Squash ensures Spindle-E–dependent heterotypic ping-pong amplification of piRNAs in the Drosophila ovary

Abstract The PIWI-interacting RNA (piRNA) pathway plays a crucial role in repressing mobile transposable elements (TEs) and protecting the integrity of the heritable genome in animal gonads. In the Drosophila ovary, piRNAs are produced in a membrane-less organelle called nuage, in which many piRNA factors are localized. Among them, Squash (Squ) has also been identified as a key component in piRNA-directed TE silencing. However, its molecular function remains largely unknown. Here, we demonstrate that loss of Squ leads to defective piRNA biogenesis, which is associated with the abnormal accumulation of precursor transcripts and the specific destabilization of Ago3, a member of the PIWI-family proteins. Reducing Ago3 results in enhanced homotypic piRNA amplification mediated by Aub itself, rather than the heterotypic ping-pong amplification between Aub and Ago3. Additionally, we demonstrate that Squ is a functional cofactor of the RNA helicase, Spindle-E (Spn-E), which plays a vital role in piRNA biogenesis. Point mutations that abrogate the interaction between Squ and Spn-E result in TE de-repression and defective precursor processing, suggesting that Squ works with Spn-E to ensure the proper piRNA biogenesis. These results identify Squ as a critical factor for Spn-E–dependent heterotypic ping-pong amplification of piRNAs in the Drosophila ovary.