Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455, USA.
RNA. 2012 Nov;18(11):2083-92. doi: 10.1261/rna.033688.112. Epub 2012 Sep 14.
Dicer cleaves double-stranded RNAs (dsRNAs) or precursor microRNAs (pre-miRNAs) to yield ≈ 22-nt RNA duplexes. The pre-miRNA structure requirement for human Dicer activity is incompletely understood. By large-scale in vitro dicing assays and mutagenesis studies, we showed that human Dicer cleaves most, although not all, of the 161 tested human pre-miRNAs efficiently. The stable association of RNAs with Dicer, as examined by gel shift assays, appears important but is not sufficient for cleavage. Human Dicer tolerates remarkable structural variation in its pre-miRNA substrates, although the dsRNA feature in the stem region and the 2-nt 3'-overhang structure in a pre-miRNA contribute to its binding and cleavage by Dicer, and a large terminal loop further enhances pre-miRNA cleavage. Dicer binding protects the terminal loop from digestion by S1 nuclease, suggesting that Dicer interacts directly with the terminal loop region.
Dicer 酶将双链 RNA（dsRNAs）或前体 microRNA（pre-miRNAs）切割成大约 22 个核苷酸的 RNA 双链体。人类 Dicer 活性的前 miRNA 结构要求尚未完全理解。通过大规模的体外切割测定和突变研究，我们发现人类 Dicer 可以有效地切割大多数（尽管不是全部）测试的 161 个人类前 miRNA。通过凝胶迁移分析检测到的 RNA 与 Dicer 的稳定结合似乎很重要，但不足以进行切割。尽管茎区的 dsRNA 特征和 pre-miRNA 中的 2 个核苷酸 3' 突出结构有助于 Dicer 的结合和切割，但较大的末端环进一步增强了 pre-miRNA 的切割。Dicer 结合保护末端环免受 S1 核酸酶的消化，表明 Dicer 直接与末端环区域相互作用。
