TRANSCRIPTION AND RNA PROCESSING IN TRYPANOSOMES

我们的研究使用了非洲锥虫作为模型系统， 人类的昏睡病和牛的长假名。我们主要关注以下几个方面 这些寄生虫特有的锥虫生物学，希望能 确定合适的化疗干预靶点。从长远来看 这项提案的目标仍然是确定以下机制的特征 控制布鲁氏锥虫的转录和RNA加工。这个 拟议的研究源于并扩展了四个主要发现，即 直接源自我们在过去资金期间的调查。 首先，剪接前导(SL)RNA帽的甲基化核苷酸 结构对于利用这种RNA进行反式剪接是必不可少的。 第二，我们建立了一个无细胞系统，它忠实地修改了 SL RNA帽结构。第三，在微管蛋白前体mRNA成熟过程中 反式剪接和反式剪接之间存在功能关系 多聚腺苷，因此，在它们各自的机器之间。 第四，反式剪接体U6小分子编码基因的表达 核(Sn)RNA、U-SnRNAB和7SL RNA受基因外调控 与A基因内部启动子区域重合的调控元件 上游tRNA基因。 在下一个资助期，我们计划： 1)阐明独特的SL RNA帽子结构是否参与了 新组装的SL的核质转运 核糖核蛋白颗粒(RNP)，有助于建立 SL RNA二级结构和/或与特定蛋白质结合 因素(S)。这些研究不仅将有助于破译 修饰核苷酸在锥虫新陈代谢中的作用 将提供关于形态发生、结构和 SL RNF的相互作用。 2)建立SL特异性RNA的纯化方案 甲基转移酶，并了解其底物需求。一旦 酶被纯化后，将有可能克隆和表达 相应的功能、结构和药理基因 学习。 3)详细分析了微管蛋白前体mRNA的顺式作用信号。 是鉴定多聚腺苷化和3‘剪接所必需的 网站。这将导致对相互作用的更好理解 反式剪接和聚腺苷酸化机制及其相互间的关系 对调节信使核糖核酸的产生有贡献。 4)研究trna基因在促进和控制中的作用。 相关的小RNA基因的表达。这些研究将探索 一种新颖的RNA聚合酶III启动子结构 在真核生物中是史无前例的，并可能提供一种新的 真核生物学中的范例。 综上所述，拟议的实验将为 对基因表达的机制和调控有详细的了解 在布氏毛滴虫细胞中。

Our studies employ as a model system the African trypanosomes, the agents of sleeping sickness in man and nagana in cattle. We focus on aspects of trypanosome biology which are unique to these parasites in the hope to identify suitable targets for chemotherapeutic intervention. The long term goal of this proposal remains the characterization of mechanisms that control transcription and RNA processing in Trypanosoma brucei. The proposed research stems and expands from four main findings which are directly derived from our investigations during the past funding period. First, the methylated nucleotides of the spliced leader (SL) RNA cap structure are essential for utilization of this RNA in trans-splicing. Second, we have established a cell-free system that faithfully modifies the SL RNA cap structure. Third, during maturation of the tubulin pre-mRNA there is a functional relationship between trans-splicing and polyadenylation and, therefore, between their respective machineries. Fourth, the expression of genes coding for the trans-spliceosomal U6 small nuclear (sn) RNA, U-snRNA B and 7SL RNA is controlled by extragenic regulatory elements which coincide with the internal promoter region of an upstream tRNA gene. In the next funding period we plan to: 1) elucidate whether the unique SL RNA cap structure is involved in nuclear-cytoplasmic trafficking of the newly-assembled SL ribonucleoprotein particle (RNP), in contributing to the establishment of the SL RNA secondary structure and/or in binding to specific protein factor(s). These studies will help decipher not only the potential function of modified nucleotides in the trypanosome metabolism but also will provide valuable information about the morphogenesis, structure and interactions of the SL RNF. 2) establish a purification scheme for the SL RNA-specific methyltransferases and understand their substrate requirements. Once the enzymes are purified it will become possible to clone and express the corresponding genes for functional, structural and pharmacological studies. 3) analyze in detail the cis-acting signals of the tubulin pre-mRNA, which are required for the identification of the polyadenylation and 3' splice sites. This will lead to a better understanding of the interactions between the trans-splicing and polyadenylation machineries and their contribution to the regulation of mRNA production. 4) investigate the function of tRNA genes in promoting and controlling expression of the associated small RNA genes. These studies will explore a promoter structure for RNA polymerase III which is novel and unprecedented among eukaryotic organisms and possibly provide a new paradigm in eukaryotic biology. Taken together, the proposed experiments will provide the basis for a detailed understanding of the mechanisms and regulation of gene expression in T. brucei cells.