Lan Lan, Xing Minli, Douglas Justin T, Gao Philip, Hanzlik Robert P, Xu Liang
Departments of Molecular Biosciences, The University of Kansas, Lawrence, KS, USA.
Bio-NMR Core Facility, The University of Kansas, Lawrence, KS, USA.
Oncotarget. 2017 Nov 20;8(63):106587-106597. doi: 10.18632/oncotarget.22540. eCollection 2017 Dec 5.
RNA-binding protein Musashi-2 (MSI2) is a key regulator in stem cells, it is over-expressed in a variety of cancers and its higher expression is associated with poor prognosis. Like Musashi-1, it contains two N-terminal RRMs (RNA-recognition Motifs, also called RBDs (RNA-binding Domains)), RRM1 and RRM2, which mediate the binding to their target mRNAs. Previous studies have obtained the three-dimensional structures of the RBDs of Musashi-1 and the RBD1:RNA complex. Here we show the binding of MSI2-RRM1 to a 15nt RNA in Fluorescence Polarization assay and time resolved Fluorescence Resonance Energy Transfer assay. Using nuclear magnetic resonance (NMR) spectroscopy we assigned the backbone resonances of MSI2-RRM1, and characterized the direct interaction of RRM1 to RNA r(GUAGU). Our NMR titration and structure modeling studies showed that MSI2-RRM1 and MSI1-RBD1 have similar RNA binding events and binding pockets. This work adds significant information to MSI2-RRM1 structure and RNA binding pocket, and contributes to the development of MSI2 specific and MSI1/MSI2 dual inhibitors.
RNA结合蛋白Musashi-2（MSI2）是干细胞中的关键调节因子，它在多种癌症中过度表达，其高表达与不良预后相关。与Musashi-1一样，它在N端含有两个RRMs（RNA识别基序，也称为RBDs（RNA结合结构域）），即RRM1和RRM2，它们介导与靶mRNA的结合。先前的研究已经获得了Musashi-1的RBDs以及RBD1:RNA复合物的三维结构。在这里，我们在荧光偏振分析和时间分辨荧光共振能量转移分析中展示了MSI2-RRM1与15nt RNA的结合。使用核磁共振（NMR）光谱，我们确定了MSI2-RRM1的主链共振，并表征了RRM1与RNA r(GUAGU)的直接相互作用。我们的NMR滴定和结构建模研究表明，MSI2-RRM1和MSI1-RBD1具有相似的RNA结合事件和结合口袋。这项工作为MSI2-RRM1结构和RNA结合口袋增添了重要信息，并有助于开发MSI2特异性和MSI1/MSI2双重抑制剂。
