A pseudohelicase-centered complex couples assembly-dependent RNA cleavage to poly(UG)ylation

Robust RNA interference in Caenorhabditis elegans requires Argonaute targeted RNAs to be cleaved by RDE 8 and poly(UG)ylated by MUT 2, generating templates for RNA dependent RNA polymerases that synthesize secondary small RNAs. RDE 8 and MUT 2 are physically coupled within the cleavage and poly(UG)ylation (CPUG) complex, together with the pseudonuclease NYN 1 and MUT 15, but how their activities are controlled and coordinated is unknown. Structural and biochemical analyses reveal that the RDE 8/NYN 1 nuclease module is autoinhibited, restricting cleavage outside CPUG. We identify MUT 15, the CPUG scaffold linking this nuclease module to MUT 2, as a pseudohelicase. MUT 15 remodels the nuclease module, licensing RDE 8 activity upon complex assembly. Reconstituted CPUG synthesizes long poly(UG) tails, with the substrate 3' end determining tailing efficiency and register. Within CPUG, RNA engagement by MUT 2 limits RDE 8 access, while poly(UG) tails themselves are not cleaved by RDE 8, preventing re cleavage. Together, CPUG couples assembly dependent RNA cleavage to downstream processing, imposing safety and directionality.