Specific requirement of DRB4, a dsRNA-binding protein, for the in vitro dsRNA-cleaving activity of Arabidopsis Dicer-like 4
Specific requirement of DRB4, a dsRNA-binding protein, for the in vitro dsRNA-cleaving activity of Arabidopsis Dicer-like 4
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DOI：
10.1261/rna.2455411
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发表时间：
2011-04-01
期刊：
影响因子：
4.5
通讯作者：
Fukuhara, Toshiyuki
中科院分区：
文献类型：
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作者：
Fukudome, Akihito;Kanaya, Akihiro;Fukuhara, Toshiyuki
拟南芥Dicer-like 4 （DCL4）通过内源性和外源性双链rna （dsRNAs）产生21-nt小干扰rna，并在体内和体外与dsrna结合蛋白DRB4相互作用。然而，DRB4在DCL4活性中的作用尚不清楚，因为DCL4的dsrna切割活性尚未得到生物化学表征。在这项研究中，我们对DCL4的Dicer活性进行了生化表征，并确定了DRB4是体外这种活性所必需的。拟南芥幼苗粗提物以DCL4/ drb4依赖的方式将长dsrna切割成21-nt小rna。免疫亲和纯化的DCL4复合物从长dsRNA中产生21-nt小rna，这些复合物具有与已知Dicer家族蛋白相似的生化特性。从DRB4 -1中纯化的DCL4复合物不切割dsRNA，并且在DRB4 -1复合物中添加重组DRB4特异性地恢复了21-nt小RNA的生成。这些结果表明，DCL4需要DRB4在体外将长dsRNA切割成21-nt小rna。DRB4的保守dsRNA结合域（dsrbd）的氨基酸替换会损害其与dsRNA的结合、与DCL4的相互作用以及促进DCL4活性。这些观察结果表明，dsrbd对DRB4功能至关重要。我们的生化方法和观察清楚地表明，DRB4是体外DCL4活性的特异性需要。
Arabidopsis thaliana Dicer-like 4 (DCL4) produces 21-nt small interfering RNAs from both endogenous and exogenous double-stranded RNAs (dsRNAs), and it interacts with DRB4, a dsRNA-binding protein, in vivo and in vitro. However, the role of DRB4 in DCL4 activity remains unclear because the dsRNA-cleaving activity of DCL4 has not been characterized biochemically. In this study, we biochemically characterize DCL4's Dicer activity and establish that DRB4 is required for this activity in vitro. Crude extracts from Arabidopsis seedlings cleave long dsRNAs into 21-nt small RNAs in a DCL4/DRB4-dependent manner. Immunoaffinity-purified DCL4 complexes produce 21-nt small RNAs from long dsRNA, and these complexes have biochemical properties similar to those of known Dicer family proteins. The DCL4 complexes purified from drb4-1 do not cleave dsRNA, and the addition of recombinant DRB4 to drb4-1 complexes specifically recovers the 21-nt small RNA generation. These results reveal that DCL4 requires DRB4 to cleave long dsRNA into 21-nt small RNAs in vitro. Amino acid substitutions in conserved dsRNA-binding domains (dsRBDs) of DRB4 impair three activities: binding to dsRNA, interacting with DCL4, and facilitating DCL4 activity. These observations indicate that the dsRBDs are critical for DRB4 function. Our biochemical approach and observations clearly show that DRB4 is specifically required for DCL4 activity in vitro.