Our research is aimed at understanding gene expression in the family Trypanosomatidae, which includes the parasitic protozoa responsible for leishmaniasis, African Sleeping Sickness, and Chagas Disease. The focus is on the genesis and function of the spliced leader (SL) RNA, a small RNA that contributes the 5'-end sequence to every nuclear messenger RNA by a trans-splicing reaction and is conserved extraordinarily at the primary sequence level throughout the Order. Trans-splicing is necessary for the conversion of polycistronic pre-messenger RNA, a likely consequence of the unusual genome organization found in kinetoplastids, into monocistronic mRNA, and is not found in the human host or insect vector, thus representing an ideal therapeutic target. This proposal outlines experiments that detail the maturation pathway of the SL RNA common to Leishmania tarentolae and Trypanosoma brucei at the level of individual proteins and interacting complexes, and probe the role of the SL in translation. 1) We will explore the role of the cap 4 2'-O-ribose methyltransferases in the bloodstream form of T. brucei, and validate their function in procyclic T. brucei using knockout strains. We will investigate the cap-binding properties of TbMTr1, and test models for the role of TbMTAP and TbMTr1 in trafficking and interacting with the pseudouridine synthase complex in the nucleus. 2) To further the characterization of complexes involved in SL RNA biogenesis and function. The trinity of PTP tagging, multidimensional protein identification technology (MudPIT), and the T. brucei genome database enables us to approach complex purification. The kinetoplastid-specific TbMTr1 complex protein TbMTAP is implicated in regulatory and structural interactions. Using the SmD3 protein, the SL RNA RNP complex and associated splicing machinery, both catalytic and core, has been captured. Of the 25 proteins identified, four novel, kinetoplastid-specific proteins will be validated for association and function along with two additional splicing homologs. These goals address discrete steps in the maturation of the SL RNA that determine its subsequent function. Specific inhibition of the trans-splicing pathway is lethal to the parasite and thus a prime target for clinical intervention.
The Order Kinetoplastida contains several pathogens of medical importance including the causative agents of human leishmaniasis, African trypanosomiasis and Chagas disease. These organisms employ unusual mechanisms of gene expression involving polycistronic transcription of protein-coding genes and trans-splicing of a universal 39-nucleotide spliced leader sequence to all nuclear mRNAs. The goal of this study is to continue the identification of spliced leader specific processing components that have the potential to be specific targets for anti-parasite chemotherapy.
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