Stress granule formation in Entamoeba histolytica: cross‐talk between EhMLBP,EhRLE3 reverse transcriptase and polyubiquitinated proteins

The Entamoeba histolytica‐methylated LINE‐binding protein (EhMLBP) binds to methylated repetitive DNA and is a positive regulator of a reverse transcriptase of a long interspersed nucleotide element (LINE). This protein protects trophozoites against heat shock by reducing protein aggregation. The presence of EhMLBP and polyubiquitinated proteins in heat shock‐induced protein aggregates raised the question whether these proteins interact. This assumption was confirmed by co‐immunoprecipitation experiments: ubiquitinated proteins were detected in the perinuclear region of non‐stressed E. histolytica trophozoites, whereas ubiquitinated proteins were detected in the perinuclear region and colocalized with EhMLBP in cytoplasmic granules in heat‐shocked trophozoites. We also observed that overexpression of the reverse transcriptase of EhRLE3 induced the upregulation of EhMLBP expression and the formation of these EhMLBP‐containing granules. Since (i) these EhMLBP‐containing granules in the cytoplasm of heat‐shocked E. histolytica trophozoites also contain polyubiquitinated proteins and poly(A)⁺ mRNA and (ii) their formation is promoted by sodium arsenate, puromycin, and pateamine A and is inhibited by cycloheximide, we propose that these cytoplasmic EhMLBP‐containing granules are stress granules. Our data also suggest that the formation of these granules is dependent upon EhMLBP and LINE.