piRNA-mediated genome defense against transposable elements operates through a conserved foundation of pervasive, non-specific cytoplasmic transcript sampling, with tissue-specific molecular modules layering adaptive specificity: in somatic cells, the DEAD-box protein Yb selectively routes uridine-rich RNAs into processing, automatically capturing antisense retrotransposon transcripts due to their intrinsically adenosine-biased genomes, while in germline cells lacking Yb, naïve piRNAs generated from basal sampling trigger autocatalytic ping-pong amplification upon encountering complementary targets produced by transposon mobility, thereby exploiting fundamental vulnerabilities of mobile elements to achieve robust, adaptable silencing without predefined genomic templates.

How piRNA-mediated genome defense achieves specificity against transposons while sampling a complex transcriptome has remained unresolved. Here we show that piRNA biogenesis operates through pervasive, non-specific sampling of cytoplasmic RNAs, with specificity imposed by tissue-specific molecular modules that exploit intrinsic vulnerabilities of transposons. In Drosophila somatic cells, the specificity factor Yb steers basal processing towards uridine-rich RNAs—automatically capturing antisense retrotransposon transcripts due to their intrinsically adenosine-biased genomes. In germline cells lacking Yb, basal sampling generates naïve piRNAs loaded into catalytically active Argonaute proteins, which trigger autocatalytic ping-pong amplification upon encountering complementary targets. In both contexts, transposon mobility facilitates the production of antisense RNAs that enable either biased processing or amplification. Thus, piRNA clusters, long associated with pathway specificity, act as sources of transposon antisense sequences, while specificity arises from layering distinct molecular mechanisms onto a shared foundation of indiscriminate transcript sampling, enabling robust and adaptable genome defense without predefined templates.