and RNA degradation. Messenger RNA (mRNA) is surveilled during translation by at least three quality control mechanisms committed to the elimination of defective mRNAs; non-sense mediated decay (NMD), no-go decay (NGD), and non-stop decay (NSD). NSD targets transcripts lacking stop codons, which not only generate aberrant proteins, but also stall ribosomes, leading to an overall decrease in translation efficiency. The mechanism of NSD in eukaryotes is not well understood. NSD is presumably triggered by the stalling of ribosomes on non-stop mRNA transcripts. In S. cerevisiae the non-stop mRNA is degraded either when the exosome-associated factor Ski7 interacts with stalled ribosomes to trigger 3' to 5' decay, or in a second pathway in which the mRNA is decapped and degraded in a 5' to 3' manner by nucleases. When eukaryotic ribosomes translate non-stop mRNA, translation presumably proceeds through the poly(A) tail creating protein products with poly-lysine C-termini that can disrupt function. These aberrant poly-Lys proteins are thought to be targeted by specific machinery. Recent studies of ?ltn knockouts in S. cerevisiae suggest that non-stop proteins may be tagged for degradation by the ribosome-associated ubiquitin ligase Ltn1. However, the precise contribution of Ltn1 to NSD is unknown. The goals of the work proposed here are to further delineate the mechanisms of mRNA the eukaryotic quality control pathway non-stop decay by studying the mechanism and targets of non-stop decay in S. cerevisiae. Specifically, these studies will investigate the targets and fundamental mechanism of NSD in eukaryotes using methods developed in the Green lab to measure the rates of peptide elongation, peptide release and ribosome release during in vitro translation. Mass spectrometry based ubiquitination assays and new deep-sequencing technologies (ribosome profiling) will be employed in addition to standard biochemical assays. The aim of these studies is to address how translation of non-stop mNRA impacts the ribosome, investigate the precise contribution of Ltn1 to NSD, and identify mRNAs targeted for NSD. This study will also address the possibility that NSD, like NMD, not only targets defective mRNAs, but also performs a broader function in the cell by regulating a subset of mRNA transcripts in order to control gene expression levels. This work will reveal the fundamental mechanism by which mRNAs lacking a stop-codon are decayed by NSD in eukaryotes and identify the in vivo of NSD targets in eukaryotes, which could provide information relevant to human health and disease. PUBLIC HEALTH RELEVANCE: Errors in mRNA processing have deleterious effects and are associated with a variety of human diseases, including ¿ - thalassemia and certain cancers. The precise mechanism and targets of non-stop decay (NSD), an mRNA quality control mechanism committed to the elimination of defective mRNAs during translation, are unknown. The work proposed here will characterize the mechanism of eukaryotic NSD and identify the full panel of NSD targets, which could provide information relevant to design of therapeutics.