Recognition and degradation of mRNA by nonsense-mediated decay.

项目总结/摘要 基因表达受到密切监测，以确保遗传信息转化为生物信息的准确性。 活动在真核细胞中，mRNA翻译过程中存在一个专门的质量控制检查点， mRNA的完整性和快速降解的转录本含有一个无义密码子在其蛋白质编码区- a 导致翻译过早终止的信号，如果不加检查，则会导致截断的 多肽。这种途径被称为无义介导的mRNA衰变（NMD），是一种重要的生物学途径。 通过保护细胞免受不完整蛋白质的有害后果而起作用。尽管超过两个 经过几十年的研究，这个过程的核心问题仍然没有得到很好的解决，包括细胞是如何 区分正常和过早的翻译终止以及随后发生的分子事件 在翻译mRNA和NMD机制之间，刺激无义密码子的加速降解， 包含文字记录。我研究的长期目标是获得一个全面的分子生物学 理解细胞识别和靶向含有无义mRNA的能力的基础事件， 快速衰变 在过去的十年中，我们在表征RNA特征方面取得了开创性的发现，这些特征对于识别 以及mRNA对NMD的有效靶向。最近，我们已经开始剖析分子事件， 通过鉴定与无义特异性相关的蛋白质的互补物来进行底物识别， 包含mRNA和表征关键NMD因子UFP 1的催化失活突变体。值得注意的是， 已经描述了NMD机制和提前终止核糖体之间的功能性相互作用 它存在于体内，并且对于决定异常转录物的命运至关重要。我们在此提议， 利用我们过去的努力，开发新的工具和试剂，i）鉴定UPF 1 RNA结合靶点 在全球范围内提供前所未有的洞察NMD因子结合如何决定mRNA靶向NMD; ii）进一步询问提前终止的核糖体和NMD之间的功能相互作用， 用于靶向底物以快速衰变所必需的机制;和iii）使用以下方法监测NMD mRNP体内动力学 酶催化邻近标记。总之，这些研究将提供前所未有的洞察力， 作为细胞RNA质量控制途径和基因表达调节因子的基础的分子事件。

PROJECT SUMMARY/ABSTRACT Gene expression is closely monitored to ensure fidelity in the conversion of genetic information into biological activity. In eukaryotic cells a specialized quality control checkpoint exists during mRNA translation to evaluate mRNA integrity and rapidly degrade transcripts containing a nonsense codon in their protein-coding region - a signal that causes premature termination of translation and, if left unchecked, the accumulation of truncated polypeptides. This pathway, referred to as nonsense-mediated mRNA decay (NMD), serves a vital biological function by protecting cells from the deleterious consequences of incomplete proteins. In spite of over two decades of study, fundamental questions central to this process remain poorly resolved, including how cells distinguish normal and premature translation termination and what molecular events occur subsequently between the translating mRNA and NMD machinery to incite accelerated degradation of the nonsense codon- containing transcript. The long term objective of my research is to gain a comprehensive molecular understanding of the events underlying the cell's ability to recognize and target nonsense-containing mRNA for rapid decay. In the last decade, we have made seminal discoveries in characterizing RNA features vital for the recognition and efficient targeting of an mRNA to NMD. Recently, we have begun to dissect molecular events subsequent to substrate recognition through identifying the complement of proteins specifically associated with nonsense- containing mRNA and characterizing a catalytically-inactive mutant of the key NMD factor, UFP1. Notably, we have described a functional interaction between the NMD machinery and prematurely terminating ribosomes that exists in vivo and which is critical for dictating the fate of the aberrant transcript. We propose here to capitalize on our past efforts and exploit novel tools and reagents to i) identify UPF1 RNA binding targets globally to provide unprecedented insight into how NMD factor binding dictates the targeting of mRNA to NMD; ii) interrogate further the functional interaction between prematurely terminating ribosomes and the NMD machinery essential for targeting substrates to rapid decay; and iii) monitor NMD mRNP dynamics in vivo using enzyme-catalyzed proximity labeling. Together, these studies will provide unprecedented insight into the molecular events underlying an integral cellular RNA quality control pathway and regulator of gene expression.