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.

Figure 1

Identification of aptamers against EhCFIm25. (A) Schematic representation of ssDNA oligonucleotides used to generate the ssRNA library for the SELEX protocol. (B) The predicted secondary structures of the C4 and C5 aptamers. (C–G) The RNA-electrophoretic mobility shift assay (REMSA). Proteins were incubated with a biotin-labelled RNA probe, and the RNA-protein complexes were resolved via PAGE and chemiluminescence assays. (C) A REMSA of the R7 aptamer population with wild-type EhCFIm25 and mutant EhCFIm25*L135T proteins. Proteinase K (1 µg), unspecific competitor (tRNA) and RNA molecules from the first round of SELEX (R0) were used as controls. The image results from the grouping of gels cropped from different parts of the same gel and a different gel. (D) A REMSA of C4 and C5 aptamers with wild-type EhCFIm25 and mutant EhCFIm25*L135T proteins. The image results from the grouping of gels cropped from two different gels. (E) A REMSA of C4 and C5 aptamers with E. histolytica protein extracts. Anti-EhCFIm25 antibodies were used as controls. (F and G) A REMSA of C4 and C5 aptamers with protein extracts from HeLa cells (F) and Trypanosoma cruzi parasites (G). Eh: E. histolytica; Tc: T. cruzi; single arrowhead: the RNA-protein complex; double arrowhead: the RNA-protein-antibody complex; asterisk: the free probe.

Figure 2

EhCFIm25 binds to the GUUG motif in vitro. (A) Sequence of the Ehthio- and Ehrib-3′UTR probes used in REMSAs. Box: the GUUG motif; circle: the stop codon: bold and underlined nucleotides: the cleavage and polyadenylation site. (B–D) The REMSA results. Proteins were incubated with a biotin-labelled RNA probe, and RNA-protein complexes were resolved via PAGE and chemiluminescence assays. (B) A REMSA of Ehthio- and Ehrib-3′UTR with E. histolytica extracts and the recombinant EhCFIm25 protein. Anti-EhCFIm25 antibodies were used as a control. (C) A REMSA of Ehthio- and Ehrib-3′UTR with the recombinant EhCFIm25 protein. The GUUG-containing oligonucleotide (GUUG-probe) was used as a competitor. (D) A REMSA of the GUUG-probe with recombinant EhCFIm25. The CAAC-containing oligonucleotide (CAAC-probe) was used as control. Eh: E. histolytica; single arrowhead: the RNA-protein complex; double arrowhead: the RNA-protein-antibody complex; asterisk: the free probe. (E) Sequence of the GUUG- and CAAC-probes. Box: the GUUG motif; the T7 promoter sequence is underlined.

Figure 3

Interactions between EhCFIm25 and the GUUG motif. (A) Graphic representation of the average structure of EhCFIm25 complexed to the GUUG motif. (B) Contact map between the aa in EhCFIm25 and the nucleotides (U2, U3 and G4) in the GUUG motif. (C) A close-up view of the interaction zone between selected aa in EhCFIm25 (in red) and U2, U3 and G4 in the GUUG fragment (in black).

Figure 4

Molecular dynamics simulations of free and GUUG-binding EhCFIm25 proteins. (A) Change over time in root mean square deviation of Cα (RMSD). Data on the mutant EhCFIm25*L135T protein were included as control. (B) Average structure of the free (left) and GUUG-binding (right) proteins. Black arrow: unstable loop; white arrow: disordered alpha helix. (C) Root mean square fluctuations of Cα coordinates (RMSF). Arrows show the most flexible regions in both systems. The box indicates the region (70–90 aa) that differs in flexibility when comparing the two systems. (D) Fluctuations of aa in the average structure of free (left) and GUUG-binding (right) protein. Black arrows indicate the 70–90 aa region with the greatest fluctuation. (E) The surface electrostatic potential of the free (left) and GUUG-binding (right) proteins. Upper panel: front view; lower panel: rear view. The circle shows the RNA-interacting area.

Figure 5

Effect of C4 and C5 aptamers on the proliferation and viability of E. histolytica trophozoites. (A) pL4440-C4/C5 plasmid constructs. (B) Detection of dsRNA in TYI-S-33 medium. (C–D) Trophozoites were treated with C4- or C5-dsRNA (100 µl/ml) and incubated at 37 °C. Each day, the cell count was determined (C), and cell viability was assessed in a Trypan blue assay (D). Trophozoites grown in the absence of dsRNA (control) or in the presence of gfp-dsRNA were also used. Data were analysed in a two-way analysis of variance or a T-test, as appropriate. **p < 0.01, ***p < 0.001 and ****p < 0.0001.