Ramos A, Grünert S, Adams J, Micklem D R, Proctor M R, Freund S, Bycroft M, St Johnston D, Varani G
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH.
EMBO J. 2000 Mar 1;19(5):997-1009. doi: 10.1093/emboj/19.5.997.
The double-stranded RNA-binding domain (dsRBD) is a common RNA-binding motif found in many proteins involved in RNA maturation and localization. To determine how this domain recognizes RNA, we have studied the third dsRBD from Drosophila Staufen. The domain binds optimally to RNA stem-loops containing 12 uninterrupted base pairs, and we have identified the amino acids required for this interaction. By mutating these residues in a staufen transgene, we show that the RNA-binding activity of dsRBD3 is required in vivo for Staufen-dependent localization of bicoid and oskar mRNAs. Using high-resolution NMR, we have determined the structure of the complex between dsRBD3 and an RNA stem-loop. The dsRBD recognizes the shape of A-form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix alpha1 interacts with the single-stranded loop that caps the RNA helix. Interactions between helix alpha1 and single-stranded RNA may be important determinants of the specificity of dsRBD proteins.
双链RNA结合结构域（dsRBD）是一种常见的RNA结合基序，存在于许多参与RNA成熟和定位的蛋白质中。为了确定该结构域如何识别RNA，我们研究了果蝇Staufen的第三个dsRBD。该结构域与含有12个不间断碱基对的RNA茎环具有最佳结合，并且我们已经鉴定出这种相互作用所需的氨基酸。通过在staufen转基因中突变这些残基，我们表明dsRBD3的RNA结合活性在体内对于bicoid和oskar mRNA的Staufen依赖性定位是必需的。使用高分辨率核磁共振，我们已经确定了dsRBD3与RNA茎环之间复合物的结构。dsRBD通过环2内的保守残基与小沟之间以及环4与相邻大沟对面的磷酸二酯主链之间的相互作用来识别A型双链RNA的形状。此外，α1螺旋与封闭RNA螺旋的单链环相互作用。α1螺旋与单链RNA之间的相互作用可能是dsRBD蛋白特异性的重要决定因素。
