Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells
Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells
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DOI：
10.1073/pnas.152327299
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发表时间：
2002-07-23
影响因子：
11.1
通讯作者：
Bishop, JM
中科院分区：
文献类型：
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作者：
Yang, D;Buchholz, F;Bishop, JM
小干扰RNA(SiRNA)已成为选择性沉默培养哺乳动物细胞基因表达的有力工具。由于同一基因的不同siRNA具有不同的沉默能力，因此RNA干扰合成的siRNA效率低且成本高，特别是在功能基因组研究中。在这里，我们报告了使用大肠杆菌RNaseIII将双链RNA(DsRNA)切割成内切核酸酶准备的siRNA(EsiRNA)，该siRNA可以靶向RNA中的多个位点。EsiRNA概括了长dsRNA对果蝇S2细胞的有效和特异性抑制。与长dsRNA不同，esiRNA在培养的哺乳动物细胞中介导有效的RNA干扰，而没有明显的非特异性作用。我们发现，在哺乳动物细胞中，esiRNA的序列特异性干扰和长dsRNA激活的非特异性干扰素反应是独立的途径。EsiRNA的工作原理是引起其同源mRNA的破坏。由于它的简单性和有效性，这种方法对于哺乳动物基因功能的分析是有用的。
Small interfering RNA (siRNA) has become a powerful tool for selectively silencing gene expression in cultured mammalian cells. Because different siRNAs of the same gene have variable silencing capacities, RNA interference with synthetic siRNA is inefficient and cost intensive, especially for functional genomic studies. Here we report the use of Escherichia coli RNase III to cleave double-stranded RNA (dsRNA) into endoribonuclease-prepared siRNA (esiRNA) that can target multiple sites within an mRNA. esiRNA recapitulates the potent and specific inhibition by long dsRNA in Drosophila S2 cells. In contrast to long dsRNA, esiRNA mediates effective RNA interference without apparent nonspecific effect in cultured mammalian cells. We found that sequence-specific interference by esiRNA and the nonspecific IFN response activated by long dsRNA are independent pathways in mammalian cells. esiRNA works by eliciting the destruction of its cognate mRNA. Because of its simplicity and potency, this approach is useful for analysis of mammalian gene functions.