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.