Department of Chemistry and Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, United States of America.
PLoS One. 2012;7(12):e51829. doi: 10.1371/journal.pone.0051829. Epub 2012 Dec 13.
One of the most exciting recent developments in RNA biology has been the discovery of small non-coding RNAs that affect gene expression through the RNA interference (RNAi) mechanism. Two major classes of RNAs involved in RNAi are small interfering RNA (siRNA) and microRNA (miRNA). Dicer, an RNase III enzyme, plays a central role in the RNAi pathway by cleaving precursors of both of these classes of RNAs to form mature siRNAs and miRNAs, which are then loaded into the RNA-induced silencing complex (RISC). miRNA and siRNA precursors are quite structurally distinct; miRNA precursors are short, imperfect hairpins while siRNA precursors are long, perfect duplexes. Nonetheless, Dicer is able to process both. Dicer, like the majority of RNase III enzymes, contains a dsRNA binding domain (dsRBD), but the data are sparse on the exact role this domain plays in the mechanism of Dicer binding and cleavage. To further explore the role of human Dicer-dsRBD in the RNAi pathway, we determined its binding affinity to various RNAs modeling both miRNA and siRNA precursors. Our study shows that Dicer-dsRBD is an avid binder of dsRNA, but its binding is only minimally influenced by a single-stranded - double-stranded junction caused by large terminal loops observed in miRNA precursors. Thus, the Dicer-dsRBD contributes directly to substrate binding but not to the mechanism of differentiating between pre-miRNA and pre-siRNA. In addition, NMR spin relaxation and MD simulations provide an overview of the role that dynamics contribute to the binding mechanism. We compare this current study with our previous studies of the dsRBDs from Drosha and DGCR8 to give a dynamic profile of dsRBDs in their apo-state and a mechanistic view of dsRNA binding by dsRBDs in general.
RNA 生物学领域最近令人兴奋的进展之一是发现了通过 RNA 干扰 (RNAi) 机制影响基因表达的小非编码 RNA。参与 RNAi 的两种主要 RNA 类别是小干扰 RNA (siRNA) 和 microRNA (miRNA)。Dicer 是一种 RNase III 酶，通过切割这两种 RNA 前体形成成熟的 siRNA 和 miRNA，然后将其装载到 RNA 诱导的沉默复合物 (RISC) 中，在 RNAi 途径中发挥核心作用。miRNA 和 siRNA 前体在结构上有很大的不同；miRNA 前体是短的、不完美的发夹，而 siRNA 前体是长的、完美的双链。尽管如此，Dicer 还是能够处理两者。Dicer 与大多数 RNase III 酶一样，含有双链 RNA 结合域 (dsRBD)，但关于该结构域在 Dicer 结合和切割机制中的具体作用的数据很少。为了进一步探索人 Dicer-dsRBD 在 RNAi 途径中的作用，我们确定了其对各种 RNA 的结合亲和力，这些 RNA 模拟 miRNA 和 siRNA 前体。我们的研究表明，Dicer-dsRBD 是 dsRNA 的强烈结合物，但它的结合仅受由 miRNA 前体中观察到的大末端环引起的单链-双链连接的微小影响。因此，Dicer-dsRBD 直接有助于底物结合，但不能区分 pre-miRNA 和 pre-siRNA 的机制。此外，NMR 自旋弛豫和 MD 模拟提供了对动态对结合机制贡献的概述。我们将本研究与我们之前对 Drosha 和 DGCR8 的 dsRBD 的研究进行了比较，以给出 dsRBD 在apo 状态下的动态概况以及 dsRBD 一般结合 dsRNA 的机制观点。
