Mechanism and targets of non-stop decay in eukaryotic translation

描述(申请人提供)：基因表达在多种水平上受到调控，包括转录、RNA加工、翻译和RNA降解。信使RNA(MRNA)在翻译过程中至少受到三种质量控制机制的监控，这些机制致力于消除有缺陷的mRNAs：无意义介导的衰退(NMD)、无意义的衰退(NGD)和不停止的衰退(NSD)。NSD针对缺乏终止密码子的转录本，这不仅会产生异常蛋白，还会使核糖体停滞，导致翻译效率的整体下降。NSD在真核生物中的作用机制尚不清楚。NSD可能是由不间断转录的核糖体停滞引起的。在酿酒酵母中，当外糖体相关因子Ski7与停滞的核糖体相互作用以触发3‘到5’衰变时，或者在第二个途径中，mRNA被核酸酶以5‘到3’的方式去帽并降解时，不间断的mRNA被降解。当真核核糖体翻译不间断的mRNA时，翻译可能通过聚(A)尾部进行，产生带有多赖氨酸C末端的蛋白质产物，这可能会破坏功能。这些异常的多聚赖氨酸蛋白被认为是特定机制的靶标。最近对酿酒酵母中？LTN基因敲除的研究表明，不间断的蛋白质可能被核糖体相关的泛素连接酶Ltd n1标记为降解。然而，Ltn1对NSD的确切贡献尚不清楚。本工作的目的是通过对酿酒酵母中mRNA不停止衰退的机制和靶点的研究，进一步揭示真核质量控制途径--mRNA不停止衰退的机制。具体地说，这些研究将利用绿色实验室开发的方法来研究NSD在真核生物中的靶标和基本机制，以测量体外翻译过程中多肽的伸长、多肽释放和核糖体释放的速率。除了标准的生化分析外，还将采用基于质谱学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翻译不间断的mNRA如何影响核糖体，研究Ltn1对NSD的确切贡献，并识别NSD的靶向mRNAs。这项研究还将探讨这样一种可能性，即NSD和NMD一样，不仅针对有缺陷的mRNAs，而且通过调节mRNA转录子集在细胞中发挥更广泛的功能，以控制基因表达水平。这项工作将揭示真核生物中缺乏终止密码子的mRNAs被NSD降解的基本机制，并在真核生物中识别NSD靶标，为人类健康和疾病提供相关信息。 公共卫生相关性：信使核糖核酸加工错误具有有害影响，并与多种人类疾病有关，包括地中海贫血和某些癌症。不停止衰变(NSD)是一种致力于在翻译过程中消除缺陷mRNAs的信使核糖核酸质量控制机制，其确切的机制和靶点尚不清楚。本文提出的工作将表征真核细胞NSD的机制，并确定NSD靶点的完整小组，这可能为治疗设计提供相关信息。

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.