Abstract
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 Entamoeba histolytica and found evidence of the existence of P-bodies. Two mRNA decay factors, namely the EhXRN2 exoribonuclease and the EhDCP2 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 EhCAF1 deadenylase and the EhAGO2-2 protein involved in RNA interference. Biochemical analysis revealed that EhCAF1 co-immunoprecipitated with EhXRN2 but not with EhDCP2 or EhAGO2-2, thus linking deadenylation to 5′-to-3′ mRNA decay. The number of EhCAF1-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) EhCAF1 colocalized with poly(A)+ RNA and (ii) during silencing of the Ehpc4 gene by RNA interference, EhAGO2-2 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 E. histolytica. Our findings should open up opportunities for deciphering the mechanisms of mRNA degradation and RNA-based gene silencing in this deep-branching eukaryote.
Highlights
The control of messenger RNA degradation plays a key role in the posttranscriptional regulation of gene expression [1]
Results messenger RNA (mRNA) degradation machineries in Entamoeba histolytica In order to identify homologous genes potentially involved in mRNA decay in E. histolytica, we screened the parasite genome at AmoebaDB by using the amino acid sequences of yeast and human proteins involved in mRNA turnover as probes
Discussion mRNA decay pathways and machineries have been extensively studied in S. cerevisiae and H. sapiens
Summary
The control of messenger RNA (mRNA) degradation plays a key role in the posttranscriptional regulation of gene expression [1]. There is growing body of evidence to show that mRNA degradation occurs in specialized cytoplasmic foci variously referred to as mRNP granules, mRNA-decay foci, GW182-bodies and mRNA processing bodies (P-bodies) These structures are enriched in RNA substrates and mRNA turnover proteins. Even though P-body components clearly play crucial roles in mRNA degradation, there is evidence to show that NMD and silencing mediated by miRNAs and siRNAs occur in cells lacking detectable P-bodies. This suggests that Pbody formation is a consequence, rather than a cause, of mRNA degradation activity [10,13]. Translational repression by miRNAs does not require P-body structures; the localization of miRNAs and RNA-induced silencing complex (RISC) proteins within P-bodies appears to be a consequence of translational repression [14]
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