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

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: RNA-seq and TT-SLAM-seq profiling of cultured Drosophila ovarian somatic cells (OSCs) to investigate the molecular logic of piRNA biogenesis and specificity. The experimental design includes: (1) RNA-seq analysis of wildtype OSCs and their derivatives following siRNA-mediated knockdown of key piRNA pathway factors, including Yb (siYB) and Armitage (siARMI), compared to control (siLUC) to determine their impact on steady-state transcript levels and piRNA precursor selection. (2) TT-SLAM-seq (Transient Transcriptome Sequencing with SLAM-seq) using 10-minute 4-thiouridine (4sU) labeling to measure nascent transcriptional output and RNA turnover rates in OSCs.