: Directed ribozyme activity will add a valuable tool to the arsenal of techniques for study of small RNAs, and has not yet been reported in the kinetoplastids. This research is aimed at understanding gene expression in the family Trypanosomatidae, which includes the parasitic protozoan responsible for leishmaniasis, African sleeping sickness, and Chagas Disease. The focus is on the genesis and function of the spliced leader RNA, a small RNA which contributes the 5'-end sequence to every nuclear messenger RNA via a trans-splicing reaction. Trans-splicing is not found in the human host or insect vector, and thus represents a possible therapeutic target. This proposal outlines two series of experiments aimed at exploring the use of hammerhead ribozymes while elucidating the maturation pathway of the spliced leader RNA in Leishmania tarentolae: 1) Ribozyme activity in cis (mono-molecular) will be used to specify the 3' end of the spliced leader RNA. Stable products will allow us to overcome known processing defects for specific mutations in the spliced leader RNA and to examine downstream processing events including 5' methylation and trans-splicing. 2) Ribozymes will be used in trans (hi-molecular) to assay for nucleolar trafficking by attaching the ribozyme to a nucleolar U3 snoRNA. The nucleolar ribozyme will be directed against the spliced leader RNA and the spliced leader associated 1 RNA. Efficient ablation of RNA targets has great potential for elucidating the role of conserved sequences, structures and subcellular Iocalization. Once the parameters of the technique are established increasingly ambitious experiments can be designed including targeting of the spliced SL or discrete mRNAs.
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