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.
Busetto, V., Brehm, M., Sgromo, A., Mager, M., Pshanichnaya, L., Becker, E. C. A., Ketting, R. F., Ameres, S. L., Falk, S.
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