Polyadenylation Machineries in Intestinal Parasites: Latest Advances in the Protozoan Parasite Entamoeba histolytica

In eukaryotic cells, nuclear cleavage and polyadenylation of mRNA precursors (pre-mRNA) generate functional protein encoding transcripts that can be exported to the cytoplasm and translated. Nevertheless, in protozoan parasites that cause intestinal infections in humans, the current knowledge on mRNA 3 -end formation is limited.We performed a genomic survey in Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum databases and predicted that polyadenylation machineries are generally well conserved in these pathogens.Notably, most parasites have the 25 kDa subunit of the heterotetrameric CFIm (CFIm25), the 77 kDa subunit of the heterohexameric CPSF (CPSF73) and the poly(A) polymerase (PAP), which are essential proteins for poly(A) site selection, RNA cleavage and poly(A) tail synthesis. However, several differences exist that may be useful to develop new methods to control these pathogens. Taking E. histolytica as a study model, we focused on the EhCFIm25 protein, because it is the unique subunit of CFIm in this pathogen, whereas active CFIm in humans is formed by two 25 kDa subunits interacting with two larger subunits. Human and parasite CFIm25 proteins only share 32% identity but they have a similar three-dimensional folding. Interestingly, trophozoites loose virulence and are induced to death when EhCFIm25 expression is silenced, which denotes the relevance of this protein for E. histolytica control. To assess this hypothesis, we obtained RNA aptamers that specifically recognize EhCFIm25 by using the SELEX (systematic evolution of ligands by exponential enrichment) procedure and showed that their ingestion by phagocytosis dramatically reduces trophozoites proliferation. Moreover, RNA-protein binding and molecular modeling assays allowed us to identify that the GUUG motif is the binding site of EhCFIm25, while it is the UGUA sequence for the human protein. All these observations led us to propose that aptamers targeting specific parasite proteins, alone or in combination with the conventional treatment, could represent a new tool for controlling the development of amoebiasis and other challenging parasitic diseases.