title: Phosphate-binding pocket in Dicer-2 PAZ domain for high-fidelity siRNA production.
abstract: The enzyme Dicer produces small silencing RNAs such as micro-RNAs (miRNAs) and small interfering RNAs (siRNAs). In Drosophila, Dicer-1 produces ∼22-24-nt miRNAs from pre-miRNAs, whereas Dicer-2 makes 21-nt siRNAs from long double-stranded RNAs (dsRNAs). How Dicer-2 precisely makes 21-nt siRNAs with a remarkably high fidelity is unknown. Here we report that recognition of the 5'-monophosphate of a long dsRNA substrate by a phosphate-binding pocket in the Dicer-2 PAZ (Piwi, Argonaute, and Zwille/Pinhead) domain is crucial for the length fidelity, but not the efficiency, in 21-nt siRNA production. Loss of the length fidelity, meaning increased length heterogeneity of siRNAs, caused by point mutations in the phosphate-binding pocket of the Dicer-2 PAZ domain decreased RNA silencing activity in vivo, showing the importance of the high fidelity to make 21-nt siRNAs. We propose that the 5'-monophosphate of a long dsRNA substrate is anchored by the phosphate-binding pocket in the Dicer-2 PAZ domain and the distance between the pocket and the RNA cleavage active site in the RNaseIII domain corresponds to the 21-nt pitch in the A-form duplex of a long dsRNA substrate, resulting in high-fidelity 21-nt siRNA production. This study sheds light on the molecular mechanism by which Dicer-2 produces 21-nt siRNAs with a remarkably high fidelity for efficient RNA silencing.
authors.affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
authors.forename: Suresh K
authors.initials: SK
authors.lastname: Kandasamy
authors.affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205 fukunaga@jhmi.edu.
authors.forename: Ryuya
authors.identifier: 0000-0002-5814-8206
authors.initials: R
authors.lastname: Fukunaga
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D029721:Drosophila Proteins / Q000378:metabolism / Q000502:physiology*; D004331:Drosophila melanogaster; D035683:MicroRNAs; D028044:Phosphate-Binding Proteins / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D020365:RNA Helicases / Q000378:metabolism / Q000502:physiology*; D034622:RNA Interference / Q000502:physiology*; D012330:RNA, Double-Stranded / Q000378:metabolism; D034741:RNA, Small Interfering / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000378:metabolism / Q000502:physiology*; D013379:Substrate Specificity
chemical_list: D029721:Drosophila Proteins; D035683:MicroRNAs; D028044:Phosphate-Binding Proteins; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; C478069:DCR-2 protein, Drosophila; D043244:Ribonuclease III; D020365:RNA Helicases
keywords: Dicer; RNA silencing; dsRNA; phosphate; siRNA