Target RNA recognition drives PIWI complex assembly for transposon silencing.

PIWI-clade Argonaute proteins and their associated PIWI-interacting RNAs (piRNAs) are essential guardians of genome integrity, silencing transposable elements through distinct nuclear and cytoplasmic pathways. Nuclear PIWI proteins direct heterochromatin formation at transposon loci, while cytoplasmic PIWIs cleave transposon transcripts to initiate piRNA amplification. Both processes rely on target RNA recognition by PIWI-piRNA complexes, yet how this leads to effector recruitment is unclear. Here, we show that target engagement triggers formation of complexes, termed PIWI-comprising a PIWI protein, a piRNA-target duplex, a GTSF family protein, and Maelstrom-that serve as molecular platforms recruiting downstream effectors. In Drosophila, nuclear Piwi engages the SFiNX complex to establish heterochromatin, while cytoplasmic Aubergine complexes recruit the helicase Spindle-E to promote piRNA biogenesis. Evolutionary analysis reveals that PIWI formation is conserved across metazoans, uncovering an ancient mechanism coupling piRNA-guided target recognition to effector function. These findings define a unifying molecular principle for PIWI-mediated silencing across cellular compartments.