RNA Clamping by Vasa Assembles a piRNA Amplifier Complex on Transposon Transcripts

Abstract

Germline-specific Piwi-interacting RNAs (piRNAs) protect animal genomes against transposons and are essential for fertility. piRNAs targeting active transposons are amplified by the ping-pong cycle, which couples Piwi endonucleolytic slicing of target RNAs to biogenesis of new piRNAs. Here, we describe the identification of a transient Amplifier complex that mediates biogenesis of secondary piRNAs in insect cells. Amplifier is nucleated by the DEAD box RNA helicase Vasa and contains the two Piwi proteins participating in the ping-pong loop, the Tudor protein Qin/Kumo and antisense piRNA guides. These components assemble on the surface of Vasa's helicase domain, which functions as an RNA clamp to anchor Amplifier onto transposon transcripts. We show that ATP-dependent RNP remodeling by Vasa facilitates transfer of 5' sliced piRNA precursors between ping-pong partners, and loss of this activity causes sterility in Drosophila. Our results reveal the molecular basis for the small RNA amplification that confers adaptive immunity against transposons.

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).…”

“…In support of this, evidence from additional Oskar-Vasa colocalization studies indicates that the eLOTUS domain might bind to the CTD only when the helicase core is in an open state. In this assay, we tested the mutation E400Q, which resides in the DEAD box of Vasa, drastically reduces the dissociation of the ATP hydrolysis products, and consequently locks the helicase in its closed conformation bound to RNA (Xiol et al 2014). While Oskar and wild-type Vasa co-localized in S2R+ cells (Fig.…”

“…Nurse cells are transcriptionally active and provide the growing oocyte with RNAs and proteins required for oocyte development and patterning of the future embryo (Johnstone and Lasko 2001). In the nuage, Vasa plays an essential role in the piRNA pathway (Malone et al 2009;Xiol et al 2014;Nishida et al 2015), a retrotransposon defense mechanism that helps maintain genome integrity (Luteijn and Ketting 2013;Sato and Siomi 2013;Czech and Hannon 2016). The Drosophila germ plasm (or pole plasm) is assembled at the posterior tip of the oocyte and specifies the Drosophila germ cell precursors called pole cells, which form at the posterior pole during early embryogenesis (for review, see Mahowald 2001).…”

Characterization of the piRNA pathway during development of the sea anemone Nematostella vectensis

“…4 The DEAD box helicase Vasa is a conserved player in this cycle facilitating the transfer of precursor piRNAs between PIWI proteins in the nuage. 6,7 Like PIWI proteins, Vasa is required for germ cell specification and maintenance. Thus, zebrafish, mouse and Drosophila mutant for piwi or vasa genes are sterile.…”

From guide to target: molecular insights into eukaryotic RNA-interference machinery

“…In insects, primary piRNAs are loaded into either Piwi or Aubergine (Aub). Piwi/Aub piRISCs slice the target transcript, the remnants of which are loaded into Ago3 with the assistance of an "amplifier" complex containing Vasa [131] and then matured. In turn, Ago3 cleaves primary transcripts that can be loaded into Piwi/Aub, thus amplifying the silencing signal.…”