Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells

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.

小干扰RNA（Small interfering RNA，siRNA）现已成为体外培养哺乳动物细胞中选择性沉默基因表达的有力工具。由于同一基因的不同siRNA其沉默能力存在差异，使用合成siRNA进行RNA干扰的效率低下且成本高昂，尤其在功能基因组学研究中更是如此。本研究报道了利用大肠杆菌核糖核酸酶III（RNase III）将双链RNA（double-stranded RNA，dsRNA）切割为可靶向mRNA内多个位点的内切核糖核酸酶制备的小干扰RNA（endoribonuclease-prepared siRNA，esiRNA）。在果蝇S2细胞中，esiRNA可重现长链dsRNA所具备的高效且特异性抑制效应。与长链dsRNA不同，esiRNA可介导有效的RNA干扰，且在体外培养的哺乳动物细胞中无明显非特异性效应。我们发现，在哺乳动物细胞中，esiRNA介导的序列特异性干扰与长链dsRNA激活的非特异性干扰素（IFN）应答属于两条独立的信号通路。esiRNA通过诱导其同源mRNA的降解发挥作用。由于该方法操作简便且效果高效，可用于哺乳动物基因功能的相关分析。