title: Single-Molecule Analysis of the Target Cleavage Reaction by the Drosophila RNAi Enzyme Complex.
abstract: Small interfering RNAs (siRNAs) direct cleavage of complementary target RNAs via an RNA-induced silencing complex (RISC) that contains Argonatute2 protein at its core. However, what happens after target cleavage remains unclear. Here we analyzed the cleavage reaction by Drosophila Argonaute2-RISC using single-molecule imaging and revealed a series of intermediate states in target recognition, cleavage, and product release. Our data suggest that, after cleavage, RISC generally releases the 5' cleavage fragment from the guide 3' supplementary region first and then the 3' fragment from the seed region, highlighting the reinforcement of the seed pairing in RISC. However, this order can be reversed by extreme stabilization of the 3' supplementary region or mismatches in the seed region. Therefore, the release order of the two cleavage fragments is influenced by the stability in each region, in contrast to the unidirectional base pairing propagation from the seed to the 3' supplementary region upon target recognition.
authors.affiliation: Graduate School of Frontier Science, The University of Tokyo, Chiba 277-8562, Japan; Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
authors.forename: Chunyan
authors.initials: C
authors.lastname: Yao
authors.affiliation: Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan.
authors.forename: Hiroshi M
authors.initials: HM
authors.lastname: Sasaki
authors.affiliation: Graduate School of Frontier Science, The University of Tokyo, Chiba 277-8562, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Ueda
authors.affiliation: Graduate School of Frontier Science, The University of Tokyo, Chiba 277-8562, Japan; Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan. Electronic address: tomari@iam.u-tokyo.ac.jp.
authors.forename: Yukihide
authors.initials: Y
authors.lastname: Tomari
authors.affiliation: Graduate School of Frontier Science, The University of Tokyo, Chiba 277-8562, Japan; Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto 606-8501, Japan. Electronic address: tadakuma.hisashi.8s@kyoto-u.ac.jp.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Tadakuma
mesh_terms: D000818:Animals; D060565:Argonaute Proteins / Q000378:metabolism*; D001483:Base Sequence; D004330:Drosophila / Q000201:enzymology / Q000235:genetics*; D029721:Drosophila Proteins / Q000378:metabolism*; D034622:RNA Interference / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000235:genetics*
chemical_list: C433807:AGO2 protein, Drosophila; D060565:Argonaute Proteins; D029721:Drosophila Proteins; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex