Gene expression in trypanosomes is controlled mainly by post-transcriptional processes. This study was designed to analyse HX1, one of the TcP2beta upstream intergenic regions. It is an efficient pre-mRNA processing region that has been widely and successfully used in Trypanosoma cruzi transfection vectors. Herein we compared its performance with other regions within the same locus, and we identified the sequence elements responsible for the HX1 efficiency in trans-splicing and protein synthesis. Our mutational analysis showed the flexibility of the branch point site selection for HX1 trans-splicing process. We demonstrated also that its 12 nt 5'UTR sequence contributes to both trans-splicing and translation efficiency. The natural insertion of the repetitive element short interspersed repetitive element (SIRE) in one of the HX1 polypyrimidine tracts decreases the translated protein level by 40%. In this report, we demonstrated that this reduction is a consequence of a decrease of five-fold in the level of processed mRNA balanced by an increased efficiency of translation due to the inclusion of a 38 nt SIRE specific sequence in the 5'UTR of the mRNA.