The dsRNA-binding protein DRB4 interacts with the Dicer-like protein DCL4 in vivo and functions in the trans-acting siRNA pathway
The dsRNA-binding protein DRB4 interacts with the Dicer-like protein DCL4 in vivo and functions in the trans-acting siRNA pathway
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DOI：
10.1007/s11103-006-9125-8
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发表时间：
2007-04-01
影响因子：
5.1
通讯作者：
Fukuhara, Toshiyuki
中科院分区：
文献类型：
--
作者：
Nakazawa, Yukihiro;Hiraguri, Akihiro;Fukuhara, Toshiyuki
拟南芥编码4个Dicer样蛋白（DCL）和5个dsRNA结合蛋白（DRB）。我们以前已经证明，DCL 4特异性地与DRB 4在体外相互作用。在这里，我们描述了DCL 4和DRB 4在体内的相互作用。DCL 4缺陷突变体（dcl 4 -2）的表型与DRB 4缺陷突变体（drb 4 -1）的表型相似：两种突变体植物都具有细长且向下卷曲的莲座叶和过量积累的花色苷。在用抗DCL 4或抗DRB 4抗体和野生型拟南芥植物粗提物的免疫沉淀实验中，检测到DCL 4和DRB 4的共免疫沉淀，表明DCL 4与DRB 4在体内相互作用。这种相互作用通过使用来自dcl 4 -2、drb 4 -1或表达血凝素标记形式的DCL 4或DRB 4的转基因植物的提取物的免疫沉淀实验来证实。免疫沉淀实验的结果还表明，大多数DCL 4与DRB 4相关，但一些DRB 4是游离的或与其他蛋白质相关。在drb 4 -1和dcl 4 -2突变体中检测到TAS 1和TAS 3反式作用siRNA（ta-siRNA）的积累减少以及其靶mRNA（At 5g 18040和生长素应答因子ARF 3和ARF 4）的过度积累。这些结果表明DRB 4与DCL 4一起在ta-siRNA生物发生中起作用。
Arabidopsis thaliana encodes four Dicer-like (DCL) proteins and five dsRNA-binding (DRB) proteins. We have previously demonstrated that DCL4 specifically interacts with DRB4 in vitro. Here we describe the interaction between DCL4 and DRB4 in vivo. The phenotype of a mutant with a defect in DCL4 (dcl4-2) was similar to that of a mutant with a defect in DRB4 (drb4-1): both mutant plants had elongated and downwardly curled rosette leaves and over-accumulated anthocyanin. In immunoprecipitation experiments with either anti-DCL4 or anti-DRB4 antibody and crude extracts of wild-type Arabidopsis plants, co-immunoprecipitation of DCL4 and DRB4 was detected, indicating that DCL4 interacts with DRB4 in vivo. This interaction was confirmed by immunoprecipitation experiments using extracts from dcl4-2, drb4-1, or transgenic plants expressing the hemagglutinin-tagged version of DCL4 or DRB4. The results of immunoprecipitation experiments also suggest that most DCL4 is associated with DRB4, but that some DRB4 is free or associated with other proteins. Reduced accumulation of the TAS1 and TAS3 trans-acting siRNA (ta-siRNA) and over accumulation of their target mRNAs (At5g18040 and auxin response factors ARF3 and ARF4) were detected in both drb4-1 and dcl4-2 mutants. These results indicate that DRB4, together with DCL4, functions in the ta-siRNA biogenesis.