Genetic nonsense and its consequences

遗传的胡言乱语及其后果

• 批准号: 9275112
• 负责人: Allan S Jacobson
• 金额: $ 45.58万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9275112
• 关键词: Address; Binding Sites; Biological Models; Bypass; C-terminal; Complex; Cryoelectron Microscopy; Development; Eukaryota; Event; Genetic; Goals; Inborn Genetic Diseases; Lead; Length; Link; Mass Spectrum Analysis; Mediating; Messenger RNA; Modality; Modeling; Molecular; Mutation; Nature; Nonsense Codon; Nonsense Mutation; Pathway interactions; Patients; Pharmaceutical Preparations; Play; Procedures; Process; Protein Biosynthesis; Proteins; Public Health; Quality Control; Regulatory Element; Regulatory Pathway; Research; Ribosomes; Role; Saccharomyces cerevisiae; Specificity; Structure; Terminator Codon; Translating; Translations; Yeasts; decapping enzyme; experimental study; follow-up; mRNA Decay; mRNA decapping; novel; novel strategies; novel therapeutics; premature; ribosome profiling

Project Summary/Abstract Nonsense-mediated mRNA decay (NMD), the destabilization of an otherwise stable mRNA by premature translation termination, is a conserved quality control pathway that exemplifies the interdependence of mRNA decay and protein synthesis. NMD has been extensively studied in multiple eukaryotes, particularly with respect to the nature of its substrates and the structures and interactions of its central regulators, Upf1, 2, and 3. In spite of these efforts many key mechanistic questions about this important regulatory pathway remain to be resolved. We have yet to understand the detailed differences between normal and premature termination or the molecular events by which the Upf proteins selectively target translating mRNAs containing premature termination codons (PTCs), promote mRNA destabilization, or enhance the disassembly of a poorly dissociable premature termination complex. In part, an understanding of these problems required new approaches that take into account the important roles played by components of the protein synthesis apparatus in implementing NMD. Using the yeast Saccharomyces cerevisiae as a model system, we have now established such approaches. We developed a selective ribosome profiling procedure that allows delineation of the specificity and timing of Upf factor association with translating ribosomes, combined the selective purification of Upf1-associated ribosomes with cryo-electron microscopy to localize Upf1 to a specific ribosomal domain, combined mass spectrometry and efficient purification of full-length proteins derived from nonsense codon readthrough to elucidate details of aberrant translation termination, and identified and characterized novel negative and positive regulatory elements, including two Upf1-binding sites, in the previously uncharacterized C- terminal domain of Dcp2, the catalytic component of the mRNA decapping enzyme. In the experiments of this proposal, we will follow up on these developments, addressing three principal research directions that seek to: i) define the mechanistic differences between premature and normal translation termination, ii) elucidate the function of ribosome-associated Upf proteins, and iii) determine the mechanism of decapping activation by Upf1 and other decapping activators. At the conclusion of these studies we anticipate being able to formulate an integrated model detailing the molecular events linking premature translational termination to targeted mRNA decay.

项目概要/摘要 无义介导的 mRNA 衰减 (NMD)，即通过以下方式使原本稳定的 mRNA 不稳定 过早翻译终止，是一种保守的质量控制途径，举例说明了 mRNA 降解和蛋白质合成的相互依赖性。 NMD 已在多个领域得到广泛研究 真核生物，特别是其底物的性质以及结构和相互作用 其中央监管机构 Upf1、2 和 3。尽管做出了这些努力，但仍存在许多关键的机制问题 重要的监管途径仍有待解决。我们尚未了解详细的差异 正常和过早终止之间或Upf蛋白选择性的分子事件之间 靶向翻译含有提前终止密码子 (PTC) 的 mRNA，促进 mRNA 不稳定，或增强解离性较差的提前终止复合物的分解。在 部分，对这些问题的理解需要新的方法，考虑到重要的 蛋白质合成装置的组件在实施 NMD 中所起的作用。使用酵母 以酿酒酵母为模型系统，我们现在已经建立了这样的方法。我们 开发了一种选择性核糖体分析程序，可以描述核糖体分析的特异性和时间 Upf因子与翻译核糖体关联，结合Upf1相关的选择性纯化 使用冷冻电子显微镜将 Upf1 定位到特定核糖体结构域的核糖体，组合质量 来自无义密码子通读的全长蛋白质的光谱测定和有效纯化 阐明异常翻译终止的细节，并识别和表征新的负面和特征 正向调控元件，包括两个 Upf1 结合位点，位于先前未表征的 C- Dcp2 的末端结构域，mRNA 脱帽酶的催化成分。在实验中 在此提案中，我们将跟进这些进展，解决三个主要研究方向 旨在： i) 定义过早翻译终止和正常翻译终止之间的机制差异， ii) 阐明核糖体相关 Upf 蛋白的功能，以及 iii) 确定其机制 通过 Upf1 和其他脱帽激活剂进行脱帽激活。在这些研究的结论中，我们 预计能够制定一个综合模型，详细说明与早产儿相关的分子事件 翻译终止导致目标 mRNA 衰变。