CINVESTAV Ciudad de Mexico, Distrito Federal, Mexico
Gretter Gonzalez Blanco (CINVESTAV)| Jesus Valdes Flores (CINVESTAV)| Maria Cristina Vélez del Valle (CINVESTAV)| Guillermina García Rivera (CINVESTAV)| Luis Ortíz Hernández (UAM)| Maria Esther Orozco Orozco (CINVESTAV)| Patricia Talmás Rohana (CINVESTAV)| Tomoyoshi Nozaki (University of Tokyo)
Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscesses. The parasite infects approximately 1% of the human population, resulting in circa 100,000 deaths annually. The determining factors resulting in amebiasis are not well understood. The proteome of in vivo assembled Early splicing complexes identified a huge set of E. histolytica proteins involved in RNA processing, among them U2AF84, which is the ortholog of the human U2AF65. The Carboxyl-terminus domain of this factor is 240 amino acids longer than U2AF65, of which the last 136 residues correspond to the KH-QUA2 acidic domain, involved in the regulation of gene expression through Intron Retention. Such domains are able to dimerize members of the STAR family of proteins, such as Splicing Factor 1 (SF1), a factor that binds to the intronic Branch point Sequence (BS) in the Early splicing complex though its own KH-QUA2. Recent transcriptomic data showed upregulated expression of U2AF84 in amoebas recovered from liver abscesses. As a hypothesis, we propose that the interaction between the U2AF84- and SF1-KH-QUA2 domains reduces splicing via intronic retention and the virulence of E. histolytica. Our objective was analyze the impact of the interaction between the U2AF84- and SF1-KH-QUA2 domains on splicing and on pathogen virulence. KH-QUA2 deleted U2AF84 amoeba transformants displayed increased splicing of target transcripts in vivo. EMSA, UV and formaldehyde crosslinking, and overlay assays showed that recombinant SF1- and U2AF84-KH-QUA2 domains interact with each other, even in the absence of a BS radioactive probe. Also, the U2AF84-KH-QUA2 domain blocks partial replacement of the SF1-KH-QUA2 by the U2 snRNA from the BS, suggesting that splicing inhibition occurs by blockage of splicing complex E to A transition. All amoeba transformants were tested for their virulent phenotypes. Changes in the U2AF84-KH-QUA2 domain resulted in more aggressive amoebas, suggesting that an increase in splicing (therefore decreased intron retention) of target transcripts is required for appropriate amebiasis outcome. To test this, we constructed a chimeric U2AF84 protein combining the amino terminus of E. histolytica U2AF84 with the longer KH-QUA2 of the non-virulent human commensal Entamoeba dispar. The transfected chimeras were less aggressive than the U2AF84-KH-QUA2 deleted or mutated transformants, confirming, at least in part, our hypothesis. We conclude that a more extensive transcriptome resulting from intron retention, and an additional layer of gene expression regulation are necessary for appropriate amebiasis establishment. Our work links for the first-time splicing downregulation and pathogen virulence.