Roth Braden M, Hennig Mirko
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, 70 President St, DD213, PO Box 250509, South Carolina 29425, USA.
Biomol NMR Assign. 2013 Oct;7(2):183-6. doi: 10.1007/s12104-012-9406-x. Epub 2012 Jul 3.
Double-stranded RNA binding domain (dsRBD) containing proteins are critical components of the microRNA (miRNA) pathway, with key roles in small RNA biogenesis, modification, and regulation. DiGeorge Critical Region 8 (DGCR8) is a 773 amino acid, dsRBD-containing protein that was originally identified in humans as a protein encoded in the region of chromosome 22 that is deleted in patients with DiGeorge syndrome. Now, it is realized that DGCR8 complements the nuclear RNase III Drosha to initiate miRNA biogenesis by promoting efficient recognition and cleavage of primary miRNAs (pri-miRNA). A pair of C-terminal tandem dsRBDs separated by a flexible linker are required for pri-miRNA substrate binding and recognition. The crystal structure of the DGCR8 core region comprising residues 493-720 revealed that each dsRBD adopts the canonical αβββα fold. However, several residues located in important flexible regions including the β1-β2-loop implicated in canonical dsRNA recognition are absent in the crystal structure and no RNA-bound structure of DGCR8 has been reported. Here we report the (1)H(N), (13)C, and (15)N backbone resonance assignments of the 24 kDa, 214 amino acid human DGCR8(core) (residues 493-706) by heteronuclear NMR spectroscopy. Our assignments lay the foundation for a detailed solution state characterization of the dynamical and RNA-binding properties of this protein in solution.
含双链RNA结合结构域（dsRBD）的蛋白质是微小RNA（miRNA）途径的关键组成部分，在小RNA生物合成、修饰和调控中起关键作用。狄乔治关键区域8（DGCR8）是一种含773个氨基酸的dsRBD蛋白，最初在人类中被鉴定为22号染色体区域编码的一种蛋白质，狄乔治综合征患者该区域会发生缺失。现在人们认识到，DGCR8与核核糖核酸酶III Drosha相互补充，通过促进对初级miRNA（pri-miRNA）的有效识别和切割来启动miRNA生物合成。pri-miRNA底物结合和识别需要一对由柔性接头隔开的C端串联dsRBD。包含493 - 720位残基的DGCR8核心区域的晶体结构表明，每个dsRBD都采用典型的αβββα折叠。然而，晶体结构中缺少位于包括参与典型dsRNA识别的β1-β2环在内的重要柔性区域的几个残基，并且尚未报道DGCR8的RNA结合结构。在此，我们通过异核核磁共振光谱报告了24 kDa、含214个氨基酸的人DGCR8（核心）（493 - 706位残基）的（1）H（N）、（13）C和（15）N主链共振归属。我们的归属为详细解析该蛋白质在溶液中的动力学和RNA结合特性奠定了基础。
