TRANSCRIPTION AND RNA PROCESSING IN TRYPANOSOMES

我们的研究采用非洲锥虫作为模型系统， 人的昏睡病和牛的长形病。我们专注于以下方面 锥虫生物学是这些寄生虫所特有的， 鉴定用于化学治疗干预的合适靶点。长期 本提案的目标仍然是确定 控制布氏锥虫的转录和RNA加工。的 拟议的研究源于并扩展了四个主要发现， 直接来自我们在过去资助期间的调查。 首先，剪接前导（SL）RNA帽的甲基化核苷酸 结构对于在反式剪接中利用该RNA是必需。 第二，我们建立了一个无细胞系统， SL RNA帽结构。第三，在微管蛋白前体mRNA的成熟过程中， 反式剪接和 聚腺苷酸化，因此，它们各自的机器之间。 第四，编码反式剪接体U6小的基因的表达 核（sn）RNA、U-snRNA B和7SL RNA受基因外调控 调控元件，其与一个启动子的内部启动子区一致， 上游tRNA基因。 在下一个融资期，我们计划： 1)阐明独特的SL RNA帽结构是否参与 新组装的SL的核质运输 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 因数。这些研究不仅有助于破译 修饰的核苷酸在锥虫代谢中的功能， 将提供有关形态发生，结构和 SL RNF的相互作用。 2)建立SL RNA特异性的纯化方案 甲基转移酶并了解其底物要求。一旦 酶被纯化，将有可能克隆和表达 相应的功能、结构和药理学基因 问题研究 3)详细分析微管蛋白前体mRNA的顺式作用信号， 是鉴定多聚腺苷酸化和3 '剪接所必需的 网站.这将有助于更好地理解 在反式剪接和多聚腺苷酸化机制之间， 对mRNA产生的调节作用。 4)研究tRNA基因在促进和控制 相关小RNA基因的表达。这些研究将探讨 一种新的RNA聚合酶III的启动子结构， 这在真核生物中是前所未有的，可能提供了一种新的 真核生物学的典范。 综上所述，拟议的实验将为 详细了解基因表达的机制和调控 于t.布氏细胞

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.