A Naïve RNA Sampling Core Enables Adaptive piRNA Specificity Against Transposable Elements [smallRNA]

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. Overall design: Sequencing of total or Argonaut bound small RNAs from wildtype and piRNA factor depleted cell lines. In addition, small RNA data from cell lines harboring reporter constructs to test piRNA biogenesis precursorSequencing of total and Argonaute-bound small RNAs from wild-type and piRNA factor–depleted cell lines. In addition, sequencing of small RNAs from cell lines harboring reporter constructs to test piRNA precursor specificity and biogenesis. specificity.