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Functional Characterization of the Translation Initiation Factor Eif4e of Echinococcus Granulosus

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Functional Characterization of the Translation Initiation Factor Eif4e of Echinococcus Granulosus Parasitology Research (2019) 118:2843–2855 https://doi.org/10.1007/s00436-019-06421-8 GENETICS, EVOLUTION, AND PHYLOGENY - ORIGINAL PAPER Functional characterization of the translation initiation factor eIF4E of Echinococcus granulosus Filipe Santos Pereira-Dutra1,2 & Martin Cancela1,2 & Bruna Valandro Meneghetti1,2 & Henrique Bunselmeyer Ferreira1,2,3 & Karina Mariante Monteiro1,2,3 & Arnaldo Zaha1,2,3 Received: 28 January 2019 /Accepted: 2 August 2019 /Published online: 10 August 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract 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 prepara- tions 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. Keywords Capped RNAs . mRNA purification . Echinococcus granulosus . eIF4E Filipe Santos Pereira-Dutra and Martin Cancela contributed equally to Introduction this work. Section Editor: Christoph G. Grevelding Hydatidosis or cystic echinococcosis caused by parasitic flat- worms of the genus Echinococcus affects humans and animals Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00436-019-06421-8) contains supplementary all over the world (WHO 2013). The metacestode or hydatid material, which is available to authorized users. cyst (HC) is the larval stage of the parasite and is located mainly in liver and lungs of the intermediate host, including * Arnaldo Zaha cattle and sheep. Once within its host, HC can grow and pro- [email protected] duce the pre-adult form, the protoscolex (PSC), the infective stage of definitive canine host. Molecular aspects associated 1 Laboratório de Biologia Molecular de Cestodeos, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, UFRGS, with parasite gene expression and regulation during develop- Avenida Bento Gonçalves, 9500, Caixa Postal 15053, Porto ment and establishment within its hosts are relevant in order to Alegre, RS CEP 91501-970, Brazil develop rational strategies for parasitic control. 2 Programa de Pós-Graduação em Biologia Celular e Molecular, Translation initiation in eukaryotes involves several pro- Centro de Biotecnologia, UFRGS, Porto Alegre, Brazil teins associated in a large protein-RNA complex that directs 3 Departamento de Biologia Molecular e Biotecnologia, Instituto de the ribosome to the initiation codon. The key and rate-limiting Biociências, UFRGS, Porto Alegre, Brazil step of this process involves the participation of the eukaryotic 2844 Parasitol Res (2019) 118:2843–2855 translation initiation factor 4E (eIF4E), a cap-binding protein phylum Platyhelminthes. Furthermore, we cloned the of about 25 kDa (Gingras et al. 1999; Prévôt et al. 2003; E. granulosus eIF4E (Eg-eIF4E) coding sequence, Goodfellow and Roberts 2008). For most eukaryotes, espe- expressed and purified the recombinant Eg-eIF4E as fu- cially for vertebrates, the canonical cap-dependent translation sion protein to GST, and tested its potential binding begins with the specific recognition of the mRNA 5′-terminal properties using total RNA from E. granulosus monomethylguanosine cap (MMGcap) by eIF4E (von der protoescoleces. Haar et al. 2004; Rutkowska-Wlodarczyk et al. 2008). In spite of the fact that the MMGcap is the most common 5′ cap structure in eukaryotes (Rutkowska-Wlodarczyk et al. 2008), Material and methods a hypermethylated cap, the trimethylguanosine cap (TMGcap) (Davis 1996; Blaxter and Liu 1996), can be added to mRNAs Sequence analyses by a trans-splicing process, present for example in several parasitic and non-parasitic nematodes and in the phylum eIF4E-like protein sequences were surveyed in the available Platyhelminthes (Davis 1996;BlaxterandLiu1996;Brehm sequence databases Wormbase ParaSite (http://parasite. et al. 2000;Hastings2005; Bitar et al. 2013). In these organ- wormbase.org/), Planmine (http://planmine.mpi-cbg.de/ isms, generally both cis- and trans-splicing processes occur planmine/begin.do), and GenBank (http://www.ncbi.nlm. simultaneously and the transcripts from both origins coexist nih.gov/) for 25 flatworms (Clonorchis sinensis, Dugesia (Davis 1996;BlaxterandLiu1996;Hastings2005). Trans- japonica, Echinococcus canadensis, Echinococcus splicing differs from cis-splicing on the origin of the tran- granulosus, Echinococcus multilocularis, Gyrodactylus scripts involved in the process. While cis-splicing is con- salaris, Hydatigera taeniaeformis, Hymenolepis diminuta, cerned with exons from a single transcript, trans-splicing can Hymenolepis microstoma, Hymenolepis nana, Macrostomum be subdivided into two groups, one involving the junction of lignano, Mesocestoides corti, Opisthorchis viverrini, exons from different pre-mRNA transcripts, and another that Protopolystoma xenopodis, Schistocephalus solidus, involves a specialized spliced leader RNA which donates a Schistosoma curassoni, Schistosoma japonicum, short exon to 5′ UTR of another RNA (Lasda and Schistosoma mansoni, Schistossoma mattheei, Schistosoma Blumenthal 2011; Bitar et al. 2013). margrebowiei, Schistosoma rodhaini, Schmidtea The vertebrate eIF4E has very low affinity for mediterranea, Taenia solium, Taenia asiatica,and TMGcap in comparison with the MMGcap Trichobilharzia regenti). To identify eIF4E-encoding genes, (Niedzwiecka et al. 2004). In contrast, some nematode we performed in silico analyses in tblastN using the previous- eIF4E isoforms, and the eIF4E from the flatworm ly identified eIF4E protein sequences from S. mansoni Schistosoma mansoni can recognize both the TMGcap (G4VCF5) and E. granulosus (U6IZR7) as queries. and MMGcap (Liu et al. 2011). In the case of Sequences from the representative species within the flatworm S. mansoni and Schistosoma japonicum, another peculiar- classes Cestoda, Trematoda, Monogenea, and Turbellaria (E. ity is the presence of only a single eIF4E isoform, atyp- granulosus, S. mansoni, P. xenopodis,andD. japonica,re- ical for metazoan (Liu et al. 2009). This characteristic spectively) were used as queries in subsequent blast searches could be explored to develop novel anthelminthic drugs to improve representation. This iterative process was repeated (Liu et al. 2009). The 5′-cap-binding proteins, including until no novel eIF4E family members were found (Jones et al. human eIF4E, and 7-methylguanosine antibodies are be- 2015). We selected only the search results that consistently ingusedastoolstodepleterRNAfromtotalRNAsam- returned pairwise alignments of > 120 aligned amino acids. ples in order to improve yield in RNA-seq experiments As an external group, sequences of Saccharomyces cerevisiae (Blower et al. 2013) and to enrich samples in full-length (P07260), Ascaris suum (Q6PKX2), Mus musculus (P63073), transcripts (Bajak and Hagedorn 2008; Papic et al. 2012). Caenorhabditis elegans eIF4E-1 (O45551), eIF4E-2 The development of a complementary and sensitive (E3MFQ1), and eIF4E-5 (P56570) were retrieved from mRNA purification methodology for parasitic flatworms Universal Protein resource, UniProt (http://www.uniprot.org). may be a key tool for elucidating important mechanisms For phylogenetic analysis, we selected only those se- associated with flatworm development and its pathoge- quences with 98% for maximum similarity and 30% for min- nicity. Based on the single ability of helminth eIF4E to imum similarity. Sequence alignment was performed using interact with both MMGcap and TMGcap, we evaluated MAFFT7 (Katoh and Standley 2013) and MEGA 7.0 in this work the potential of the eIF4E protein (Tamura et al. 2013). To reconstruct the phylogenetic tree, E. granulosus as a tool for enrichment of mRNA sam- we performed the maximum likelihood method using ples from total parasite RNA. Based on the above, in the PhyML v3.0 (Guindon et al. 2010) and bootstrap (1000 rep- present work, we analyzed the presence and distribution licates) methods for branch support, and the tree was visual- of eIF4E genes in E. granulosus and other species of the ized and edited using Evolview (http://www.evolgenius.info). Parasitol Res (2019) 118:2843–2855 2845 Parasite material of the purified recombinant
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