Wang Bingbing, Li Shuqiang, Qi Hank H, Chowdhury Dipanjan, Shi Yang, Novina Carl D
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.
Nat Struct Mol Biol. 2009 Dec;16(12):1259-66. doi: 10.1038/nsmb.1712. Epub 2009 Nov 29.
Argonaute (AGO) proteins bind to small RNAs and mediate small RNA-induced silencing in eukaryotes. Using a minimal in vitro system, we show that bacterially expressed human AGO1 and AGO2 but not AGO3 and AGO4 possess strand-dissociating activity of microRNA (miRNA) duplexes. Both AGO1 and AGO2 function as RNA chaperones, capable of performing multiple rounds of strand dissociation. Unexpectedly, both AGO1 and AGO2 demonstrate passenger strand cleavage activity of a small interfering RNA (siRNA) duplex, but only AGO2 has target RNA cleavage activity. These observations indicate that passenger strand and mRNA endonuclease activities are mechanistically distinct. We further validate these observations in mammalian extracts and cultured mammalian cells, in which we demonstrate that AGO1 uses only miRNA duplexes when assembling translational repression-competent complexes, whereas AGO2 can use both miRNA and siRNA duplexes. We show that passenger strand cleavage and RNA chaperone activities that are intrinsic to both AGO1 and AGO2 are sufficient for RNA-induced silencing complex (RISC) loading.
在真核生物中，AGO蛋白（Argonaute）与小RNA结合并介导小RNA诱导的基因沉默。利用一个最小化的体外系统，我们发现细菌表达的人AGO1和AGO2具有微小RNA（miRNA）双链体的链解离活性，而AGO3和AGO4则没有。AGO1和AGO2均作为RNA伴侣发挥作用，能够进行多轮链解离。出乎意料的是，AGO1和AGO2均表现出小干扰RNA（siRNA）双链体的过客链切割活性，但只有AGO2具有靶RNA切割活性。这些观察结果表明，过客链和mRNA内切核酸酶活性在机制上是不同的。我们在哺乳动物提取物和培养的哺乳动物细胞中进一步验证了这些观察结果，在这些细胞中我们证明，AGO1在组装具有翻译抑制能力的复合物时仅使用miRNA双链体，而AGO2既可以使用miRNA双链体也可以使用siRNA双链体。我们表明，AGO1和AGO2所固有的过客链切割和RNA伴侣活性足以用于RNA诱导沉默复合体（RISC）的装载。
