Mechanism and targets of non-stop decay in eukaryotic translation

真核翻译不间断衰变的机制和靶点

• 批准号: 8638787
• 负责人: Kristin S Koutmou
• 金额: $ 5.89万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8638787
• 关键词: Address; Bacteria; Biochemical; Biological Assay; C-terminal; Cells; Characteristics; Commit; Disease; Dissociation; Eukaryota; Gene Expression; Gene Expression Regulation; Genetic Transcription; Goals; Health; Homeostasis; Human; In Vitro; Knock-out; Lysine; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Mediating; Messenger RNA; Methods; Non-Stop Decay; Pathway interactions; Peptides; Poly(A) Tail; Proteins; Quality Control; RNA Degradation; RNA Processing; Ribosomes; Saccharomyces cerevisiae; Signal Transduction; System; Tail; Technology; Terminator Codon; Thalassemia; Therapeutic; Transcript; Translating; Translations; Ubiquitination; Work; base; deep sequencing; design; human disease; in vivo; mutant; nuclease; protein degradation; tmRNA; ubiquitin ligase

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.

描述（由申请人提供）：基因表达在许多水平上受到调控，包括转录、RNA加工、翻译和RNA降解。信使RNA（mRNA）在翻译过程中受到至少三种质量控制机制的监控，这些机制致力于消除缺陷mRNA;无义介导的衰变（NMD），不进行衰变（NGD）和不停止衰变（NSD）。NSD靶向缺乏终止密码子的转录物，其不仅产生异常蛋白质，而且使核糖体停滞，导致翻译效率的总体降低。NSD在真核生物中的机制还不清楚。NSD可能是由核糖体在不停止的mRNA转录物上的停滞触发的。In S.在酿酒酵母中，当外泌体相关因子Ski 7与停滞的核糖体相互作用以触发3'至5'衰变时，或者在第二途径中，其中mRNA被核酸酶以5'至3'的方式去帽和降解时，非终止mRNA被降解。当真核生物核糖体不间断地翻译mRNA时，翻译可能通过poly（A）尾进行，产生具有多聚赖氨酸C末端的蛋白质产物，其可以破坏功能。这些异常的多聚赖氨酸蛋白被认为是特定机制的目标。最近的研究？在S.酿酒酵母表明，不停止蛋白质可以被标记为通过核糖体相关的泛素连接酶Ltn 1降解。然而，Ltn 1对NSD的确切贡献尚不清楚。本研究的目的是通过对S.啤酒。具体而言，这些研究将使用绿色实验室开发的方法研究真核生物中NSD的靶标和基本机制，以测量体外翻译过程中肽延伸、肽释放和核糖体释放的速率。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停止mNRA的翻译如何影响核糖体，研究Ltn 1对NSD的精确贡献，并确定NSD靶向的mRNA。这项研究还将探讨NSD和NMD一样不仅针对缺陷mRNA，而且通过调节mRNA转录物的子集以控制基因表达水平来在细胞中发挥更广泛功能的可能性。本工作将揭示真核生物中缺少终止密码子的mRNA被NSD降解的基本机制，并确定真核生物中NSD的体内靶点，这将为人类健康和疾病提供相关信息。