Recognition and degradation of mRNA by nonsense-mediated decay.

项目摘要/摘要 密切监测基因表达，以确保将遗传信息转化为生物信息的保真度 活动。在真核细胞中，在mRNA翻译过程中存在一个专门的质量控制检查点来评估 MRNA的完整性和快速降解在其蛋白编码区含有无意义密码子的转录本-a 导致提前终止转换的信号，如果不进行检查，则会累积截断的 多肽。这一途径被称为无意义介导的信使核糖核酸衰变(NMD)，在生物上起重要作用。 通过保护细胞免受不完全蛋白质的有害后果而发挥作用。尽管有两个多人 经过几十年的研究，这一过程的核心基本问题仍然没有得到很好的解决，包括细胞如何 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，刺激无意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种全面的分子 了解细胞识别和靶向含有无义的信使核糖核酸的能力的基础事件 迅速腐烂。 在过去的十年里，我们在表征对识别至关重要的rna特征方面取得了开创性的发现。 以及有效靶向NMD的mRNA。最近，我们已经开始剖析后续的分子事件。 通过识别与废话相关的蛋白质的补体来识别底物- 含有mRNA，并鉴定了关键的NMD因子UFP1的催化失活突变体。值得注意的是，我们 已经描述了NMD机制和过早终止的核糖体之间的功能相互作用 它存在于体内，对决定异常转录本的命运至关重要。我们在这里提议 利用我们过去的努力并开发新的工具和试剂来i)识别UPF1 RNA结合靶点 在全球范围内提供前所未有的洞察力，了解NMD因子结合如何决定将mRNA靶向NMD； 二)进一步询问过早终止的核糖体与NMD之间的功能相互作用 靶向底物以快速腐烂所必需的机械；以及iii)监测体内NMD mRNP的动态 酶催化的邻近标记。总而言之，这些研究将提供前所未有的洞察力 一个完整的细胞RNA质量控制途径和基因表达调节的潜在分子事件。

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.