mRNA Decay Proteins Are Targeted to poly(A)⁺ RNA and dsRNA-Containing Cytoplasmic Foci That Resemble P-Bodies in <em>Entamoeba histolytica</em>

<div><p>In higher eukaryotes, mRNA degradation and RNA-based gene silencing occur in cytoplasmic foci referred to as processing bodies (P-bodies). In protozoan parasites, the presence of P-bodies and their putative role in mRNA decay have yet to be comprehensively addressed. Identification of P-bodies might provide information on how mRNA degradation machineries evolved in lower eukaryotes. Here, we used immunofluorescence and confocal microscopy assays to investigate the cellular localization of mRNA degradation proteins in the human intestinal parasite <em>Entamoeba histolytica</em> and found evidence of the existence of P-bodies. Two mRNA decay factors, namely the <em>Eh</em>XRN2 exoribonuclease and the <em>Eh</em>DCP2 decapping enzyme, were localized in cytoplasmic foci in a pattern resembling P-body organization. Given that amoebic foci appear to be smaller and less rounded than those described in higher eukaryotes, we have named them “P-body-like structures”. These foci contain additional mRNA degradation factors, including the <em>Eh</em>CAF1 deadenylase and the <em>Eh</em>AGO2-2 protein involved in RNA interference. Biochemical analysis revealed that <em>Eh</em>CAF1 co-immunoprecipitated with <em>Eh</em>XRN2 but not with <em>Eh</em>DCP2 or <em>Eh</em>AGO2-2, thus linking deadenylation to 5′-to-3′ mRNA decay. The number of <em>Eh</em>CAF1-containing foci significantly decreased after inhibition of transcription and translation with actinomycin D and cycloheximide, respectively. Furthermore, results of RNA-FISH assays showed that (i) <em>Eh</em>CAF1 colocalized with poly(A)⁺ RNA and (ii) during silencing of the <em>Ehpc4</em> gene by RNA interference, <em>Eh</em>AGO2-2 colocalized with small interfering RNAs in cytoplasmic foci. Our observation of decapping, deadenylation and RNA interference proteins within P-body-like foci suggests that these structures have been conserved after originating in the early evolution of eukaryotic lineages. To the best of our knowledge, this is the first study to report on the localization of mRNA decay proteins within P-body-like structures in <em>E. histolytica</em>. Our findings should open up opportunities for deciphering the mechanisms of mRNA degradation and RNA-based gene silencing in this deep-branching eukaryote.</p>