Preliminary map of the trans-spliceosome/polyadenylation complex in the kinetoplatid Trypanosoma cruzi.

BERCOVICH, NATALIA; ATORRASAGASTI, CATALINA; VAZQUEZ, MARTIN; LEVIN, MARIANO J

Iguazú, Misiones, Argentina

Congreso; RNA processing.; 2003

Protein-coding genes transcribed by RNA polymerase II in trypanosomes are not subjected to regulation at the level of trasncription initation. Therefore, the main regulatory steps are at the post-transcriptional level. Long polycistronic transcription units are processed by trans-splicing and polyadenylation, two physically coupled and dependent mechanisms, to render functional monocistronic mRNAs. Using data mining tools we screened the Tcruzi whole genome shotgun database to find putative trans-splicing and polyadenylation factors. The complete dataset of the human spliceosome was used as a probe. We identified and cloned 10 putative splicing factors and 8 polyadenylation factors so far. Our initial analysis is focused on the E complex (U2AF heterodimer and SF1/BBP proteins) where we found significant and surprising differences with an additional CCHC type zinc finger and most important it lacks the conserved Trp residue at position 134. This residue, which is absolutely necessary for the reciprocal “tongue in grove” heterodimerization with U2AF65, is changed to Lys in the Tcruzi protein. In fact, TcU2AF35 did not interact with human U2AF65 in yeast two hybrid assays. Based on this evidence, we expected to find a modified version of the U2AF65 orthologue to accommodate the change in its partner. Indeed, we were unable to find a U2AF65 protein in the Tcruzi genome neither using blast searches nor HMM profile searches. Nevertheless, we selected a putative and largely modified protein os 842 aminoacids with reminiscence of the human U2AF65 architectural domains. Also the candidate for the Tcruzi SF1/BBP factor is a singular protein with a modified KH domain and it lacks the additional zinc finger motifs that are characteristics of the human and yeast proteins. In overall, Tcruzi splicing factors present several differences in comparison with other eukaryotes.