Human Dicer Binds Short Single-strand and Double-strand RNA with High Affinity and Interacts with Different Regions of the Nucleic Acids
Human Dicer Binds Short Single-strand and Double-strand RNA with High Affinity and Interacts with Different Regions of the Nucleic Acids
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DOI：
10.1074/jbc.m803748200
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发表时间：
2009-01-23
影响因子：
4.8
通讯作者：
Crooke, Stanley T.
中科院分区：
文献类型：
--
作者：
Lima, Walt F.;Murray, Heather;Crooke, Stanley T.
Human Dicer 是 RNA 干扰途径的一个组成部分。 Dicer 将前微 RNA 和双链 RNA 分别加工成成熟微 RNA 和短干扰 RNA (siRNA)，并将加工后的产物转移到 RNA 诱导的沉默复合物中。为了更好地了解对 siRNA 链的结合、易位和选择性识别重要的因素，我们测定了人 Dicer 对加工产品 (siRNA) 和短单链 RNA (ssRNA) 的结合亲和力。 siRNA 和 ssRNA 竞争性抑制人 Dicer 活性，表明它们与酶的活性位点相互作用。与 27 核苷酸双链 RNA 底物相比，未修饰 siRNA 的解离常数 (K-d) 弱 5-11 倍。化学修饰的 siRNA 对 Dicer 的结合亲和力与底物相当。 siRNA 中的 3'-二核苷酸突出端影响人 Dicer 对 siRNA 的结合亲和力以及加载到 RNA 诱导的沉默复合物中的偏向链。 ssRNA 的 K-d 值比底物的 K-d 弱 3 至 40 倍。 ssRNA 3'-末端核苷酸的序列组成对 Dicer 结合表现出最大的影响。 Dicer 切割含有短 siRNA 样双链区域和相邻 ssRNA 区域中延伸的 3' 或 5' ssRNA 突出端的底物。值得注意的是，观察到的切割位点与从延伸的 3' ssRNA 末端进入底物的酶一致。这些数据表明 siRNA 和 ssRNA 主要与酶的 PAZ 结构域相互作用。最后，结合最紧密的 siRNA 也是更有效的基因表达抑制剂。
Human Dicer is an integral component of the RNA interference pathway. Dicer processes premicro-RNA and double-strand RNA to, respectively, mature micro-RNA and short interfering RNA (siRNA) and transfers the processed products to the RNA-induced silencing complex. To better understand the factors that are important for the binding, translocation, and selective recognition of the siRNA strands, we determined the binding affinities of human Dicer for processed products (siRNA) and short single-strand RNAs (ssRNA). siRNAs and ssRNAs competitively inhibited human Dicer activity, suggesting that they are interacting with the active site of the enzyme. The dissociation constants (K-d) for unmodified siRNAs were 5-11-fold weaker compared with a 27-nucleotide double-strand RNA substrate. Chemically modified siRNAs exhibited binding affinities for Dicer comparable with the substrate. 3'-Dinucleotide overhangs in the siRNA affected the binding affinity of human Dicer for the siRNA and biased strand loading into RNA-induced silencing complex. The K-d values for the ssRNAs ranged from 3- to 40-fold weaker than the K-d for the substrate. Sequence composition of the 3'-terminal nucleotides of the ssRNAs exhibited the greatest effect on Dicer binding. Dicer cleaved substrates containing short siRNA-like double-strand regions and extended 3' or 5' ssRNA overhangs in the adjacent ssRNA regions. Remarkably, cleavage sites were observed consistent with the enzyme entering the substrate from the extended 3' ssRNA terminus. These data suggest that the siRNAs and ssRNAs interact predominantly with the PAZ domain of the enzyme. Finally, the tightest binding siRNAs were also more potent inhibitors of gene expression.