Genetic nonsense and its consequences

项目摘要/摘要 无意义介导的信使核糖核酸衰变(NMD)，即通过 过早终止翻译，是一种保守的质量控制途径，它例证了 信使核糖核酸衰变与蛋白质合成的相互依赖。NMD已经在多个领域得到了广泛的研究 真核生物，特别是关于其底物的性质以及其结构和相互作用 它的中央调节器，UPF1，2和3。尽管做出了这些努力，但关于这一点的许多关键机械问题 重要的调控途径仍有待解决。我们还没有弄清楚细节上的区别。 在正常终止和过早终止之间，或者UPF蛋白选择性地通过分子事件 靶向翻译含有提前终止密码子(PTCs)的mRNAs，促进mRNA 不稳定，或加强分解不良的提前终止复合体。在……里面 要理解这些问题，需要新的方法来考虑重要的 蛋白质合成装置的部件在实施国家导弹防御系统中的作用。使用酵母菌 酿酒酵母作为一个模型系统，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图谱程序，可以描绘出特异性和时间 UPF因子结合翻译核糖体，联合选择性纯化UPF1相关蛋白 用冷冻电子显微镜将UPF1定位到特定的核糖体结构域，结合质量 从无义密码子通读到的全长蛋白质的光谱和高效纯化 阐明异常翻译终止的细节，并确定和表征小说的负面和 积极的调控元件，包括两个UPF1结合位点，在以前未知的C- Dcp2的末端结构域，是信使核糖核酸解离酶的催化成分。在……的实验中 这项建议，我们将跟进这些发展，解决三个主要研究方向 其寻求：i)定义提前翻译终止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 UPF1和其他去包裹激活剂的去包裹激活。在这些研究的结论中，我们 预计能够制定一个完整的模型，详细说明与早熟有关的分子事件 靶向信使核糖核酸的翻译终止。

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.