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.

项目概要/摘要 该提案解决了 mRNA 的翻译和降解问题 翻译终止。此类 mRNA 通常被认为源自含有无意义基因的基因 突变，还包括失败或选择性剪接的细胞质产物、转录物 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 启动或意外的移码。这些转录物的翻译延伸通常会导致 核糖体识别提前终止密码子 (PTC) 并触发加速 mRNA 衰变过程称为无义介导的 mRNA 衰变 (NMD)。本次中央监管机构 mRNA 质量控制途径是三种 Upf 蛋白（Upf1、Upf2 和 Upf3）。虽然NMD已经 在多种真核生物中进行了广泛的研究，Upf 蛋白识别 核糖体经历非典型终止事件并通过触发加速降解来对其做出反应 相关 mRNA 的含量仍未知。在最近的几项研究中，我们已经开始获得显着的成果 深入了解酵母模型系统中的 NMD 并辨别至少四种 可定义的步骤，其中：1) Upf1 在监测时随机结合至延长核糖体 翻译终止，2) Upf1 与参与提前终止的核糖体的关联是 Upf2 和 Upf3 结合促进了 NMD 的稳定和承诺，3) Upf1 的激活 ATP 水解产生的解旋酶活性促进终止复合物的解离和招募 mRNA 脱帽酶，4) 脱帽后，mRNA 主体被核酸外切酶消化 由Xrn1。在本提案的实验中，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该模型的几个关键方面： 寻求：阐明 Upf1 对 NMD 底物脱帽激活的机制，确定其作用 以及将提前终止与 mRNA 脱帽联系起来的因素的功能顺序，并定义基础 对于Pab1翻译终止效率的增强，可能是区分早熟的关键步骤 从正常终止。在这项研究结束时，我们预计能够制定更多 集成模型详细描述了过早翻译终止的非典型方面，其机制为 三种 Upf 蛋白的目标是过早终止，以及连接这些蛋白的分子事件 Upf 蛋白对目标 mRNA 降解的功能。我们预计我们的酵母 NMD 改进模型将 一般适用于高等生物中 NMD 的分子事件，并将提供见解 改进由无义突变引起的人类疾病的治疗方法。