In Vitro Assembly of Plant RNA-Induced Silencing Complexes Facilitated by Molecular Chaperone HSP90
In Vitro Assembly of Plant RNA-Induced Silencing Complexes Facilitated by Molecular Chaperone HSP90
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DOI：
10.1016/j.molcel.2010.05.014
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发表时间：
2010-07-30
期刊：
影响因子：
16
通讯作者：
Ishikawa, Masayuki
中科院分区：
文献类型：
--
作者：
Iki, Taichiro;Yoshikawa, Manabu;Ishikawa, Masayuki
RNA诱导的沉默复合物（RISC）在转录后基因沉默中起着重要作用。在植物中，RISC组装的机制仍然难以捉摸，因为缺乏无细胞系统来概括这一过程。在这份报告中，我们证明了植物AGO1蛋白合成的体外翻译，使用抽空烟草原生质体提取物纳入合成的小干扰RNA（siRNA）和microRNA（miRNA）双链体，形成RISC的螯合单链siRNA引导链和miRNA链，分别。形成的RISC能够识别和切割互补的靶RNA。在该系统中，siRNA双链体被掺入到HSP90结合的AGO1中，随后通过HSP90的ATP水解触发乘客链的去除。siRNA乘客链的去除需要AGO 1的核糖核酸酶活性，而miRNA星星链的去除则不需要。在此基础上，对植物RISC的形成机理进行了探讨。
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.