Transposon and mRNA are distinguished by piRNA's pairing and piRNA's expression levels in Ovarian Somatic Cell.

Piwi-interacting RNAs (piRNAs) are the main repressors of transposable elements (TEs) in animal germlines. In Drosophila, Piwi-piRNA complexes associate with nascent TE transcripts to drive heterochromatin formation and TE repression. However, previous studies have shown that Piwi also associates with large amounts of mRNAs, raising the question of how Piwi discriminates between mRNAs and TEs. To answer this question, we performed a comprehensive analysis of Piwi-associated RNAs, compositionally and functionally, to decipher the targeting rules of Piwi-piRNA complexes. While Piwi initially identifies its targets through the seed sequence, it requires pairing well beyond the seed, nearly a perfect match, to elicit a repressive response. In addition to the complementarity of piRNAs to their targets, their abundance must reach a certain threshold to be functional. Together, these findings explain large differences in target repression of Piwi-associated RNAs and reveals how Piwi efficiently distinguishes TEs from mRNAs despite associating with both. Overall design: ChIP-seq for H3K9me3 and H3K4me3 in pMT- Ovarian Somatic Cell (OSC), Piwi CLASH in WT OSC, CAGE in EGFP KD and Piwi KD OSC, Iso-seq in EGFP KD and Piwi KD OSC, Pacbio Genome sequensing in OSC, scRNA-seq in OSC