RNA METABOLISM IN TRYPANOSOMES

我们的研究使用非洲锥虫作为模型系统，非洲锥虫是人类昏睡病和牛昏睡病的病原体。在过去的几年里，布鲁氏锥虫和利什曼原虫正在进行的基因组测序项目表明，锥虫原虫已经达到了单细胞病原体模式系统的地位。此外，对锥虫RNA代谢的研究有助于发现真核RNA生物学中的新概念，如反式剪接、帽4修饰、线粒体RNA编辑、反式剪接和多聚腺苷化的耦合，以及最近的RNA干扰(RNAi)。由于锥虫主要在转录后水平调节基因表达，我们预计控制mRNA代谢的酶对这些寄生虫是必不可少的。这项建议侧重于锥虫RNA代谢的一些方面，这些方面要么是这些寄生虫所特有的(CAP 4的生物合成和功能，反式剪接和多聚腺苷的耦合)，要么是真核生物学中的新概念(RNA干扰)。拟议的研究源于我们在上一个资助期的调查中直接得出的三个主要结论。首先，剪接前导RNA独特的4帽结构的形成是由一个核多提交复合体完成的，其成分在整个真核进化过程中都是保守的。第二，反式剪接和聚腺苷酸化在功能上是偶联的，并受3‘剪接点的序列和外显子增强子序列的调控。第三，双链RNA可诱导布氏支原体的mRNA降解。这项建议的长期目标是描述参与RNA新陈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.通过克隆剩余的亚基来进一步鉴定CAP4甲基转移酶复合体，并研究它们的组装和CAP4修饰的功能。2.通过结合遗传和生化方法产生合适的试剂，进一步表征聚腺苷酸化和反式剪接之间的偶联以及它们与转录机制的潜在相互作用。3.研究布氏毛滴虫的RNA干扰机制，优化基因表达调控体系。

Our studies employ as a model system the African trypanosomes, the agents of sleeping sickness in man and nagana in cattle. In the last years, trypanosomatid protozoa have attained the status of model systems for unicellular pathogens, as underscored by the ongoing genome sequencing projects of Trypanosoma brucei and Leishmania. Furthermore, the study of trypanosomatid RNA metabolism has been instrumental in the discovery of new concepts in eukaryotic RNA biology, like trans-splicing, cap 4 modification, mitochondrial RNA editing, coupling of trans-splicing and polyadenylation, and more recently RNA interference (RNAi). As trypanosomes regulate gene expression mainly at the post-transcriptional level, we anticipate that enzymes governing mRNA metabolism are essential for these parasites. This proposal focuses on aspects of trypanosome RNA metabolism that are either unique to these parasites (cap 4 biosynthesis and function, coupling of trans-splicing and polyadenylation) or are novel concepts in eukaryotic biology (RNA interference). The proposed research stems and expands from three main findings that are directly derived from our investigations during the last funding period. First, formation of the unique cap 4 structure of the spliced leader RNA is carried out by a nuclear multisubmit complex with components conserved throughout eukaryotic evolution. Second, trans- splicing and polyadenylation are functionally coupled and modulated by sequences at the 3' splice site and by exonic enhancer sequences. Third, double-stranded RNA induces mRNA degradation in T brucei. The long term goal of this proposal is the characterization of molecular mechanisms involved in RNA metabolism and their relevance in trypanosome biology. In the next funding period we plan to: 1.Further characterize the cap 4 methyltransferase complex by cloning the remaining subunits and study their assembly and function of cap 4 modifications. 2.Further characterize the coupling between polyadenylation and trans- splicing and their potential interaction with the transcriptional machinery by generating appropriate reagents in combination with genetic and biochemical approaches. 3.Investigate the mechanism of RNA interference in T.brucei and optimize the system for modulating gene expression.