Identificação e caracterização da argonauta TcPIWI e de pequenos RNAs em Trypanosoma cruzi

Abstract

Trypanosoma cruzi is an intracellular parasite, etiological agent of Chagas disease, with a complex life cycle between two hosts and displays different life stages. In order to adapt to the different challenges that undergoes during its life cycle, T. cruzi needs a fast and efficient molecular response. Since T. cruzi has a polycistronic transcription, the regulation of gene expression occurs mainly at the post-transcriptional level. In many eukaryotes one of the most important pathways toregulate gene expression at post-transcriptional level is the RNA interference pathway (RNAi). RNAi pathway utilizes small non-coding RNAs (ncRNAs) associated with Argonaute proteins to regulate gene expression. The ncRNA within the Argonaute complex serves as guide to find complementary sequences within RNA targets. In trypanosomatids two Argonaute proteins were described, AGO and PIWI. The AGO protein is associated with the gene silencing pathway induced by doublestranded RNA, knows as classic RNAi pathway. But the AGO protein and the classic RNAi pathway are not present in T. cruzi. Unlike this, the PIWI protein is conserved in all trypanosomatids. Despite of the presence of the PIWI protein, named TcPIWI, and ncRNAs in T. cruzi, the functions of these components are not well understood. The existence of the TcPIWI indicates the presence of at least one functional RNAi pathway. The goal of this work was the study of this potential RNAi pathway in T. cruzi through the characterization of the TcPIWI and ncRNAs. We observed adecrease in the expression of TcPIWI during macrophages infection and TcPIWI knockout parasites show higher infection rate in macrophages. On the other hand, we observed that TcPIWI has an increase in its expression when the parasite is exposed to gamma radiation and TcPIWI knockout parasites have a lower capacity to recover after the exposure. We observed that the protein TcPIWI is localized diffused in the cytoplasm like others Argonauts. In order to identify potential population of ncRNAs that could be associated with the TcPIWI, we sequenced T. cruzi small RNA libraries in different conditions. We observed that the most consistent ncRNAs population is derived from tRNAs and presents average size of 33 nt. These ncRNAs are derived mainly of the 5 region of the tRNA from glutamic acid and valine. These small RNAs derived from tRNA do not seem associated with the Argonaute protein because their abundance dont change when TcPIWI has different expression or isdeleted. Our results suggest that the TcPIWI is important to regulate the response of trypanosomatids to different conditions but the mechanisms of action of this new RNAi pathway stills unclear. It seems that the ncRNAs populations associated with the Argonaute protein were not identified in several studies including ours. These results suggest that TcPIWI would be associated with a new population of ncRNAs orit works without any association with ncRNAs. In both cases, this would be a new mechanism of action not described in any RNAi pathway yet.