Structural and Functional Studies of Mtr4 and Mtr4-mediated Complexes

 描述(由申请人提供) 细胞RNA的完整性和可用性受到各种RNA加工的严格控制， 周转率和监控路径。真核细胞外切体在细胞核和胞浆中对多种RNA的加工和降解起着重要作用。几种蛋白质因子与外切体相关，以识别合适的RNA底物并激活外切体活性。一个主要的挑战是了解这些蛋白质如何与RNA底物相互作用以及如何相互作用以实现底物特异性的分子细节。值得注意的是，核外切体所有已知活动所需的唯一蛋白质因子是RNA解旋酶MTR4。鉴于MTR4在外切体激活中的核心作用，迫切需要了解MTR4如何发挥作用，它如何与RNA底物相互作用，以及它如何调节其他外切体激活因子的组装并受到其影响。最近的研究表明，MTR4的ARCH结构域在解开RNA底物方面起着以前未被认识的作用。这一观察的潜在基础尚不清楚，但被认为是连接ARCH和核心解旋酶结构域的底物相互作用的函数。MTR4与其他复合体，如TRAMP复合体之间的详细相互作用也鲜为人知。这个项目试图通过使用各种生化和结构方法，结合体内遗传分析来确定mtr4功能的分子基础。本项目的具体目标是(1)描述ARCH结构域在RNA识别和解离中的作用，以及(2)表征MTR4与蛋白质结合伙伴的相互作用。

 DESCRIPTION (provided by applicant) The integrity and availability of cellular RNA is tightly regulated by a variety of RNA processing, turnover, and surveillance pathways. The eukaryotic exosome plays a major role in the processing and degradation of a wide variety of RNAs in both the nucleus and cytosol. Several protein factors associate with the exosome to identify appropriate RNA substrates and activate exosome activity. A major challenge is to understand the molecular details of how these proteins interact with RNA substrates and with each other to achieve substrate specificity. Notably, the only protein factor that is required for all known activities of the nuclear exosome i the RNA helicase, Mtr4. Given this central role in exosome activation, there is a critical need to understand how Mtr4 functions, how it interacts with RNA substrates, and how it mediates and is influenced by assembly of other exosome activating factors. Recent studies indicate that the arch domain of Mtr4 plays a previously unrecognized role in unwinding RNA substrates. The underlying basis for this observation is unclear, but is proposed to be a function of substrate interactions that connect the arch to the core helicase domains. The detailed interactions between Mtr4 and other complexes, such as the TRAMP complex, are also poorly understood. This project seeks to define the molecular basis for Mtr4 function by employing a variety of biochemical and structural approaches, combined with in vivo genetic analysis. The specific aims of this project are to (1) describe the role of the arch domain in RNA recognition and unwinding, and (2) characterize Mtr4 interactions with protein binding partners.