: Chagas' Disease is globally ranked behind only malaria and schistosomiasis as the most serious parasitic diseases. Other kinetoplastids, including Leishmania and other Trypanosoma also have a strong impact on health and economic development in tropical and subtropical regions of the world. Research over the past decades has demonstrated that these organisms diverged early from other eukaryotes and consequently exhibited biochemical features that differ significantly from those of higher eukaryotes. Differences mechanisms of gene expression are quite striking. Thus, protein-coding genes are transcribed into polycistronic RNA that are matured into mRNAs via the trans-splicing. Although trans-splicing resembles cis-splicing of higher eukaryotes, it differs in that two primary transcripts, the SL RNA and the pre-mRNA, participate in a bimolecular splicing reaction to generate the final mature mRNA. The mechanisms by which these two RNAs are juxtaposed to permit their cleavage and joining are only poorly understood. Several U RNAs (U2, U4, U5, U6, and more recently Ul) and SL RNA associated RNAs (SLA RNAs) participate. However, relatively few protein components of trans splicing are known. Elucidation of the protein components of trans-splicing will identify novel processes that are potential targets for therapeutic intervention in diseases caused by all kinetoplastid protozoa. We have developed an approach, based on the yeast three-hybrid system, to identify and clone genes expressing proteins that bind to the Sl RNA. Herein, we propose to exploit this system in parallel with genetic systems that we have established in T. cruzi to identify and characterize these protein factors.
Our specific aims are to: I. Identify and clone SL RNA binding proteins from T. cruzi using the yeast three-hybrid system. 2. Identify sequence domains and structures of the SL RNA that mediate these interactions. 3. Identify domains and structures of the SL RNA binding proteins that recognize and bind the SL RNA. 4. Identify the function(s) of these SL RNA binding factors. The long-term goals of this work are two-fold: 1) to characterize the trans-splicing reaction in the kinetoplastid protozoa; and 2) to identify features of the T. cruzi mRNA maturation apparatus that vary sufficiently from mammalian systems to permit development of new therapeutic strategies.
| Valente, Andre X C N; Roberts, Seth B; Buck, Gregory A et al. (2009) Functional organization of the yeast proteome by a yeast interactome map. Proc Natl Acad Sci U S A 106:1490-5 |
