Department of Chemistry, The Pennsylvania State University, 104 Chemistry Building, University Park, Pennsylvania 16802, United States.
Biochemistry. 2010 Dec 21;49(50):10728-36. doi: 10.1021/bi1015716. Epub 2010 Nov 22.
MicroRNAs (miRNAs) affect gene regulation by base pairing with mRNA and contribute to the control of cellular homeostasis. The first step in miRNA maturation is conducted in the nucleus by the "microprocessor" complex made up of an RNase III enzyme, Drosha, that contains one dsRNA binding domain (dsRBD), and DGCR8, that contains two dsRBDs in tandem. The crystal structure of DGCR8-Core (493-720), containing both dsRBDs, and the NMR solution structure of Drosha-dsRBD (1259-1337) have been reported, but the solution dynamics have not been explored for any of these dsRBDs. To better define the mechanism of dsRNA binding and thus the nuclear maturation step of miRNA processing, we report NMR spin relaxation and MD simulations of Drosha-dsRBD (1259-1337) and DGCR8-dsRBD1 (505-583). The study was motivated by electrophoretic mobility shift assays (EMSAs) of the two dsRBDs, which showed that Drosha-dsRBD does not bind a representative miRNA but isolated DGCR8-dsRBD1 does (K(d) = 9.4 ± 0.4 μM). Our results show that loop 2 in both dsRBDs is highly dynamic but the pattern of the correlations observed in MD is different for the two proteins. Additionally, the extended loop 1 of Drosha-dsRBD is more flexible than the corresponding loop in DGCR8-dsRBD1 but shows no correlation with loop 2, which potentially explains the lack of dsRNA binding by Drosha-dsRBD in the absence of the RNase III domains. The results presented in this study provide key structural and dynamic features of dsRBDs that contribute to the binding mechanism of these domains to dsRNA.
微小 RNA（miRNA）通过与 mRNA 碱基配对来影响基因调控，并有助于控制细胞内稳态。miRNA 成熟的第一步是在细胞核中由“微处理器”复合物完成的，该复合物由一种 RNA 酶 III 酶 Drosha 组成，Drosha 含有一个 dsRNA 结合域（dsRBD），以及 DGCR8，它含有两个串联的 dsRBD。DGCR8-Core（493-720）的晶体结构和 Drosha-dsRBD（1259-1337）的 NMR 溶液结构已经被报道，但这些 dsRBD 的溶液动力学尚未被探索。为了更好地定义 dsRNA 结合的机制，从而更好地定义 miRNA 加工的核成熟步骤，我们报告了 Drosha-dsRBD（1259-1337）和 DGCR8-dsRBD1（505-583）的 NMR 自旋弛豫和 MD 模拟。这项研究的动机是对这两个 dsRBD 的电泳迁移率变动分析（EMSA），结果表明 Drosha-dsRBD 不结合代表性的 miRNA，但分离的 DGCR8-dsRBD1 结合（K(d) = 9.4 ± 0.4 μM）。我们的结果表明，两个 dsRBD 中的环 2 高度动态，但在 MD 中观察到的相关性模式对于两种蛋白质是不同的。此外，Drosha-dsRBD 的扩展环 1 比 DGCR8-dsRBD1 中的相应环更灵活，但与环 2 没有相关性，这可能解释了 Drosha-dsRBD 在没有 RNA 酶 III 结构域的情况下缺乏 dsRNA 结合的原因。本研究中提出的结果提供了 dsRBD 的关键结构和动态特征，这些特征有助于这些结构域与 dsRNA 的结合机制。
