title: In vitro assembly of plant RNA-induced silencing complexes facilitated by molecular chaperone HSP90.
abstract: RNA-induced silencing complexes (RISCs) play central roles in posttranscriptional gene silencing. In plants, the mechanism of RISC assembly has remained elusive due to the lack of cell-free systems that recapitulate the process. In this report, we demonstrate that plant AGO1 protein synthesized by in vitro translation using an extract of evacuolated tobacco protoplasts incorporates synthetic small interfering RNA (siRNA) and microRNA (miRNA) duplexes to form RISCs that sequester the single-stranded siRNA guide strand and miRNA strand, respectively. The formed RISCs were able to recognize and cleave the complementary target RNAs. In this system, the siRNA duplex was incorporated into HSP90-bound AGO1, and subsequent removal of the passenger strand was triggered by ATP hydrolysis by HSP90. Removal of the siRNA passenger strand required the ribonuclease activity of AGO1, while that of the miRNA star strand did not. Based on these results, the mechanism of plant RISC formation is discussed.
authors.affiliation: Division of Plant Sciences, National Institute of Agrobiological Sciences (NIAS), 2-1-2 Kannondai Tsukuba, Ibaraki 305-8602, Japan.
authors.forename: Taichiro
authors.initials: T
authors.lastname: Iki
authors.forename: Manabu
authors.initials: M
authors.lastname: Yoshikawa
authors.forename: Masaki
authors.initials: M
authors.lastname: Nishikiori
authors.forename: Mauren C
authors.initials: MC
authors.lastname: Jaudal
authors.forename: Eiko
authors.initials: E
authors.lastname: Matsumoto-Yokoyama
authors.forename: Ichiro
authors.initials: I
authors.lastname: Mitsuhara
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Meshi
authors.forename: Masayuki
authors.initials: M
authors.lastname: Ishikawa
mesh_terms: D001483:Base Sequence; D002474:Cell-Free System / Q000378:metabolism; D039642:Eukaryotic Initiation Factors / Q000235:genetics / Q000378:metabolism*; D020868:Gene Silencing / Q000502:physiology; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011523:Protoplasts / Q000166:cytology / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000166:cytology / Q000235:genetics / Q000378:metabolism*
chemical_list: D039642:Eukaryotic Initiation Factors; D018841:HSP90 Heat-Shock Proteins; D035683:MicroRNAs; D046912:Multiprotein Complexes; D010940:Plant Proteins; D018749:RNA, Plant; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex