Enzymes and enzyme complexes in RNA metabolism

 描述(申请人提供)：计划中的研究旨在定量了解关键酶及其相关复合体在RNA代谢中的功能。我们重点研究了酿酒酵母和人DDX3X中的DEAD-box RNA解旋酶Ded1p及其在真核细胞翻译起始中的作用。这项研究对于了解DEAD-box RNA解旋酶的功能和调控以及阐明真核生物的翻译启动是至关重要的。这项工作对于描述几种肿瘤类型的分子基础以及了解多种传染病的宿主-病原体相互作用具有重要意义。我们还研究了酿酒酵母的TRAMP-外切体机制及其在核RNA代谢中的作用。这条探索路线对于理解核RNA质量控制、非编码RNA表达的调节以及几种RNA类型的核处理至关重要。这项研究对某些神经退行性疾病和自身免疫性疾病的分子基础以及几种肿瘤类型具有重要意义。使用生化、生物物理和下一代测序方法的组合，我们检查了Ded1p是如何被招募到细胞中的特定位置并在其上发挥功能的，它与广泛的RNA混杂相互作用。此外，我们还确定了翻译后修饰如何在体外和细胞内影响Ded1p的功能。对于人的DDX3X，我们建立了一个机制框架，并研究了与酶相互作用的两种病毒蛋白--丙型肝炎病毒核心蛋白和痘苗病毒K7在体外和细胞内如何影响DDX3X的功能。对于TRAMP-Exosome机制，我们确定了整个酵母外切体在单核苷酸分辨率下的降解动力学，并定量地测量了TRAMP-Exosome机制中多聚腺苷基化和腐烂是如何协调的。最后，我们在细胞内的动力学水平上研究了流动型外切体机械的功能。我们期待着我们的工作为酶复合体在体外和细胞内的功能提供前所未有的定量知识，我们期待着对真核细胞翻译启动和核RNA代谢的全新见解。

 DESCRIPTION (provided by applicant): The planned research aims to quantitatively understand the function of key enzymes and their associated complexes in RNA metabolism. We focus on the DEAD-box RNA helicases Ded1p from S. cerevisiae and human DDX3X and their role in eukaryotic translation initiation. This research is critical for understanding functin and regulation of DEAD-box RNA helicases and for illuminating eukaryotic translation initiation. The work is important for delineating the molecular basis of several tumor types and for understanding host- pathogen interactions of multiple infectious diseases. We also investigate the TRAMP-exosome machinery from S. cerevisiae and its role in nuclear RNA metabolism. This line of inquiry is critical for understanding nuclear RNA quality control, regulation of non-coding RNA expression, and nuclear processing of several RNA types. This research has implications for the molecular basis of certain neurodegenerative and autoimmune diseases and for several tumor types. Using a combination of biochemical, biophysical and next generation sequencing approaches, we examine how Ded1p, which interacts promiscuously with a broad range of RNAs, is recruited to and functions at specific sites in the cell. In addition, we determine how post-translational modifications impact the function of Ded1p in vitro and in the cell. For human DDX3X, we establish a mechanistic framework and examine how two viral proteins that interact with the enzyme, HCV core and vaccinia virus K7, affect the function of DDX3X in vitro and in the cell. For the TRAMP-exosome machinery, we determine degradation kinetics at single nucleotide resolution for the entire yeast exosome and quantitatively measure how polyadenylation and decay are coordinated in the TRAMP-exosome machinery. Finally, we examine the function of the TRAMP-exosome machinery on a kinetic level in the cell. We anticipate our work to provide unprecedented quantitative knowledge about the function of enzyme complexes in vitro and in the cell and we expect fundamentally novel insight into eukaryotic translation initiation and nuclear RNA metabolism.