Translation, targeting, and decay of yeast nonsense-containing mRNAs

含有无义酵母的 mRNA 的翻译、靶向和衰变

• 批准号: 10550367
• 负责人: Allan S Jacobson
• 金额: $ 57.8万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-15 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10550367
• 关键词: ATP Hydrolysis; Acceleration; Address; Alternative Splicing; Binding; Complex; Cryoelectron Microscopy; Cytoplasm; Dissociation; Eukaryota; Event; Gene Rearrangement; Genes; Genetic Code; Genetic Diseases; Goals; Health Benefit; Hereditary Disease; Link; Mediating; Messenger RNA; Modeling; Molecular; Molecular Biology; Monitor; Mutation; Nonsense Mutation; Open Reading Frames; Organism; Pathway interactions; Process; Proteins; Pseudogenes; Public Health; Quality Control; Ribosomes; Role; Terminator Codon; Therapeutic; Transcript; Transcription Initiation; Translation Initiation; Translations; Work; Yeast Model System; Yeasts; decapping enzyme; experimental study; helicase; human disease; improved; insight; mRNA Decay; mRNA Transcript Degradation; mRNA decapping; novel therapeutic intervention; premature; recruit; tool; translational genetics; yeast genetics

Project Summary/Abstract This proposal addresses the translation and degradation of mRNAs subject to premature translation termination. Such mRNAs are usually thought to be derived from genes harboring nonsense mutations, but also include cytoplasmic products of failed or alternative splicing, transcripts of pseudogenes or unproductive gene rearrangements, and mRNAs with upstream open reading frames, as well as mRNAs arising from non-standard transcription initiation or undergoing out-of-frame translation initiation or unexpected frameshifting. Translational elongation on these transcripts generally leads to ribosomal recognition of a premature termination codon (PTC) and to the triggering of accelerated mRNA decay in a process known as nonsense-mediated mRNA decay (NMD). The central regulators of this mRNA quality control pathway are the three Upf proteins (Upf1, Upf2, and Upf3). Although NMD has been studied extensively in multiple eukaryotes, the precise mechanisms by which the Upf proteins recognize a ribosome undergoing an atypical termination event and respond to it by triggering accelerated degradation of the associated mRNA remain unknown. In several recent studies we have begun to gain significant mechanistic insight into NMD in the yeast model system and to discern what appear to be at least four definable steps during which: 1) Upf1 binds stochastically to elongating ribosomes while monitoring translational termination, 2) Upf1 association with a ribosome engaged in premature termination is stabilized and a commitment to NMD is promoted by Upf2 and Upf3 binding, 3) activation of the Upf1 helicase activity by ATP hydrolysis promotes dissociation of the termination complex and recruitment of the mRNA decapping enzyme, and 4) post-decapping, the body of the mRNA is digested exonucleolytically by Xrn1. In the experiments of this proposal we will use the tools of yeast genetics, molecular biology, and cryo-electron microscopy to pursue several critical aspects of this model by addressing three goals that seek to: elucidate the mechanism of decapping activation of NMD substrates by Upf1, determine the roles and functional order for factors that link premature termination to mRNA decapping, and define the basis for Pab1 enhancement of translation termination efficiency, likely a key step for distinguishing premature from normal termination. At the conclusion of this study we anticipate being able to formulate a more integrated model detailing the atypical aspects of premature translation termination, the mechanism by which premature termination is targeted by the three Upf proteins, and the molecular events linking the functions of Upf proteins to targeted mRNA decay. We expect that our refined model for yeast NMD will be generally applicable to the molecular events of NMD in higher organisms and will provide insights to improving therapeutic approaches to human diseases caused by nonsense mutations.

项目总结/摘要 该提案解决了受过早表达影响的mRNA的翻译和降解。 翻译终止这种mRNA通常被认为是来自含有无意义的基因 突变，但也包括失败或选择性剪接的细胞质产物， 假基因或非生产性基因重排，以及具有上游开放阅读框的mRNA，如 以及由非标准转录起始或进行框外翻译产生的mRNA 启动或意外的框架转移。这些转录本上的翻译延伸通常导致 提前终止密码子（PTC）的核糖体识别和加速mRNA的触发 无义介导的mRNA衰变（NMD）。这其中的中央监管机构 mRNA质量控制途径是三种Upf蛋白（Upf 1，Upf 2和Upf 3）。虽然NMD已经被 在多个真核生物中广泛研究，Upf蛋白识别一种 核糖体经历非典型终止事件，并通过触发加速降解对其作出反应 相关的mRNA仍然未知。在最近的几项研究中， 在酵母模型系统中对NMD的机械洞察，并辨别出似乎至少有四种 可定义的步骤，在此期间：1）Upf 1将stochlycerin结合到延长的核糖体上，同时监测 翻译终止，2）Upf 1与参与提前终止的核糖体的关联， 通过Upf 2和Upf 3结合促进稳定化并致力于NMD，3）Upf 1的激活 通过ATP水解的解旋酶活性促进终止复合物的解离和 mRNA脱帽酶，和4）脱帽后，mRNA体被外切核酸消化 在Xrn 1在这项建议的实验中，我们将使用酵母遗传学、分子生物学和 冷冻电子显微镜通过解决三个目标来追求这个模型的几个关键方面， 目的：阐明Upf 1对NMD底物的脱帽活化机制，确定Upf 1在NMD底物脱帽活化中的作用， 和功能顺序的因素，链接提前终止mRNA脱帽，并定义的基础 对于Pab 1，翻译终止效率的提高，可能是区分早熟的关键步骤， 正常终止。在这项研究的结论，我们预计能够制定一个更 综合模型详细介绍了翻译过早终止的非典型方面， 这三种Upf蛋白靶向哪一种提前终止，以及连接这三种蛋白的分子事件。 Upf蛋白对靶向mRNA降解的功能。我们期望我们的酵母NMD的改进模型将 一般适用于高等生物中的NMD分子事件，并将为 改进治疗由无义突变引起的人类疾病的方法。