Coexpression of Argonaute-2 enhances RNA interference toward perfect match binding sites
Coexpression of Argonaute-2 enhances RNA interference toward perfect match binding sites
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DOI：
10.1073/pnas.0800803105
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发表时间：
2008-07-08
影响因子：
11.1
通讯作者：
Habert, Daniel A.
中科院分区：
文献类型：
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作者：
Diederichs, Sven;Jung, Stephanie;Habert, Daniel A.
RNAi被广泛应用于抑制特定基因的表达，但它受到经验设计的siRNA或shRNA构建体的可变效率和特异性的限制。这使得针对单个基因的研究变得复杂，并显着损害了使用全基因组敲低文库的大规模筛选。在这里，我们表明RISC切割器Argonaute-2（Ago 2，eIF 2C 2）的异位表达显著增强了特异性针对具有完全匹配结合位点的mRNA靶点的RNAi。这种作用取决于其内切酶活性，并且与其对microRNA表达的调节解偶联。为了模拟Ago 2与shRNA敲低共表达的应用，我们靶向了表现出对EGFR癌基因成瘾的肺癌细胞中的EGF受体（EGFR）。尽管多个经验设计的shRNA构建体在介导EGFR敲低和细胞杀伤方面表现出高度不同的效率，但Ago 2的共表达导致EGFR依赖性细胞中均匀且高度特异性的靶基因抑制和凋亡。因此，Ago 2与shRNA构建体或siRNA双链体的共递送提供了在实验和潜在治疗环境中增强RNAi的功效和特异性的策略。
RNAi is widely applied to inhibit expression of specific genes, but it is limited by variable efficiency and specificity of empirically designed siRNA or shRNA constructs. This complicates studies targeting individual genes and significantly impairs large-scale screens using genome-wide knockdown libraries. Here, we show that ectopic expression of the RISC slicer Argonaute-2 (Ago2, eIF2C2) dramatically enhances RNAi specifically for mRNA targets with perfectly matched binding sites. This effect depends on its endonuclease activity and is uncoupled from its regulation of microRNA expression. To model the application of Ago2 coexpression with shRNA knockdown, we targeted the EGF receptor (EGFR) in lung cancer cells exhibiting oncogene addiction to EGFR. Whereas multiple empirically designed shRNA constructs exhibited highly divergent efficiencies in mediating EGFR knockdown and cell killing, coexpression of Ago2 resulted in uniform and highly specific target gene suppression and apoptosis in EGFR-dependent cells. Codelivery of Ago2 with shRNA constructs or siRNA duplexes thus provides a strategy to enhance the efficacy and the specificity of RNAi in experimental and potentially therapeutic settings.