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UNIVERSITY OF CALIFORNIA, IRVINE

Trypanosomatids are protozoan hemoflagellates responsible for a variety of human diseases, including Chagas' disease, leishmaniasis, and African sleeping sickness, which account for nearly one million deaths per year. The unique genetic phenomena occurring in parasites giant mitochondria have been extensively studied, producing major advances in the understanding of kinetoplastid DNA kDNA replication and uridine insertion-deletion mRNA editing. However, the molecular mechanisms governing mRNA stability and turnover in mitochondria remain mostly unknown. Our goal is to set the stage for molecular analysis of the regulatory mechanisms controlling mitochondrial mRNA polyadenylation, and better understanding of life cycle-correlated changes in kDNA expression. The specific hypothesis underlying the proposed research is that polyadenylation in trypanosomal mitochondria is essential for the stability of translationally-competent mRNA and, therefore, mitochondrial function. We suggest that polyadenylation is accomplished by two divergent Kinetoplastid Poly A Polymerases acting as catalytic subunits of multi-protein complexes. The candidate kPAP enzymes have been identified by homology to RNA uridylyltransferases, further advancing our concept of the crucial roles played by TUTase-like structural scaffolds in mitochondrial RNA processing. We propose to: 1. Characterize the roles of kPAP1 and 2 in polyadenylation of non edited, pre-edited, and edited mRNAs. RNA interference will be used to down regulate expression of both kPAPs in procyclic and bloodstream forms, and their significance for mitochondrial function will be addressed by comparative analysis of cell viability, inner membrane potential, and RNA transcripts. 2. Elucidate the protein composition of the polyadenylation machinery in trypanosomal mitochondria. The kPAP-containing complexes will be isolated by tandem affinity chromatography, tested for enzymatic activity, and analyzed by mass spectrometry.

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