MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is specifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer's DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the complete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicer•-miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleavage-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways.

CEITEC Central European Institute of Technology Masaryk University 625 00 Brno Czech Republic

CEITEC Central European Institute of Technology Masaryk University 625 00 Brno Czech Republic; Institute of Molecular Genetics of the Czech Academy of Sciences v v i Videnska 1083 142 20 Prague 4 Czech Republic

CEITEC Central European Institute of Technology Masaryk University 625 00 Brno Czech Republic; National Centre for Biomolecular Research Faculty of Science Masaryk University 625 00 Brno Czech Republic

Centre for Regenerative Medicine Institute for Regeneration and Repair Institute for Stem Cell Research School of Biological Sciences University of Edinburgh 5 Little France Drive Edinburgh EH16 4UU UK; European Molecular Biology Laboratory Mouse Biology Unit Via Ramarini 32 Monterotondo Scalo 00015 Italy

Centre for Regenerative Medicine Institute for Regeneration and Repair Institute for Stem Cell Research School of Biological Sciences University of Edinburgh 5 Little France Drive Edinburgh EH16 4UU UK; European Molecular Biology Laboratory Mouse Biology Unit Via Ramarini 32 Monterotondo Scalo 00015 Italy; Wellcome Centre for Cell Biology School of Biological Sciences University of Edinburgh Edinburgh EH9 3BF UK

Czech Centre for Phenogenomics and Laboratory of Transgenic Models of Diseases Institute of Molecular Genetics of the Czech Academy of Sciences v v i Prumyslova 595 252 50 Vestec Czech Republic

Institute of Molecular Genetics of the Czech Academy of Sciences v v i Videnska 1083 142 20 Prague 4 Czech Republic

Institute of Molecular Genetics of the Czech Academy of Sciences v v i Videnska 1083 142 20 Prague 4 Czech Republic; Bioinformatics Group Department of Biology Faculty of Science University of Zagreb Horvatovac 102a 10000 Zagreb Croatia

Institute of Science and Technology Austria Am Campus 1 3400 Klosterneuburg Austria

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Enhanced RNAi does not provide efficient innate antiviral immunity in mice

Functional canonical RNAi in mice expressing a truncated Dicer isoform and long dsRNA

Dicer structure and function: conserved and evolving features

Structural basis of microRNA biogenesis by Dicer-1 and its partner protein Loqs-PB