Drosophila Piwi distinguishes transposons from mRNAs by piRNA complementarity and abundance.

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 numbers 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 the target repression of Piwi-associated RNAs and reveal how Piwi efficiently distinguishes TEs from mRNAs despite associating with both.