Regulation of mRNA Turnover in Trypanosomes

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DESCRIPTION (provided by applicant): Trypanosomiasis and Leishmaniasis are major diseases in developing countries throughout the world. In these areas 100,000 people are currently infected with trypanosomes. Human illness includes African trypanosomiasis ("sleeping sickness"), Chagas' disease (endemic in regions of South and Central America) and cutaneous, mucocutaneous and visceral Leishmaniasis (endemic in parts of the Middle East and the Indian Subcontinent). African trypanosomiasis is spread to humans by the bite of tsetse flies that harbor the Trypanosoma brucei subspecies. The disease burden is estimated by the World Health Organization to be 2.05 million Disability Adjusted Life Years. The cost of treatment is high, and untreated infection usually results in death. However, trypanosomatids share many metabolic pathways among themselves that differ enough from human pathways to be exploited in the development of therapeutics that are more efficacious than those currently available. One possible therapeutic approach is suggested by the observation that trypanosomatids occur in different forms in their human and insect hosts. These differing forms at various stages of the life cycle may reflect differences in mRNA stability. The purpose of this proposal is to gain an understanding of how mRNAs are metabolized in trypanosomes. There appears to be little transcriptional regulation of steady state levels mRNA in these parasites. Therefore, the differences in mRNA stability that exist among different mRNAs in one life cycle form may be regulated, in part, by differential mRNA decay. Differences in mRNA expression patterns among the different life cycle forms is also likely to be controlled, in part, by differential mRNA turnover. We propose to identify and characterize two crucial enzymatic activities important in mRNA decay. Our aims are (1) to purify and clone the decapping enzyme from trypanosomes; (2) to purify and clone the deadenylating nuclease from trypanosomes; and (3) to assess the roles of the decapping enzyme and deadenylase in mRNA turnover in vivo. We have recently developed a cell-free system that is competent for both of these enzymatic activities. This work is a key first step in our proposed experimental plan.