Functional characterization of the translation initiation factor eIF4E of Echinococcus granulosus
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The eukaryotic initiation factor 4E (eIF4E) specifically recognizes the 5′ mRNA cap, a rate-limiting step in the translation initiation process. Although the 7-methylguanosine cap (MMGcap) is the most common 5′ cap structure in eukaryotes, the trans-splicing process that occurs in several organism groups, including nematodes and flatworms, leads to the addition of a trimethylguanosine cap (TMGcap) to some RNA transcripts. In some helminths, eIF4E can have a dual capacity to bind both MMGcap and TMGcap. In the present work, we evaluated the distribution of eIF4E protein sequences in platyhelminths and we showed that only one gene coding for eIF4E is present in most parasitic flatworms. Based on this result, we cloned the Echinococcus granulosus cDNA sequence encoding eIF4E in Escherichia coli, expressed the recombinant eIF4E as a fusion protein to GST, and tested its ability to capture mRNAs through the 5′ cap using pull-down assay and qPCR. Our results indicate that the recombinant eIF4E was able to bind preferentially 5′-capped mRNAs compared with rRNAs from total RNA preparations of E. granulosus. By qPCR, we observed an enrichment in MMG-capped mRNA compared with TMG-capped mRNAs among Eg-eIF4E-GST pull-down RNAs. Eg-eIF4E structural model using the Schistosoma mansoni eIF4E as template showed to be well preserved with only a few differences between chemically similar amino acid residues at the binding sites. These data showed that E. granulosus eIF4E can be used as a potential tool to study full-length 5′-capped mRNA, besides being a potential drug target against parasitic flatworms.
KeywordsCapped RNAs mRNA purification Echinococcus granulosus eIF4E
Co-wrote and edited the manuscript, retrieved sequences, performed phylogenetic analysis, and performed the experiments: FSPD and MC; performed the structural and docking analyses of proteins: BVM; conceived and designed the experiments: FSPD, MC, KMM, and AZ. Contributed reagents/materials/analysis tools: HBF and AZ. Wrote the paper: FSPD, MC, BVM, and AZ. All authors read and approved the final manuscript.
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior–Brasil (CAPES) (Finance Code 001 and grant number 1278/2011) and by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (grant number 472316/2013–3). FSPD and BVM were recipients of a CAPES MSc. fellowship and MC was recipient of a CAPES postdoctoral fellowship.
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