Targeting the polyadenylation factor EhCFIm25 with RNA aptamers controls survival in Entamoeba histolytica

Abstract

Messenger RNA 3′-end polyadenylation is an important regulator of gene expression in eukaryotic cells. In our search for new ways of treating parasitic infectious diseases, we looked at whether or not alterations in polyadenylation might control the survival of Entamoeba histolytica (the agent of amoebiasis in humans). We used molecular biology and computational tools to characterize the mRNA cleavage factor EhCFIm25, which is essential for polyadenylation in E. histolytica. By using a strategy based on the systematic evolution of ligands by exponential enrichment, we identified single-stranded RNA aptamers that target EhCFIm25. The results of RNA-protein binding assays showed that EhCFIm25 binds to the GUUG motif in vitro, which differs from the UGUA motif bound by the homologous human protein. Accordingly, docking experiments and molecular dynamic simulations confirmed that interaction with GUUG stabilizes EhCFIm25. Incubating E. histolytica trophozoites with selected aptamers inhibited parasite proliferation and rapidly led to cell death. Overall, our data indicate that targeting EhCFIm25 is an effective way of limiting the growth of E. histolytica in vitro. The present study is the first to have highlighted the potential value of RNA aptamers for controlling this human pathogen.

mRNA Polyadenylation Machineries in Intestinal Protozoan Parasites

“…Interestingly, the absence of certain conserved residues in the Nudix domain does not significantly affect the folding neither the RNA binding of CFIm25 in E. histolytica (Ospina‐Villa et al. ,b; Pezet‐Valdez et al. ), highlighting the relevance of the domain folding.…”

“…; Ospina‐Villa et al. ,b; Zamorano et al. ) and the conservation of the polyadenylation process through the evolutionary scale, we proposed a refined model in which the EhCPSF complex binds the consensus poly(A) signal UA(A/U)UU through the EhWDR33 polypeptide, while EhCstF complex recognizes the U‐rich motif downstream the poly(A) site, tethering other factors to RNA.…”

“…Moreover, we recently demonstrated that the 25 kDa subunit of cleavage factor Im (EhCFIm25) is necessary for trophozoites survival, indicating that polyadenylation factors are valuable targets for parasite control (Ospina‐Villa et al. , ,b) as it has been reported for other protozoan parasites (Hendriks et al. ; Palencia et al.…”

Life and Death of mRNA Molecules in Entamoeba histolytica

“…Despite the absence of a classical RBD, EhCFIm25 is able to bind the 3′ UTR of E. histolytica transcripts and this interaction involves the participation of the conserved Leu135 and Tyr236 residues (Ospina-Villa et al, 2015 ). EhCFIm25 recognizes the GUUG motif in mRNA 3′end, while the human protein binds the UGUA sequence; consequently, aptamers containing this motif specifically identify the parasite protein, suggesting that they could be used as molecular tools for diagnosis or therapeutic purposes (Ospina-Villa et al, 2018 ). Interestingly, EhCFIm25 interacts with EhPAP (Pezet-Valdez et al, 2013 ), which indicates that it may be introduced into the processing complex in the early steps of the cleavage/polyadenylation reaction.…”

“…Interestingly, EhCFIm25 interacts with EhPAP (Pezet-Valdez et al, 2013 ), which indicates that it may be introduced into the processing complex in the early steps of the cleavage/polyadenylation reaction. EhCFIm25 controls the selection of the distal poly(A) site during mRNA 3′ end formation; consequently, its silencing by dsRNA or its blocking by aptamers alter parasite proliferation and virulence, demonstrating for the first time the relevance of polyadenylation factors as biochemical targets in E. histolytica (Ospina-Villa et al, 2017 , 2018 ). Other works have also recently reported that targeting polyadenylation factors represents a valuable strategy for the control of the protozoan parasites Trypanosoma brucei (CPSF-30), Toxoplama gondii , and Plasmodium falciparum (CPSF-73) (Hendriks et al, 2003 ; Sidik et al, 2016 ; Palencia et al, 2017 ; Sonoiki et al, 2017 ).…”