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.

项目摘要/摘要 该建议涉及mRNA的翻译和降解 翻译终止。这种mRNA通常被认为是源于胡说八道的基因 突变，但还包括失败或替代剪接的细胞质产物， 假基因或非生产性基因重排和带有上游开放式阅读框的mRNA， 以及由非标准转录启动或进行框外翻译引起的mRNA 启动或意外的框架。这些成绩单上的翻译伸长通常会导致 过早终止密码子（PTC）的核糖体识别和加速mRNA的触发 在称为废话介导的mRNA衰变（NMD）的过程中衰变。此的中心监管机构 mRNA质量控制途径是三种UPF蛋白（UPF1，UPF2和UPF3）。虽然NMD一直 在多个真核生物中进行了广泛的研究，UPF蛋白识别的精确机制 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 相关的mRNA仍然未知。在最近的一些研究中，我们已经开始获得大量 在酵母模型系统中对NMD的机械洞察力，并辨别出至少四个 可确定的步骤：1）upf1在监测时随机结合与伸长核糖体绑定 翻译终止，2）与从事过早终止的核糖体的UPF1关联是 稳定和对NMD的承诺由UPF2和UPF3结合促进，3）激活UPF1 通过ATP水解进行解旋酶活性促进终止复合物的解离并募集 mRNA decapping酶和4）在解剖后，mRNA的身体被消化外核酸溶液 由XRN1。在该提案的实验中，我们将使用酵母遗传学，分子生物学和 低温电子显微镜通过解决三个目标，以追求该模型的几个关键方面 寻求：通过UPF1阐明NMD底物激活激活的机制，确定角色 和功能顺序，以将过早终止与mRNA破坏联系起来并定义基础的功能顺序 为了提高翻译终止效率的PAB1，可能是区分过早的关键步骤 从正常终止。在这项研究结束时，我们预计能够制定更多 综合模型详细介绍了过早翻译终止的非典型方面 哪种过早终止是由三种UPF蛋白靶向的，而分子事件将 UPF蛋白在靶向mRNA衰减中的功能。我们希望我们的酵母NMD模型将 通常适用于高等生物中NMD的分子事件，并将提供见解 改善因胡说八道突变引起的人类疾病的治疗方法。