RNA METABOLISM IN TRYPANOSOMES

我们的研究采用非洲锥虫作为模型系统，锥虫是人类昏睡病和牛那加纳病的病原体。在过去几年中，正如正在进行的布鲁氏锥虫和利什曼原虫基因组测序项目所强调的那样，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生物学的新概念，如反式剪接、cap 4修饰、线粒体RNA编辑、反式剪接和聚腺苷酸化的偶联，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至关重要。本研究的重点是锥虫RNA代谢的各个方面，这些方面要么是这些寄生虫所特有的（第4章生物合成和功能，反式剪接和聚腺苷化的偶联），要么是真核生物学中的新概念（RNA干扰）。拟议的研究源于并扩展了三个主要发现，这些发现直接来自我们在上一个资助期间的调查。首先，剪接的先导RNA的独特cap 4结构的形成是由核多提交复合体进行的，其成分在真核生物进化过程中是保守的。其次，反式剪接和聚腺苷化是由3'剪接位点和外显子增强子序列在功能上偶联和调节的。第三，双链RNA诱导T细胞mRNA降解。本提案的长期目标是表征参与RNA代谢的分子机制及其在锥虫生物学中的相关性。在下一个融资期，我们计划：通过克隆剩余亚基进一步表征cap 4甲基转移酶复合物，并研究其组装和cap 4修饰的功能。2.通过结合遗传和生化方法生成合适的试剂，进一步表征聚腺苷酸化和反式剪接之间的耦合以及它们与转录机制的潜在相互作用。3.研究布鲁氏体RNA干扰的机制，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.