Abstract

The PIWI-interacting RNA (piRNA) pathway represses mobile transposable elements (TEs) to protect genome integrity in animal gonads. In Drosophila ovaries, piRNAs are generated in the nuage, a membrane-less organelle enriched with piRNA factors. Squash (Squ) is a key component for piRNA-mediated TE silencing, but its molecular function remains unclear. Here, we show that loss of Squ impairs piRNA biogenesis, causing abnormal accumulation of precursor transcripts and destabilization of Ago3. Reduction of Ago3 enhances Aub-Aub homotypic ping-pong instead of Aub-Ago3 heterotypic piRNA amplification. Furthermore, we demonstrate that Squ acts as a cofactor of the RNA helicase spindle-E (Spn-E), which is crucial for piRNA biogenesis. Disrupting the Squ-Spn-E interaction leads to TE de-repression and defective precursor processing, indicating that Squ cooperates with Spn-E to ensure proper piRNA biogenesis. Together, these findings identify Squ as a critical factor for Spn-E-dependent heterotypic piRNA amplification in the Drosophila ovary.