Transcriptomic analysis of N-terminal mutated Trypanosoma cruzi UBP1 knockdown underlines the importance of this RNA-binding protein in parasite development.

Autor: Sabalette, Karina B., Campo, Vanina A., Sotelo-Silveira, José R., Smircich, Pablo, De Gaudenzi, Javier G.
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Zdroj: PLoS Neglected Tropical Diseases; 5/17/2024, Vol. 18 Issue 5, p1-29, 29p
Abstrakt: Background: 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. Methods: 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. Results: 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. Conclusion: 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. Author summary: In the intriguing world of Trypanosoma cruzi, the parasite causing Chagas disease in humans, a key factor known as TcUBP1 helps the pathogen navigate its different life stages. This microorganism undergoes a complex life cycle in which it alternates between a triatomine bug ("kissing bug") and a mammalian host. Our research demonstrated that increasing TcUBP1 levels in insect-dwelling forms prompts a gene expression pattern akin to infective forms, enhancing their ability to infect. Attempts to turn off TcUBP1 through gene editing resulted in TcUBP1mut, a variant with reduced expression closely resembling the native protein. TcUBP1mut cells exhibited distinct gene expression profiles compared to infective forms, according to the results of RNA-Seq analysis. This study corroborates our earlier findings on TcUBP1 overexpression, pinpointing genes with opposing expression patterns in both scenarios. These affected genes govern the production of infective-specific glycoproteins and ribosomal proteins, offering valuable insights into TcUBP1's regulatory role and unraveling mRNA targets crucial for parasite differentiation and infectivity. [ABSTRACT FROM AUTHOR]
Databáze: Complementary Index
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