Knockdown of the RNA-binding protein TcUBP1 in epimastigote cells reveals its essential role in trypanosome life cycle progression

SABALETTE, KB; CAMPO, VA; DE GAUDENZI, JG

Mendoza

Congreso; XI Congreso SAP 2022; 2022

Sociedad Argentina de Protozoología

Throughout its life cycle, Trypanosoma cruzi must adapt to dissimilar environments in a quickly and dynamically manner. Here, gene expression variation of the regulatory RNA-binding protein TcUBP1 plays a crucial role. We have previously shown that the over-expression of TcUBP1 in epimastigotes triggers an expression profile that resembles infective forms and increases twice the infectivity of cell-derived trypomastigotes. At this time, we intended to obtain knockout parasites for TcUBP1 by using CRISPR/Cas9 technology. In an attempt to do so, we were able to generate an endogenous genome editing of the coding sequence by removing the first 6 residues corresponding to the N-terminal region of the protein. Interestingly, while the expression of ΔN-TcUBP1 was reduced by 70% compared to the wild type form, immunofluorescence analysis with specific antibodies revealed its typical cytoplasmic subcellular localization pattern. Although these TcUBP1-knockdown parasites were able to differentiate from epimastigotes to metacyclic trypomastigotes and infect mammalian cells normally, no cell-derived trypomastigotes were obtained from in vitro infection assays. This result, in concordance with previous studies, suggest a specific role of TcUBP1 in the differentiation processes from replicative to infective forms. In order to exhaustively elucidate the specific contribution of TcUBP1 on the T. cruzi transcriptome, total RNA samples from wild type, TcUBP1-overexpressing or -knockdown epimastigotes were purified and subjected to high-throughput RNA-seq analysis. Comparison of the modulated mRNAs (up/downregulated) that we expect to find in this study, will expand our current knowledge of TcUBP1, allowing us to identify the genes engaged among the different expression profiles.