Methods to pick up overlapping small fragment pairs from two libraries

PIWI-interacting (pi)RNA pathways control transposable elements (TEs) and endogenous genes, playing important roles in animal gamete formation. However, the underlying piRNA biogenesis mechanisms remain elusive. Here, we show endogenous protein-coding sequences (CDSs), that are normally used for translation, serve as origins of non-coding piRNA biogenesis in Drosophila melanogaster testes. The product, namely CDS-piRNAs, formed silencing complexes with Aubergine (Aub) in germ cells. Proximity proteome and functional analyses show CDS-piRNAs and cluster/TE-piRNAs are distinct species occupying Aub, the former loading selectively relies on chaperone Cyclophilin 40. Moreover, Argonaute-2 (Ago2) and Dicer-2 activities were found critical for CDS-piRNA production. We provide evidence that Ago2-bound short interfering (si)RNAs and micro(mi)RNAs specify precursors to be processed into piRNAs. We further demonstrate that Aub is crucial in spermatid differentiation, regulating chromatins through mRNA cleavage. Collectively, our data illustrate a unique strategy employed by male germline, expanding piRNA repertoire for silencing of endogenous genes during spermatogenesis.