title: Phosphate and R2D2 restrict the substrate specificity of Dicer-2, an ATP-driven ribonuclease.
abstract: Drosophila Dicer-2 generates small interfering RNAs (siRNAs) from long double-stranded RNA (dsRNA), whereas Dicer-1 produces microRNAs (miRNAs) from pre-miRNA. What makes the two Dicers specific for their biological substrates? We find that purified Dicer-2 can efficiently cleave pre-miRNA, but that inorganic phosphate and the Dicer-2 partner protein R2D2 inhibit pre-miRNA cleavage. Dicer-2 contains C-terminal RNase III domains that mediate RNA cleavage and an N-terminal helicase motif, whose function is unclear. We show that Dicer-2 is a dsRNA-stimulated ATPase that hydrolyzes ATP to ADP; ATP hydrolysis is required for Dicer-2 to process long dsRNA, but not pre-miRNA. Wild-type Dicer-2, but not a mutant defective in ATP hydrolysis, can generate siRNAs faster than it can dissociate from a long dsRNA substrate. We propose that the Dicer-2 helicase domain uses ATP to generate many siRNAs from a single molecule of dsRNA before dissociating from its substrate.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology and Howard Hughes Medical Institute, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA.
authors.forename: Elif Sarinay
authors.initials: ES
authors.lastname: Cenik
authors.forename: Ryuya
authors.initials: R
authors.lastname: Fukunaga
authors.forename: Gang
authors.initials: G
authors.lastname: Lu
authors.forename: Robert
authors.initials: R
authors.lastname: Dutcher
authors.forename: Yeming
authors.initials: Y
authors.lastname: Wang
authors.forename: Traci M
authors.initials: TM
authors.lastname: Tanaka Hall
authors.forename: Phillip D
authors.initials: PD
authors.lastname: Zamore
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D035683:MicroRNAs / Q000378:metabolism; D008954:Models, Biological; D010710:Phosphates / Q000378:metabolism*; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012330:RNA, Double-Stranded / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D029721:Drosophila Proteins; D035683:MicroRNAs; D010710:Phosphates; C478068:R2D2 protein, Drosophila; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C478069:DCR-2 protein, Drosophila; C515033:Dcr-1 protein, Drosophila; D043244:Ribonuclease III; D020365:RNA Helicases