Function of the TRAMP Complex at the Molecular Level

描述（由申请人提供）：拟议研究的总体目标是获得对TRAMP（Trf 4/Air2/Mtr 4聚腺苷酸化）和核外泌体形成的多组分复合物的详细机制理解。这种TRAMP-外泌体机制在真核基因表达的调控中起关键作用。TRAMP由在真核生物中高度保守的三个亚基组成，即非典型的多聚（A）聚合酶、锌关节蛋白和RNA解旋酶。真核细胞核外泌体由11个单元组成，它们在真核细胞中也是高度保守的，并且包括两个功能性3'至5'核酸酶，Rrp 6p和Rrp 44 p。TRAMP标记具有短poly（A）尾的RNA，并解析RNA二级结构，而外泌体降解TRAMP标记的RNA。尽管在RNA代谢中起着关键作用，但尚不清楚TRAMP-外泌体机制的组分如何在RNA加工过程中协调其功能，以及如何鉴定RNA底物。 在我们的建议中，我们使用生物化学手段来解决这些问题。使用重建的TRAMP-外泌体系统，我们将描绘哪些外泌体和TRAMP组分和活性对于TRAMP的外泌体刺激以及外泌体对TRAMP功能的影响是至关重要的。为了理解RNA加工的TRAMP-外泌体机制在各个反应步骤的水平，我们将设计一个机制，定量框架的反应。然后，我们将研究TRAMP-外泌体机制如何识别RNA靶标。为此，我们将研究如何将低修饰的tRNAiMet与正确修饰的tRNAiMet区分开来，TRAMP-外泌体机制如何展开RNA，以及RNA长度和二级结构是否以及如何影响TRAMP-外泌体机制。 我们期望我们的研究提供一个新的水平的机制理解的TRAMP-外泌体机制，和新的，重要的洞察核RNA代谢的分子基础。 公共卫生相关性：RNA加工缺陷与许多疾病有关，包括自身免疫缺陷、神经退行性疾病和癌症。为了研究这些疾病的分子基础并指导潜在治疗药物的开发，我们建议描述TRAMP复合物的分子功能及其与核外泌体的相互作用，这对于许多细胞RNA的质量控制和正确加工至关重要。

DESCRIPTION (provided by applicant): The overall goal of the proposed research is to obtain detailed mechanistic understanding of the multi-component complex formed by TRAMP (Trf4/Air2/Mtr4 Polyadenylation) and the nuclear exosome. This TRAMP-exosome machinery plays key roles in the regulation of eukaryotic gene expression. TRAMP consists of three subunits that are highly conserved in eukaryotes, a non-canonical poly(A) polymerase, a Zn-knuckle protein, and an RNA helicase. The eukaryotic nuclear exosome consists of eleven units, which are also highly conserved in eukaryotes, and include two functional 3' to 5' nucleases, Rrp6p and Rrp44p. TRAMP marks RNAs with short poly(A) tails, and resolves RNA secondary structures, while the exosome degrades the RNAs marked by TRAMP. Despite pivotal roles in RNA metabolism, it is not understood how the components of the TRAMP-exosome machinery coordinate their functions during RNA processing, and how RNA substrates are identified. In our proposal, we address these problems using biochemical means. Using a reconstituted TRAMP-exosome system, we will delineate which exosome and TRAMP components and activities are critical for the exosome stimulation by TRAMP, and for effects of the exosome on TRAMP function. To understand RNA processing by the TRAMP-exosome machinery at the level of individual reaction steps, we will devise a mechanistic, quantitative framework for the reaction. We will then examine how the TRAMP-exosome machinery identifies RNA targets. To this end we will investigate how hypomodified tRNAiMet is distinguished from correctly modified tRNAiMet, how the TRAMP-exosome machinery unfolds the RNAs, and whether and how RNA length and secondary structure affect the TRAMP-exosome machinery. We anticipate our studies to provide a new level of mechanistic understanding of the TRAMP-exosome machinery, and novel, significant insight into the molecular basis of nuclear RNA metabolism. PUBLIC HEALTH RELEVANCE: Defects in RNA processing have been linked to many diseases including autoimmune defects, neurodegenerative diseases, and cancer. To examine the molecular basis for these diseases and to guide the development of potential therapeutic agents, we propose to delineate the molecular function for the TRAMP complex and its interaction with the nuclear exosome, which are pivotal for quality control and correct processing of many cellular RNAs.