Functional relevance of trans-spliceosomal and ribosomal trypanosome proteins as assessed using two-hybrid and RNAi systems

MARIANO J. LEVIN; V.M. PABLO; NATALIA BERCOVICH; MAXIMILIANO JURI AYUB; CATALINA ATORRASAGASTI; FLORENCE CARO; BENSON NYAMEGA; M. DANIEL; SHULAMIT MICHAELI

Tallinn, Estonia

Congreso; Meeting of International Research Scholars; 2004

Both the trypanosomes Trypanosoma brucei and T. cruzi have evolved specific variations of common eukaryotic mechanisms, such as trans-splicing and translation. Given that 1) trypanosome versions of ubiquitous proteins harbor significant differences with respect to their mammalian counterparts and that 2) protein-protein interactions are crucial for the integrity of multicomponent enzymatic machines, we reasoned that key parasite proteins involved in essential steps of spliceosomal and/or ribosomal assembly would be appropriate targets for a functional genomic generation of antiparasitic drugs. Initially, we identified, in trypanosome genomic data banks, T. cruzi and T. brucei orthologues of the components of the above-mentioned protein complexes using as query sequences those of human and/or yeast proteins. To date, more than 45 proteins linked to trans-splicing, polyadenylation, or RNA degradation have been identified for T. cruzi. More than 30 have been cloned into different vectors of the Gateway system. Similarly, more than 40 ribosomal proteins of T. cruzi have been identified and cloned. Already, certain protein-protein interactions have been mapped, and the construction of a partial protein-protein interaction map for these complexes is under way. The genes selected for T. cruzi have been used to find and clone the corresponding ones in T. brucei. The functional relevance of these proteins is being tested by analyzing the loss-of-function phenotypes generated using the inducible RNAi system developed for T. brucei. This approach is required because the RNA-mediated interference mechanism is not present in T. cruzi.