In vivo mapping of the functional regions of the DEAD-box helicase Vasa

Abstract

The maternally expressed Drosophila melanogaster DEAD-box helicase Vasa (Vas) is necessary for many cellular and developmental processes, including specification of primordial germ cells (pole cells), posterior patterning of the embryo, piRNA-mediated repression of transposon-encoded mRNAs, translational activation of gurken (grk) mRNA, and completion of oogenesis itself. Vas protein accumulates in the perinuclear nuage in nurse cells soon after their specification, and then at stage 10 Vas translocates to the posterior pole plasm of the oocyte. We produced a series of transgenic constructs encoding eGFP-Vas proteins carrying mutations affecting different regions of the protein, and analyzed in vivo which Vas functions each could support. We identified novel domains in the N- and C-terminal regions of the protein that are essential for localization, transposon repression, posterior patterning, and pole cell specification. One such functional region, the most C-terminal seven amino acids, is specific to Vas orthologues and is thus critical to distinguishing Vas from other closely related DEAD-box helicases. Surprisingly, we also found that many eGFP-Vas proteins carrying mutations that would be expected to abrogate DEAD-box helicase function localized to the nuage and posterior pole, and retained the capacity to support oogenesis, although they did not function in embryonic patterning, pole cell specification, grk activation, or transposon repression. We conclude from these experiments that Vas, a multifunctional protein, uses different domains and different molecular associations to carry out its various cellular and developmental roles.

The LOTUS domain is a conserved DEAD-box RNA helicase regulator essential for the recruitment of Vasa to the germ plasm and nuage

“…However, compared with endogenous Vasa, the levels of expression of the transgenic protein were reduced (Fig. 5B), as reported previously for other vasa transgenes (e.g., Johnstone and Lasko 2004;Xiol et al 2014;Dehghani and Lasko 2015).…”

piRNA Biogenesis in Drosophila melanogaster

“…Qin - another Tudor domain protein - has a role in maintaining heterotypic ping-pong by preventing the loading of Aub cleavage products into Piwi or Aub [74, 97]. Qin forms a complex with Vasa [98, 99], a member of the DEAD box helicase family, with ATPase, RNA binding and RNA unwinding activity [100–102]. Two roles were proposed for Vasa in piRNA processing.…”

Germ Plasm Biogenesis—An Oskar-Centric Perspective

“…While homologs of these two proteins are widely conserved as universal germ cell markers, oskar is a novel gene that evolved within the order of insects (Ewen-Campen et al, 2012). Like osk , females mutant for vasa or tudor produce embryos that lack germ cells and fail to form an abdomen (Boswell and Mahowald, 1985; Dehghani and Lasko, 2015). Vasa encodes a DEAD-box RNA helicase and has been proposed to be involved in translational activation of RNA localized to the posterior pole (Lasko and Ashburner, 1988).…”