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 n amed Tc52 sharing structural and functional properties with the thioredoxin and glutaredoxin protein family involved in thiol-disulphide redox reactio ns. Furthermore, we reported that Tc52 also plays a role in T. cruzi-associ ated immunosuppression observed during Chagas' disease. Moreover, Tc52 gene targeting deletion strategy allowed us to demonstrate that monoallelic dis ruption 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 addition al open reading frames (ORF-A and C). The ORFs are likely to have protein c oding function by a number of criteria, including reverse transcriptase pol ymerase chain reaction (RT-PCR), Western blot and immunofluorescence analys es. 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 i nteract with RNA, interspersed with a high density of glycine with regular spacing of tryptophan (WX9-10) in which X is often a glycine. Moreover, the C-terminal part of the ORF-C (aa 253-289) contains a motif that is strikin gly similar (7-35% identity, 14-46% similarity over 28 aa) to a short seque nce (RNP1) comprising the consensus sequence RNA binding domain (CS-RBD) fo und in a number of proteins that interact with RNA. The aa sequence from th e ORF-C localized downstream of the Tc52 gene showed significant homology t o human adenosine deaminase acting on RNA (hADAT1) that specifically deamin ates adenosine 37 to inosine in eukaryotic tRNA(Ala) and to its homologue y east protein (Tad1p) (22-25% identity and an additional 38-40% similarity o ver 177 aa). Moreover, highly similar motifs of the deaminase domain are pr esent in the T. cruzi ORF-C. Furthermore, the 5' flanking regions of the ge nes contained repeat TATA and CAAT nucleotide sequences which resemble the motifs found upstream of the transcription initiation sites in eukaryotic p romoters. Therefore, the characterization of novel T, cruzi genes encoding proteins which show similarity to components of RNA processing reactions pr ovides new tools to investigate the gene expression regulation in these par asitic organisms. Moreover, our recent findings on the Tc52 encoding gene u nderline 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. (C) 2000 Elsevier Science B.V. All rig hts reserved.