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bioRxiv · 10.64898/2026.09.04.749402

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

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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Abstract

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.

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Busetto, V., Brehm, M., Sgromo, A., Mager, M., Pshanichnaya, L., Becker, E. C. A., Ketting, R. F., Ameres, S. L., Falk, S.. 2026-09-08. A pseudohelicase-centered complex couples assembly-dependent RNA cleavage to poly(UG)ylation. https://doi.org/10.64898/2026.09.04.749402

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