Transcriptomic analysis of N-terminal mutatedTrypanosoma cruziUBP1 knockdown underlines the importance of this RNA-binding protein in parasite development

Abstract

During its life cycle, the human pathogen Trypanosoma cruzi must quickly adapt to different environments, in which the variation in the gene expression of the regulatory U-rich RNA-binding protein 1 (TcUBP1) plays a crucial role. We have previously demonstrated that the overexpression of TcUBP1 in insect-dwelling epimastigotes orchestrates an RNA regulon to promote differentiation to infective forms. In an attempt to generate TcUBP1 knockout parasites by using CRISPR-Cas9 technology, in the present study, we obtained a variant transcript that encodes a protein with 95% overall identity and a modified N-terminal sequence. The expression of this mutant protein, named TcUBP1mut, was notably reduced compared to that of the endogenous form found in normal cells. TcUBP1mut-knockdown epimastigotes exhibited normal growth and differentiation into infective metacyclic trypomastigotes and were capable of infecting mammalian cells. We analyzed the RNA-Seq expression profiles of these parasites and identified 276 up- and 426 downregulated genes with respect to the wildtype control sample. RNA-Seq comparison across distinct developmental stages revealed that the transcriptomic profile of these TcUBP1mut-knockdown epimastigotes significantly differs not only from that of epimastigotes in the stationary phase but also from the gene expression landscape characteristic of infective forms. This is both contrary to and consistent with the results of our recent study involving TcUBP1-overexpressing cells. Together, our findings demonstrate that the genes exhibiting opposite changes under overexpression and knockdown conditions unveil key mRNA targets regulated by TcUBP1. These mostly encompass transcripts that encode for trypomastigote-specific surface glycoproteins and ribosomal proteins, supporting a role for TcUBP1 in determining the molecular characteristics of the infective stage.

RNA-seq data exploration after trypanosome RNA-binding protein UBP1 expression is altered by CRISPR-Cas9 gene editing and overexpression

“…In addition, the oppositely affected genes after UBP1 overexpression and knockdown were recently reported by Sabalette et al (see Ref. [4] ). Raw data is available through NCBI's Sequence Read Archive at http://www.ncbi.nlm.nih.gov/sra.cgi with accession numbers: PRJNA907231 and PRJNA949967.…”

“…Control wild-type UBP1, four-day tetracycline-induced UBP1 parasites [6] , and UBP1-CRISPR/Cas9 edited cells [4] were cultured in epimastigote form conditions at 28 ºC. Total RNA from three biological replicates was prepared from approximately 10 7 epimastigote cells of each sample (WT, UBP1-OE, and UBP1mut-KD) using the TRIzol reagent following the manufacturer's instructions (Invitrogen).…”

“…This data describes a transcriptomic experiment to investigate the differential gene expression profiles between the T. cruzi N-terminal mutated UBP1 knockdown [4] and full-length protein overexpression in epimastigote cells. The experimental design consists of three high-throughput sequencing samples with three biological replicates each of wild-type RNA-binding protein TcUBP1 (WT), UBP1-GFP tetracycline-induced epimastigotes for four days (UBP1-OE), and CRISPR/Cas edited epimastigotes (UBP1mut-KD).…”

“…To further investigate the regulatory role of this protein, we used the CRISPR-Cas9 technology to generate a population of parasites that did not express this protein. However, we obtained an N-terminal mutated protein that is significantly less expressed than the endogenous form found in normal cells [4] .…”