Abstract
The DEAD-box RNA helicase Vasa (Vas, also known as DDX4) is required for germ cell development. In Drosophila, analysis of hypomorphic mutations has implicated maternally expressed Vas in germ cell formation and posterior embryonic patterning. vas-null females, which rarely complete oogenesis, exhibit defects in mitotic progression of germline stem cells, Piwi-interacting RNA (piRNA)-mediated transposon silencing, and translation of Gurken (Grk), an EGFR ligand. The carboxy-terminal region of Vas orthologs throughout the animal kingdom consists of several acidic residues as well as an invariant tryptophan in the penultimate or ultimate position (Trp660 in Drosophila melanogaster). Using CRISPR/Cas9 gene editing, we made a substitution mutant in this residue. Replacing Trp660 by Glu (W660E) abolishes the ability of Vas to support germ cell formation and embryonic patterning and greatly reduces Vas activity in piRNA biogenesis, as measured by transposon silencing, and in activating Grk translation. A conservative substitution (W660F) has much milder phenotypic consequences. In addition, females expressing only a form of Vas in which the seven C-terminal amino acids were replaced with the corresponding residues from Belle (Bel, also known as DDX3) show defects in perinuclear nuage assembly and transposon silencing. Oogenesis in females expressing only the chimeric Vas arrests early; however, in a vas 1 background, in which early expression of endogenous Vas supports oogenesis, the chimeric protein supports posterior patterning and germ cell specification. These results indicate that the unique C-terminus of Vas is essential for its function in piRNA biogenesis and that the conserved Trp660 residue has an important functional role.
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Acknowledgments
We are grateful to Beili Hu for microinjection into the embryos. All the images were taken using the Cellular Imaging and Analysis Network (CIAN) facility at McGill University. We would also like to thank Fillip Port for his technical support during CRISPR gene editing and for sharing some of his unpublished observations. This work was supported by NSERC Discovery grant RGPIN-2014-06340 to P. L.
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Communicated by Angelika Stollewerk
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Fig. S1
Interactions of Vas with Osk, Gus and eIF5B are not abolished by ∆655-661 or W660E. A. A β-Galactosidase filter assay shows similar results for vas +, vas ∆655-661 and vas W660E in terms of interactions with Osk, Gus and eIF5B. B. Positive interactions would enable yeast to grow on the quadruple drop-out (-Trp, -Leu, -His and –Ade) media. This test also confirmed that the interactions with Osk, Gus and eIF5B are not notably different among vas +, vas ∆655-661 and vas W660E. (GIF 169 kb)
Table S1
The list of the proteins that have been previously found to associate with Vas through different assays, including yeast two-hybrid (Y2H), GST pull-down or co-immunoprecipitation (co-IP). The latter has been performed using the endogenous or tagged proteins from Drosophila ovaries or Bombyx mori BmN4 cell lines. We expressed each one of these candidates in yeast, either as a full length protein or a fragment. Our Y2H assays only showed a direct interaction between full-length Vas and Osk, Gus and eIF5B, consistent with the previous studies. 1: (Breitwieser et al. 1996), 2: (Anne 2010), 3: (Styhler et al. 2002), 4: (Kugler et al. 2010), 5: (Carrera et al. 2000), 6: (Webster et al. 1997), 7: (Lerit and Gavis 2011), 8: (Patil and Kai 2010), 9: (Megosh et al. 2006), 10: (Pek and Kai 2011a), 11: (Anand and Kai 2012), 12: (Xiol et al. 2014) (GIF 78 kb)
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Dehghani, M., Lasko, P. C-terminal residues specific to Vasa among DEAD-box helicases are required for its functions in piRNA biogenesis and embryonic patterning. Dev Genes Evol 226, 401–412 (2016). https://doi.org/10.1007/s00427-016-0560-5
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DOI: https://doi.org/10.1007/s00427-016-0560-5