RNA METABOLISM IN TRYPANOSOMES

描述(申请人提供)：在过去的几年里，布鲁氏锥虫的生物学研究见证了令人难以置信的增长，这要归功于两个重大进展：布鲁氏锥虫基因组序列的完成和我们实验室发现的RNA干扰(RNAi)。布鲁氏锥虫是人类昏睡病和牛昏睡病的病原体。通过RNAi将基因组信息与下调基因功能相结合，现在使用全球方法研究复杂的细胞机制是现实的。由于转录调控在布氏毛滴虫中的作用较小，因此对基因表达的调控主要集中在转录后调控机制上。因此，我们推测，控制mRNA新陈代谢的酶对这些寄生虫是必不可少的。这项建议侧重于RNA代谢的一些方面，这些方面要么是锥体所特有的(第4帽生物合成和功能)，要么是真核生物学中的新概念(反式剪接和多聚腺苷酸化与RNAi途径的耦合)。 这项提议的长期目标仍然是描述参与RNA代谢的分子机制及其与锥虫生物学的相关性。在下一个资助期，我们计划： 1.鉴定参与CAP-4生物合成的特定RNA甲基转移酶，并探讨CAP-4的形成与多聚腺苷酸化之间是否存在机制联系。 2.通过使用多种方法，包括监测活细胞中加工因子与转录基因的关联，加深了我们对反式剪接和聚腺苷酸化偶联的理解。 3.进一步研究RNAi途径的组成以及TbAGO1与翻译核糖体的相互作用。

DESCRIPTION (provided by the applicant): In the last few years the study of the biology of Trypanosoma brucei, the agent of sleeping sickness in humans and nagana in cattle, has witnessed an incredible growth due to two major advances: the completion of the T. brucei genome sequence and the discovery by our laboratory of RNA interference (RNAi). By interfacing genome information with down-regulating gene function by RNAi, it is now realistic to investigate complex cellular mechanisms using global approaches. As transcription regulation plays a minor role in T. brucei, the emphasis on regulation of gene expression is primarily on post-transcriptional regulatory mechanisms. Thus, we anticipate that the enzymes governing mRNA metabolism are essential for these parasites. This proposal focuses on aspects of RNA metabolism that are either unique to trypanosomes (cap 4 biosynthesis and function) or are novel concepts in eukaryotic biology (coupling of trans-splicing and polyadenylation and the RNAi pathway). The long-term goal of this proposal remains the characterization of molecular mechanisms involved in RNA metabolism and their relevance to trypanosome biology. In the next funding period we plan to: 1. Characterize the specific RNA methyltransferases involved in cap 4 biosynthesis and investigate whether there is a mechanistic link between cap 4 formation and polyadenylation. 2. Further our understanding of the coupling of trans-splicing and polyadenylation by using a variety of approaches, including monitoring the association of processing factors along transcribed genes in living cells. 3. Further characterize the RNAi pathway in terms of its components and how the interaction of TbAGO1 with translating ribosomes is brought about.