In vitro assembly of plant RNA-induced silencing complexes facilitated by molecular chaperone HSP90

Mol Cell. 2010 Jul 30;39(2):282-91. doi: 10.1016/j.molcel.2010.05.014. Epub 2010 Jun 3.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cell-Free System / metabolism
  • Eukaryotic Initiation Factors / genetics
  • Eukaryotic Initiation Factors / metabolism*
  • Gene Silencing / physiology
  • HSP90 Heat-Shock Proteins / genetics
  • HSP90 Heat-Shock Proteins / metabolism*
  • MicroRNAs / genetics
  • MicroRNAs / metabolism
  • Molecular Sequence Data
  • Multiprotein Complexes / genetics
  • Multiprotein Complexes / metabolism*
  • Plant Proteins / genetics
  • Plant Proteins / metabolism*
  • Protoplasts / cytology
  • Protoplasts / metabolism
  • RNA, Plant / genetics
  • RNA, Plant / metabolism
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • RNA-Induced Silencing Complex / genetics
  • RNA-Induced Silencing Complex / metabolism*
  • Tobacco / cytology
  • Tobacco / genetics
  • Tobacco / metabolism*

Substances

  • Eukaryotic Initiation Factors
  • HSP90 Heat-Shock Proteins
  • MicroRNAs
  • Multiprotein Complexes
  • Plant Proteins
  • RNA, Plant
  • RNA, Small Interfering
  • RNA-Induced Silencing Complex

Associated data

  • GENBANK/AB542739