Bombyx Vasa sequesters transposon mRNAs in nuage via phase separation requiring RNA binding and self-association.

Bombyx Vasa (BmVasa) assembles non-membranous organelle, nuage or Vasa bodies, in germ cells, known as the center for Siwi-dependent transposon silencing and concomitant Ago3-piRISC biogenesis. However, details of the body assembly remain unclear. Here, we show that the N-terminal intrinsically disordered region (N-IDR) and RNA helicase domain of BmVasa are responsible for self-association and RNA binding, respectively, but N-IDR is also required for full RNA-binding activity. Both domains are essential for Vasa body assembly in vivo and droplet formation in vitro via phase separation. FAST-iCLIP reveals that BmVasa preferentially binds transposon mRNAs. Loss of Siwi function derepresses transposons but has marginal effects on BmVasa-RNA binding. This study shows that BmVasa assembles nuage by phase separation via its ability to self-associate and bind newly exported transposon mRNAs. This unique property of BmVasa allows transposon mRNAs to be sequestered and enriched in nuage, resulting in effective Siwi-dependent transposon repression and Ago3-piRISC biogenesis.

Maelstrom functions in the production of Siwi-piRISC capable of regulating transposons in Bombyx germ cells.

PIWI-interacting RNAs (piRNAs) bind to PIWI proteins to assemble the piRISC, which represses germline transposons. Maelstrom (Mael) is necessary for piRISC biogenesis in germ cells, but its function remains unclear. Here, we show that Mael interconnects Spindle-E (Spn-E), a key piRISC biogenesis factor, with unloaded Siwi, one of two silkworm PIWI members. Mael also assembles a subset of nuage, a non-membranous organelle involved in piRISC biogenesis. Loss of Mael abrogated the Spn-E-Siwi interaction and Ago3-piRISC biogenesis, but Siwi-piRISC was produced. Bioinformatic analysis showed that Siwi-bound piRNAs in Mael-lacking cells were rich in transposon-targeting piRNAs as in normal cells but were biased toward transposons that are marginally controlled by Siwi-piRISC. This explains the impairment in Ago3-piRISC production because transposon mRNAs cleaved by Siwi are the origin of Ago3-loaded piRNAs. We argue that Mael plays a role in the production of primary Siwi-piRISC capable of regulating transposon expression in germ cells.

Crystal Structure and Activity of the Endoribonuclease Domain of the piRNA Pathway Factor Maelstrom.

PIWI-interacting RNAs (piRNAs) protect the genome from transposons in animal gonads. Maelstrom (Mael) is an evolutionarily conserved protein, composed of a high-mobility group (HMG) domain and a MAEL domain, and is essential for piRNA-mediated transcriptional transposon silencing in various species, such as Drosophila and mice. However, its structure and biochemical function have remained elusive. Here, we report the crystal structure of the MAEL domain from Drosophila melanogaster Mael, at 1.6 Å resolution. The structure reveals that the MAEL domain has an RNase H-like fold but lacks canonical catalytic residues conserved among RNase H-like superfamily nucleases. Our biochemical analyses reveal that the MAEL domain exhibits single-stranded RNA (ssRNA)-specific endonuclease activity. Our cell-based analyses further indicate that ssRNA cleavage activity appears dispensable for piRNA-mediated transcriptional transposon silencing in Drosophila. Our findings provide clues toward understanding the multiple roles of Mael in the piRNA pathway.