title: Single-molecule fluorescence measurements reveal the reaction mechanisms of the core RISC, composed of human Argonaute 2 and a guide RNA.
abstract: In eukaryotes, small RNAs play important roles in both gene regulation and resistance to viral infection. Argonaute proteins have been identified as a key component of the effector complexes of various RNA-silencing pathways, but the mechanistic roles of Argonaute proteins in these pathways are not clearly understood. To address this question, we performed single-molecule fluorescence experiments using an RNA-induced silencing complex (core-RISC) composed of a small RNA and human Argonaute 2. We found that target binding of core-RISC starts at the seed region of the guide RNA. After target binding, four distinct reactions followed: target cleavage, transient binding, stable binding, and Argonaute unloading. Target cleavage required extensive sequence complementarity and accelerated core-RISC dissociation for recycling. In contrast, the stable binding of core-RISC to target RNAs required seed-match only, suggesting a potential explanation for the seed-match rule of microRNA (miRNA) target selection.
authors.affiliation: Department of Physics and Astronomy, Institute of Applied Physics, National Center of Creative Research Initiatives, Seoul National University, Seoul 08826, Korea.
authors.forename: Myung Hyun
authors.initials: MH
authors.lastname: Jo
authors.affiliation: Department of Biological Sciences, KAIST, Daejeon 34141, Korea.
authors.forename: Ji-Joon
authors.initials: JJ
authors.lastname: Song
authors.affiliation: Department of Physics and Astronomy, Institute of Applied Physics, National Center of Creative Research Initiatives, and Department of Biophysics and Chemical Biology, Seoul National University, Seoul 08826, Korea.
authors.forename: Sungchul
authors.initials: S
authors.lastname: Hohng
mesh_terms: D060565:Argonaute Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism; D061848:Optical Imaging / Q000379:methods; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D060565:Argonaute Proteins; D035683:MicroRNAs; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; D000094704:RNA, Guide, CRISPR-Cas Systems; C466422:AGO2 protein, human