Elucidating the role of microprocessor protein DGCR8 in bending RNA structures.
Elucidating the role of microprocessor protein DGCR8 in bending RNA structures.
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DOI：
10.1016/j.bpj.2020.10.038
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发表时间：
2020-11
影响因子：
3.4
通讯作者：
S. Pabit;Yen-Lin Chen;E. T. Usher;E. Cook;L. Pollack;S. Showalter
中科院分区：
文献类型：
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作者：
S. Pabit;Yen-Lin Chen;E. T. Usher;E. Cook;L. Pollack;S. Showalter
尽管RNA分子的构象动力学在microRNA（miRNA）加工中具有潜在的重要性，但蛋白质结合伴侣在促进必要的结构变化中的作用还没有得到很好的理解。在以前的工作中，我们和其他人已经证明，非双链体结构元件和构象的灵活性，他们支持的是必要的有效的RNA结合和切割的蛋白质与两个主要阶段的miRNA加工。然而，最近的研究表明，DGCR 8蛋白以相似的亲和力结合初级miRNA和双链体RNA。在这里，我们研究了DGCR 8蛋白的小重组构建体的RNA结合以及由此产生的RNA构象变化。该构建体DGCR 8核心包含两个双链RNA结合结构域（dsRBD）和C末端尾。为了评估由结合引起的构象变化，我们应用具有对比度变化的小角X射线散射来检测与DGCR 8核心复合的初级-miR-16-1的构象变化。该方法仅报告复合物内的RNA构象，并表明蛋白质在结合时使RNA弯曲。使用smFRET研究结合到核心的RNA双链体的构象的支持工作也显示出弯曲。总之，这些研究阐明了DGCR 8在miRNA加工的早期阶段与RNA相互作用的作用。
Although conformational dynamics of RNA molecules are potentially important in microRNA (miRNA) processing, the role of the protein binding partners in facilitating the requisite structural changes is not well understood. In previous work, we and others have demonstrated that nonduplex structural elements and the conformational flexibility they support are necessary for efficient RNA binding and cleavage by the proteins associated with the two major stages of miRNA processing. However, recent studies showed that the protein DGCR8 binds primary miRNA and duplex RNA with similar affinities. Here, we study RNA binding by a small recombinant construct of the DGCR8 protein and the RNA conformation changes that result. This construct, the DGCR8 core, contains two double-stranded RNA-binding domains (dsRBDs) and a C-terminal tail. To assess conformational changes resulting from binding, we applied small-angle x-ray scattering with contrast variation to detect conformational changes of primary-miR-16-1 in complex with the DGCR8 core. This method reports only on the RNA conformation within the complex and suggests that the protein bends the RNA upon binding. Supporting work using smFRET to study the conformation of RNA duplexes bound to the core also shows bending. Together, these studies elucidate the role of DGCR8 in interacting with RNA during the early stages of miRNA processing.