Identification and molecular characterization of two novel Trypanosoma cruzi genes encoding polypeptides sharing sequence motifs found in proteins involved in RNA editing reactions.

We have previously identified a Trypanosoma cruzi cDNA encoding a protein named Tc52 sharing structural and functional properties with the thioredoxin and glutaredoxin protein family involved in thiol-disulphide redox reactions. Furthermore, we reported that Tc52 also plays a role in T. cruzi-associated immunosuppression observed during Chagas' disease. Moreover, Tc52 gene targeting deletion strategy allowed us to demonstrate that monoallelic disruption of Tc52 resulted in the alteration of the metacyclogenesis process and the production of less virulent parasites. Sequence analysis of a 7358 bp genomic fragment containing the Tc52 encoding gene revealed two additional open reading frames (ORF-A and C). The ORFs are likely to have protein coding function by a number of criteria, including reverse transcriptase polymerase chain reaction (RT-PCR), Western blot and immunofluorescence analyses. The deduced amino-acid (aa) sequence of the ORF-A localized upstream of the Tc52 gene revealed that it contains within its N-terminus (aa 1 to 170) four RGG boxes known to act as RNA binding motifs in some proteins that interact with RNA, interspersed with a high density of glycine with regular spacing of tryptophan (WX(9-10)) in which X is often a glycine. Moreover, the C-terminal part of the ORF-C (aa 253-289) contains a motif that is strikingly similar (7-35% identity, 14-46% similarity over 28aa) to a short sequence (RNP1) comprising the consensus sequence RNA binding domain (CS-RBD) found in a number of proteins that interact with RNA. The aa sequence from the ORF-C localized downstream of the Tc52 gene showed significant homology to human adenosine deaminase acting on RNA (hADAT1) that specifically deaminates adenosine 37 to inosine in eukaryotic tRNA(Ala) and to its homologue yeast protein (Tad1p) (22-25% identity and an additional 38-40% similarity over 177aa). Moreover, highly similar motifs of the deaminase domain are present in the T. cruzi ORF-C. Furthermore, the 5' flanking regions of the genes contained repeat TATA and CAAT nucleotide sequences which resemble the motifs found upstream of the transcription initiation sites in eukaryotic promoters. Therefore, the characterization of novel T. cruzi genes encoding proteins which show similarity to components of RNA processing reactions provides new tools to investigate the gene expression regulation in these parasitic organisms. Moreover, our recent findings on the Tc52 encoding gene underline the interest of genetic manipulation of T. cruzi, not only making it possible to use more closely an in vitro approach to find out how genes function, but also to obtain 'attenuated' strains that could be used in the development of vaccinal strategies.