The Roles of Individual Mammalian Argonautes in RNA Interference In Vivo

Argonaute 2 (Ago2) is the only mammalian Ago protein capable of mRNA cleavage. It has been reported that the activity of the short interfering RNA targeting coding sequence (CDS), but not 3′ untranslated region (3′UTR) of an mRNA, is solely dependent on Ago2 in vitro. These studies utilized extremely high doses of siRNAs and overexpressed Ago proteins, as well as were directed at various highly expressed reporter transgenes. Here we report the effect of Ago2 in vivo on targeted knockdown of several endogenous genes by siRNAs, targeting both CDS and 3′UTR. We show that siRNAs targeting CDS lose their activity in the absence of Ago2, whereas both Ago1 and Ago3 proteins contribute to residual 3′UTR-targeted siRNA-mediated knockdown observed in the absence of Ago2 in mouse liver. Our results provide mechanistic insight into two components mediating RNAi under physiological conditions: mRNA cleavage dependent and independent. In addition our results contribute a novel consideration for designing most efficacious siRNA molecules with the preference given to 3′UTR targeting as to harness the activity of several Ago proteins.Argonaute 2（Ago2）是哺乳动物中唯一能够介导mRNA切割的Argonaute家族蛋白。已有研究报道，体外实验中靶向mRNA编码区（CDS）而非3'非翻译区（3′UTR）的小干扰RNA（siRNA）的活性，完全依赖于Ago2。但此类研究均使用了极高剂量的siRNA、过表达的Argonaute蛋白，且靶向的均为多种高表达的报告转基因。 本研究探讨了体内Ago2对siRNA靶向敲低多种内源基因的调控作用，所使用的siRNA分别靶向CDS与3′UTR。实验结果显示，靶向CDS的siRNA在Ago2缺失时会完全丧失活性；而在小鼠肝脏中，当Ago2缺失时，剩余的靶向3′UTR的siRNA介导的基因敲低效应，则由Ago1与Ago3蛋白共同参与介导。 本研究为生理条件下介导RNA干扰（RNAi）的两种核心途径提供了机制层面的解析：依赖mRNA切割的途径与不依赖该途径的途径。此外，本研究还为高效siRNA分子的设计提供了全新思路：优先选择靶向3′UTR的siRNA，以调动多种Argonaute蛋白的活性，从而获得最优的基因敲低效果。