Vasa-Like DEAD-Box RNA Helicases of Schistosoma mansoni

Abstract

Genome sequences are available for the human blood flukes, Schistosoma japonicum, S. mansoni and S. haematobium. Functional genomic approaches could aid in identifying the role and importance of these newly described schistosome genes. Transgenesis is established for functional genomics in model species, which can lead to gain- or loss-of-functions, facilitate vector-based RNA interference, and represents an effective forward genetics tool for insertional mutagenesis screens. Progress toward routine transgenesis in schistosomes might be expedited if germ cells could be reliably localized in cultured schistosomes. Vasa, a member of the ATP-dependent DEAD-box RNA helicase family, is a prototypic marker of primordial germ cells and the germ line in the Metazoa. Using bioinformatics, 33 putative DEAD-box RNA helicases exhibiting conserved motifs that characterize helicases of this family were identified in the S. mansoni genome. Moreover, three of the helicases exhibited vasa-like sequences; phylogenetic analysis confirmed the three vasa-like genes-termed Smvlg1, Smvlg2, and Smvlg3-were members of the Vasa/PL10 DEAD-box subfamily. Transcripts encoding Smvlg1, Smvlg2, and Smvlg3 were cloned from cDNAs from mixed sex adult worms, and quantitative real time PCR revealed their presence in developmental stages of S. mansoni with elevated expression in sporocysts, adult females, eggs, and miracidia, with strikingly high expression in the undeveloped egg. Whole mount in situ hybridization (WISH) analysis revealed that Smvlg1, Smvlg2 and Smvlg3 were transcribed in the posterior ovary where the oocytes mature. Germ cell specific expression of schistosome vasa-like genes should provide an informative landmark for germ line transgenesis of schistosomes, etiologic agents of major neglected tropical diseases.

Figure 1. Multiple sequence alignment of DEAD-box RNA helicases from Schistosoma mansoni.

Panel A: Schematic representation of ten motifs conserved in DEAD-box helicases; the EARKF motif, also shown, is conserved in Vasa and PL10 DEAD-box helicases. For each motif, with the exception of the Q motif, the capital case letters in the consensus sequence indicate highly conserved amino acids (in >80% enzymes) whereas lower case letters represent amino acids conserved in 50–79% of helicases . The capital case letters of the Q motif indicate amino acids conserved between 49%–99% of helicases. The lower case letters represent groups of amino acids, where a represents an aromatic residue, c is a charged residue, o is an alcohol, and l is an aliphatic residue . Panel B: Multiple sequence alignment of DEAD-box RNA helicases from Schistosoma mansoni, informative Vasa-like enzymes from other flatworms, Caenorhabditis elegans and Homo sapiens. DEAD box motifs are indicated with colored lines above and below the sequence that corresponds with the colors in the schematic representation (panel A). Accession numbers of aligned orthologues: Smvlg1 of Schistosoma mansoni (JQ619869), Smvlg2 of S. mansoni (JQ619870), Smvlg3 of S. mansoni (a consensus of Smp_068440 and JQ619871), Ngvlg1 of Neobenedenia girellae (BAF44659), Ngvlg2 of N. girellae (BAF44660), DjvlgA of Dugesia japonica (BAA34993), GLH-1 of Caenorhabditis elegans (P34689.3), and Vasa of Homo sapiens (AAF72705).

Figure 2. Phylogenetic tree and gene structure relationships of Schistosoma mansoni DEAD-box RNA helicases.

Panel A: Evolutionary relationships of Vasa, PL10, p68, and eIF4A proteins among informative species were inferred using the Neighbor-Joining method based on entire protein sequences. The S. mansoni vasa like genes are shown in red ink. Among DEAD-box helicases p68 is closely related to Vasa and PL10. IF4A served as the outgroup. Branch names indicate the common name of the species displayed; GenBank and GeneDB accessions are provided. Colored boxes are used to highlight families of DEAD box helicases, e.g. the blue region includes vasa orthologues, and the teal boxes include platyhelminth Vasa-like enzymes, etc. Panel B: Exon-intron boundary map revealed evolutionary relationships among Smvlg1, Smvlg2 and Smvlg3. Grey blocks represent exons and the black dashed-lines represent introns. Numbers below blocks denote the exon number. Vertical bars above the exons indicate sites encoding the DEAD-box characteristic motifs. Colors of the vertical bars correspond with the colors in the schematic representation of the DEAD-box motifs presented in Figure 1A. Scale bar, 1 kb.

Figure 3. Developmental expression of Vasa-like DEAD-box helicases in Schistosoma mansoni.

Expression levels of Smvlg1 (Panel A), Smvlg2 (B) and Smvlg3 (C) of developmental stages of S. mansoni were determined by quantitative real time PCR. Normalized fold expression of the vasa-like genes is presented; bars represent one standard deviation of the mean.

Figure 4. Localization of Smvlg1 and Smvlg2in adult Schistosoma mansoni females.

Riboprobes specific for Smvlg1 and Smvlg2 localized to the posterior ovary of S. mansoni. A and B: Female worms hybridized with Smvlg1 antisense riboprobe showing staining in the posterior region of the ovary. B, higher magnification view of the boxed area in image A. C and D: Female worms hybridized with Smvlg1 sense riboprobe revealing absence of staining; D, higher magnification view of the boxed area in panel C. E and F: Female worms hybridized with Smvlg2 antisense riboprobe showing staining in the posterior region of the ovary. F, higher magnification view of the boxed area in panel E. G and H: Female worms hybridized with Smvlg2 sense riboprobes. H, higher magnification view of the boxed area in panel G. Abbreviations: AO, anterior ovary; E, egg; PO, posterior ovary. Scale bars, 100 µm.