Enzymes and enzyme complexes in RNA metabolism

 描述（由申请人提供）：计划的研究旨在定量了解RNA代谢中关键酶及其相关复合物的功能。我们重点研究了S.酿酒酵母和人DDX 3X及其在真核生物翻译起始中的作用。该研究对于理解DEAD盒RNA解旋酶的功能和调控以及阐明真核生物翻译起始具有重要意义。这项工作对于阐明几种肿瘤类型的分子基础和理解多种感染性疾病的宿主-病原体相互作用具有重要意义。我们还研究了S.酿酒酵母及其在核RNA代谢中的作用。这一系列的研究对于理解核RNA质量控制、非编码RNA表达的调节以及几种RNA类型的核加工至关重要。这项研究对某些神经退行性疾病和自身免疫性疾病以及几种肿瘤类型的分子基础有影响。 使用生物化学，生物物理学和下一代测序方法的组合，我们研究了Ded 1 p如何与广泛的RNA相互作用，并在细胞中的特定位点发挥作用。此外，我们还确定了翻译后修饰如何影响Ded 1 p在体外和细胞中的功能。对于人DDX 3X，我们建立了一个机制框架，并研究了两种病毒蛋白与酶，HCV核心和牛痘病毒K7相互作用，影响DDX 3X在体外和细胞中的功能。对于TRAMP-外泌体机器，我们确定了整个酵母外泌体在单核苷酸分辨率下的降解动力学，并定量测量了TRAMP-外泌体机器中聚腺苷酸化和衰变是如何协调的。最后，我们研究了TRAMP-外泌体机器在细胞中动力学水平上的功能。 我们期望我们的工作提供前所未有的定量知识酶复合物在体外和细胞中的功能，我们期望从根本上新颖的见解真核翻译起始和核RNA代谢。

 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.