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

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

Taichiro Iki  1 , Manabu Yoshikawa, Masaki Nishikiori, Mauren C Jaudal, Eiko Matsumoto-Yokoyama, Ichiro Mitsuhara, Tetsuo Meshi, Masayuki Ishikawa
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Free article

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

Taichiro Iki et al. Mol Cell. .
Free article

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.

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Comment in

  • Chaperones get RISC loaded.
    Landthaler M. Landthaler M. Mol Cell. 2010 Jul 30;39(2):161-2. doi: 10.1016/j.molcel.2010.07.009. Mol Cell. 2010. PMID: 20670883 No abstract available.

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