Identificação e caracterização de proteínas de ligação a RNA diferencialmente expressas em Trypanosoma cruzi

Abstract

Trypanosoma cruzi, the causative agent of Chagas disease, has three biochemically and morphologically distinct developmental stages that are programed to rapidly respond to all environmental changes the parasite faces during its life cycle. Unlike other eukaryotes, the T. cruzi genome contains protein-coding genes that are transcribed into polycistronic pre-mRNAs before they are processed into mature mRNAs through coupled trans-splicing and poly-adenylation reactions. Because of this, control of gene expression relies mainly on post-transcriptional mechanisms that must be mediated by RNA binding proteins (RBP) that control steady-state levels and/or translation rates of mRNAs. After searching for motifs present in eukaryotic RBPs, we identified in the T. cruzi CL Brener genome, 253 sequences encoding proteins containing RNA recognition motif (RRM), PABP, Alba, Pumillio and zinc finger motifs. Using RNA-seq data generated with mRNA present in epimastigotes, tissue culture trypomastigotes and amastigotes extracted at two time points during infection of human fibroblasts, we analyzed the expression of all T. cruzi RBPs throughout the life cycle of this parasite. Among the five genes up-regulated in CL Brener epimastigotes compared with amastigotes and trypomastigotes, we found the gene TcCLB.506739.99 which encodes a RBP containing a zinc finger motif. The importance of the protein encoded for this RBP was revealed by knockdown parasites which showed decrease in the end of logarithmic phase of growth. Null mutants also reveals high capacity of differentiation in metacyclic trypomastigotes compared with wild type epimastigotes. Global gene expression were analyzed by RNA-Seq using mRNA of null mutants and reveals 12 genes with differential expression, but no gene were up-regulated in null mutants compared with wild type parasites. The capacity of this RBP to bind a mRNA encoding for a protein associated with differentiation, found in RNA-Seq data, were confirmed by immunoprecipitation assays. The T. cruzi population is highly heterogeneous with strains presenting different biological, biochemical and molecular characteristics. One example of this is CL Brener and CL-14 cloned T. cruzi, that are virulent and avirulent clones respectively. To investigate the regulatory mechanisms controlling the distinct transcriptional programs that drive the development from intracellular amastigotes to the extracellular trypomastigote stage, we searching for RBPs that are differentially expressed throughout the infection of the host cell by CL Brener and CL-14 cloned T. cruzi. Among the seven transcripts differently expressed during the intracellular life cycle, the RBP encoded by the TcCLB.507611.300 gene, containing RRM motif, is the only RBP with differential expression between CL Brener and CL-14. This RBP has a 3-fold increased expression of in CL-14 trypomastigotes when compared to CL Brener, suggesting that this RBP may have has a regulatory role related to the for non-virulent phenotype of CL-14.