title: Structural determinants of miRNAs for RISC loading and slicer-independent unwinding.
abstract: MicroRNAs (miRNAs) regulate expression of their target mRNAs through the RNA-induced silencing complex (RISC), which contains an Argonaute (Ago) family protein as a core component. In Drosophila melanogaster, miRNAs are generally sorted into Ago1-containing RISC (Ago1-RISC). We established a native gel system that can biochemically dissect the Ago1-RISC assembly pathway. We found that miRNA-miRNA* duplexes are loaded into Ago1 as double-stranded RNAs in an ATP-dependent fashion. In contrast, unexpectedly, unwinding of miRNA-miRNA* duplexes is a passive process that does not require ATP or slicer activity of Ago1. Central mismatches direct miRNA-miRNA* duplexes into pre-Ago1-RISC, whereas mismatches in the seed or guide strand positions 12-15 promote conversion of pre-Ago1-RISC into mature Ago1-RISC. Our findings show that unwinding of miRNAs is a precise mirror-image process of target recognition, and both processes reflect the unique geometry of RNAs in Ago proteins.
authors.affiliation: Institute of Molecular and Cellular Biosciences, The University of Tokyo, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Kawamata
authors.forename: Hervé
authors.initials: H
authors.lastname: Seitz
authors.forename: Yukihide
authors.initials: Y
authors.lastname: Tomari
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D060565:Argonaute Proteins; D020137:Base Pair Mismatch; D001483:Base Sequence; D019281:Dimerization; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism; D039642:Eukaryotic Initiation Factors; D035683:MicroRNAs / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation / Q000187:drug effects; D011485:Protein Binding; D020365:RNA Helicases / Q000378:metabolism; D012326:RNA Splicing; D034743:RNA-Induced Silencing Complex / Q000378:metabolism*; D043244:Ribonuclease III / Q000378:metabolism
chemical_list: C489654:AGO1 protein, Drosophila; D060565:Argonaute Proteins; D029721:Drosophila Proteins; D039642:Eukaryotic Initiation Factors; D035683:MicroRNAs; D034743:RNA-Induced Silencing Complex; D000255:Adenosine Triphosphate; C515033:Dcr-1 protein, Drosophila; D043244:Ribonuclease III; D020365:RNA Helicases
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