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.

项目总结/摘要 无义介导的mRNA衰变（NMD），即通过 过早的翻译终止，是一个保守的质量控制途径， mRNA降解和蛋白质合成的相互依赖性。NMD在多个国家得到了广泛的研究， 真核生物，特别是关于其底物的性质和结构和相互作用， 其中央监管机构Upf 1、2和3。尽管这些努力，许多关键的机械问题， 重要调控途径仍有待解决。我们还没有弄清楚 正常和过早终止之间的关系，或者Upf蛋白选择性地 靶向翻译含有提前终止密码子（PTC）的mRNA，启动子mRNA 去稳定化，或增强可不良解离的过早终止复合物的分解。在 部分，理解这些问题需要新的方法，考虑到重要的 蛋白质合成装置的组成部分在实施NMD中所起的作用。使用酵母 酿酒酵母作为模型系统，我们现在已经建立了这样的方法。我们 开发了一种选择性的核糖体分析程序，可以描绘出特异性和时间， Upf因子与翻译核糖体的关联，结合Upf 1相关蛋白的选择性纯化， 核糖体与冷冻电子显微镜定位Upf 1到一个特定的核糖体结构域，结合质量 无义密码子通读衍生的全长蛋白质的光谱分析和有效纯化， 阐明异常翻译终止的细节，并确定和表征了新的阴性和 阳性调控元件，包括两个Upf 1结合位点，在以前未表征的C- Dcp 2的末端结构域，mRNA脱帽酶的催化组分。实验中 我们会跟进这些发展，并致力于三个主要的研究方向 试图：i）定义过早和正常翻译终止之间的机械差异， ii）阐明核糖体相关Upf蛋白的功能，以及iii）确定 通过Upf 1和其他去盖激活剂进行去盖激活。在这些研究结束时，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， 翻译终止到靶mRNA衰变。