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.
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