Caracterização da proteína RBP42 do parasito Trypanosoma cruzi em condições normais e de estresse celular induzido pela radiação gama

Abstract

RNA-binding proteins (RBPs) are essential factors for gene expression regulation in trypanosomatids, which occurs mostly at the post-transcriptional level. In this work, we studied the RNA-binding protein 42 from the T. cruzi CL Brener reference strain (TcRBP42) by characterizing it under both normal and stress conditions induced by 1000 Gy of gamma radiation. This species can withstand high doses of ionizing radiation by employing largely unknown mechanisms that may involve RBPs. In this sense, a transcriptome analysis of irradiated epimastigotes detected increased mRNA levels for TcRBP42 in response to gamma radiation. We started the functional characterization of this protein by analyzing its structure computationally. In the most recent CL Brener genome, this protein is coded by a single-copy gene with two alleles. Furthermore, this protein contains two conserved domains, NTF2-like and RRM, interconnected by a long and unstructured region. This modular organization is also shared with other RBP42 orthologs. For the in vitro experiments, parasites overexpressing or lacking TcRBP42 gene expression were generated. While the overexpression was well tolerated by T. cruzi, this gene knockout caused the appearance of round-shaped epimastigotes with a shortened flagellum and reduced mobility. Mild proliferation defects were only observed in late culture growth. The knockout parasite's viability contrasts with the lethal phenotype observed in Trypanosoma brucei. Concerning protein localization, it was determined by detecting a TcRBP42 His-tagged version inserted into epimastigotes overexpressing this gene. At normal conditions, TcRBP42 was found distributed throughout the whole parasite's cytoplasm. However, exposure to gamma radiation produced a partial relocalization of this protein to the nucleus. It suggests the importance of TcRBP42 for a stress response based on the translation control exerted by transcript compartmentalization. Regarding epimastigotes proliferation, gamma irradiation did not produce significant differences in the growth of the assayed parasite lines (Wild type, GFP, Overexpressor and Knockout). Real-time PCR experiments were carried out to evaluate the expression of energy metabolism transcripts bound by the T. brucei RBP42. Our results suggest TcRBP42 contributes to target transcript stabilization only under normal conditions. Studying gene expression after gamma radiation exposure also exposed for the first time a possible connection between the proline metabolism and the radioresistance of T. cruzi. This amino acid has also been linked to the parasite's resistance to nutritional and oxidative stress. In summary, this work was the first to generate and characterize TcRBP42 knockout epimastigotes, thus expanding the literature for the T. cruzi RBPs.