Distinct and Collaborative Functions of Yb and Armitage in Transposon-Targeting piRNA Biogenesis

Abstract

PIWI-interacting RNAs (piRNAs) repress transposons to maintain germline genome integrity. Previous studies showed that artificial tethering of Armitage (Armi) to reporter RNAs induced piRNA biogenesis. However, the lack of female sterile (1) Yb (Yb) in Drosophila ovarian somatic cells (OSCs) impaired the production of transposon-targeting piRNAs, even in the presence of Armi. Here, we show that the specific interaction of Armi with RNA transcripts of the flamenco piRNA cluster, the primary source of transposon-targeting piRNAs in OSCs, is strictly regulated by Yb. The lack of Yb allowed Armi to bind RNAs promiscuously, leading to the production of piRNAs unrelated to transposon silencing. The ATP hydrolysis-defective mutants of Armi failed to unwind RNAs and were retained on them, abolishing piRNA production. These findings shed light on distinct and collaborative requirements of Yb and Armi in transposon-targeting piRNA biogenesis. We also provide evidence supporting the direct involvement of Armi but not Yb in Zucchini-dependent piRNA phasing.

Armitage determines Piwi−piRISC processing from precursor formation and quality control to inter‐organelle translocation

“…The Gasz-Armi association in S2 cells was recapitulated in this study ( Fig EV3C). We also demonstrate that recombinant Armi (purified from S2 cells under harsh conditions, preventing co-purification of other proteins [31]) bound with recombinant Gasz lacking the C-terminal TM region (DC113), which was bacterially purified, without any other factors (Figs 3E and EV3D); this supports their ability to directly interact. These findings more strongly support the first scenario that Armi resists leaving Yb bodies unless Piwi localizes to Yb bodies to become pre-piRISC there.…”

“…Even under conditions where Piwi-pre-piRISC was properly formed at Yb bodies, when Armi lacked RNA-binding activity, both proteins remained at the bodies. One possible reason is that Yb, but not Armi, Zuc, or even Piwi, has a strong sequence preference in RNA binding [31]. Why is such multilayered, elaborate regulation required in piRISC processing (Fig 7)?…”

“…Another potential reason for relying on Armi's RNA-binding activity might be related to Armi's promiscuous RNA binding [31,33]. This type of regulation relying on the RNA-binding activity of Armi is biologically relevant because Armi without RNA-binding activity would fail to unwind (relax) piRNA precursors for Zuc cleavage, blocking piRNA biogenesis.…”

“…Expression vector of Armi-Flag WT was produced by inverse PCR and infusion using the RNAi-resistant Flag-Armi WT vector [31] as a template. Expression vector of Armi-Flag WT was produced by inverse PCR and infusion using the RNAi-resistant Flag-Armi WT vector [31] as a template.…”

“…Briefly, GST-fused proteins were expressed in E. coli and were resuspended with lysis buffer [20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1.0% Triton X-100, 1× cOmplete TM ULTRA Tablets, EDTA-free, glass vials Protease Inhibitor Cocktail (Sigma), 1 mM DTT] and lysed by sonication. Recombinant Armi protein was purified as previously described [31]. The proteins were eluted with elution buffer [20 mM Tris-HCl (pH 9.0), 150 mM NaCl, 1.0% Triton X-100, 1 mM DTT, 10 mM glutathione, 10% glycerol].…”