Molecular and biochemical probing of yeast, RNA surveillance complex TRAMP

描述（由申请人提供）： 项目摘要：核RNA监测是一种基本的质量保证机制，对于功能RNA的准确表达和及时破坏本质上是调节性的RNA或加工副产物至关重要。在核RNA监视中发生的任何不平衡都有可能导致异常的RNA产生和积累，这可能具有病理后果。因此，重要的是通过研究其组成部分来全面了解核RNA监视机制。不正确形成的RNA被TRAMP复合物聚腺苷酸化，TRAMP复合物由polyA聚合酶（Trf 4p/Trf 5 p）、RNA解旋酶（Mtr 4p）和Zn结合蛋白（Air 1 p/Air2 p）组成，并被核外泌体靶向降解。尽管TRAMP亚基在核RNA监测中具有关键地位，但我们对每个TRAMP亚基对RNA识别、腺苷酸化/调节、结构改变和与外泌体通信的作用的理解还很不发达。为了克服这一点，并发展一个更完整的理解TRAMP如何区分缺陷的功能RNA，我们将着手发现基本问题的答案，TRAMP如何作为一个三成员复合物，每个亚基如何影响其他成员在RNA识别，修饰，并与降解机制，核外泌体通信。这将使用遗传学中已建立的方法以及蛋白质、蛋白质复合物和RNA加工和降解的生物化学分析来完成。利用具有新目的的成熟技术，我们正在开发一种方法来表征细胞中TRAMP功能丧失的腺苷酸化和RNA解旋。 公共卫生相关性： 项目叙述：缺陷RNA的表达可能会通过蛋白质的异位或失败表达产生病理影响。我们的研究解决了关于降解不正确形成的RNA所需机制的基本问题，因此我们的研究将增加对细胞控制发病机制的认识。

DESCRIPTION (provided by applicant): Project Summary: Nuclear RNA surveillance is a fundamental quality assurance mechanism critical for the accurate expression of functional RNAs and the timely destruction of RNAs that are either regulatory in nature or byproducts of processing. Any imbalance that occurs in nuclear RNA surveillance has the potential to cause aberrant RNA production and accumulation, which can have pathological consequences. It is therefore important to develop a complete understanding of the nuclear RNA surveillance mechanism by studying its components. Incorrectly formed RNAs are polyadenylated by the TRAMP complex, composed of a polyA polymerase (Trf4p/Trf5p), RNA helicase (Mtr4p) and Zn-binding protein (Air1p/Air2p), and targeted for degradation by the nuclear exosome. Our understanding of what each TRAMP subunit contributes to RNA recognition, adenylation/modulation, structure alterations and communication with exosome is poorly developed despite its pivotal position in nuclear RNA surveillance. To overcome this and develop a more complete understanding of how TRAMP distinguishes defective from functional RNA, we will undertake to discover answers to basic questions about, how TRAMP works as a three member complex, how each subunit influences the other members in RNA recognition, modification, and communication with the degrading machinery, the nuclear exosome. This will be accomplished using established methodologies in genetics, and biochemical analyses of proteins, protein-complexes and RNA processing and degradation. Capitalizing on proven technology with a new purpose, we are developing a method to characterize loss of TRAMP function in cells with respect to adenylation and RNA unwinding. PUBLIC HEALTH RELEVANCE: Project Narrative: The expression of defective RNAs can have pathological effects through ectopic or failed expression of proteins. Our study addresses fundamental questions about a mechanism required for the degradation of improperly formed RNAs, thereby our research will increase knowledge of cellular efforts to control pathogenesis.