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.
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