Pachytene piRNAs define a conserved program of meiotic gene regulation

PIWI-interacting RNAs (piRNAs) safeguard genome integrity and fertility across animals and in humans ^1-3 . In mammals, pre-pachytene piRNAs execute the ancestral program of transposon silencing, whereas a second, mammalian-specific class functions during meiosis ⁴ . These pachytene piRNAs are indispensable for spermatogenesis ³ , yet their sequence diversity, rapid evolution, and lack of obvious complementarity have long obscured their primary targets and mode of action ⁵ . Here, we show that groups of pachytene piRNAs converge on single mRNAs with near-perfect complementarity, evoking the specificity and efficacy of classical RNA interference ⁶ . These mRNA-targeting piRNAs originate from pseudogene fragments within discrete piRNA clusters, establishing an unexpected one-to-one relationship: a given cluster encodes thousands of piRNAs that collectively silence a single gene. Deleting such piRNA clusters or their embedded pseudogene fragments, eliminates the gene-targeting piRNAs, derepresses the target mRNA, and causes spermatogenic defects. Comparative genomics reveals a simple, unifying logic, whereby rapidly evolving cluster–target pairs coexist with a conserved, primate-specific regulon maintained over tens of millions of years. These findings provide a mechanistic framework for pachytene piRNA action, repositioning them as precise gene regulators during meiosis and highlighting a uniquely preserved piRNA–target pair in primates.