Cloning and functional analysis of trans-splicing factors in Trypanosoma brucei

Abstract

Trypanosomes are parasitic protozoa that cause trypanosomosis. The disease is a threat to human population and a major impediment to livestock production and economic development in many countries in sub-Sahara Afiica, where it is endemic. Presently, it is controlled by reduction of tsetse fly vector population, chemotherapy and chemoprophylaxis. However, none of these approaches is completely eifective and thus the need for development of new approaches. The splicing machinery in trypanosomes presents a potential target for anti-trypanosome drug development. The development of new dmgs is needed because of increasing incidents of resistance to available drugs. In trypanosomes, the mature messenger RNA is derived from independent pre-mRNA molecules in a process called trans-splicing. This is a variation from the more common eis-splicing which occurs in the mammalian hosts of trypanosomes, where the mature RNA is derived from one pre- mRNA molecule. The process is important in regulation of gene expression in trypanosomes that is predominantly post-transcriptional. ln this study, thirteen homologs of Trypanosoma brucei genes for trans-splicing and polyadenylation were identified in silico using Trypanosoma cruzi, yeast and/or human splicing and polyadenylation factors to query GeneDB, the repository of genome data for T23/panosoma brucei, Trypanosoma cruzi and Leishmania major. Degenerate PCR approach was used to clone the Factors, which were subsequently sequenced. The amino acid sequences generated were used to query public protein databases and were also compared to homologous sequences from II cruzi, L. major and Homo sapiens. Conserved RNA binding proteins domains and domains of proteins involved in multi-protein complex assemblies were identified. The kinetoplastid sequences were similar to each other, but were individually significantly dififerent from human homologs. Sequence specific gene silencing of three factors (P14, CPSF 30 and Ul-70k) using RNA interference (RN/Xi) technique was lethal suggesting the importance of the three factors in viability. Protein depletion was detected in the silencing of the P14 gene. Significant variations of the kinetoplastid sequences from human and importance of the factors in parasite viability suggest that some components of the trypanosome spliceosome are targets for the design of novel dmgs.