University of Regensburg, 93053 Regensburg, Germany.
RNA. 2011 Apr;17(4):737-49. doi: 10.1261/rna.2348111. Epub 2011 Mar 2.
Argonaute (Ago) proteins form the core of RNA-induced silencing complexes (RISCs) and mediate small RNA-guided gene silencing. In RNAi, short interfering RNAs (siRNAs) guide RISCs to complementary target RNAs, leading to cleavage by the endonuclease Ago2. Noncatalytic Ago proteins, however, contribute to RNAi as well but cannot cleave target RNA and often generate off-target effects. Here we show that synthetic siRNA duplexes interact with all Ago proteins, but a functional RISC rapidly assembles only around Ago2. By stabilizing the siRNA duplex, we show that the noncatalytic Ago proteins Ago1, -3, and -4 can be selectively blocked and do not form functional RISCs. In addition, stabilized siRNAs form an Ago2-RISC more efficiently, leading to increased silencing activity. Our data suggest novel parameters for the design of siRNAs with selective activation of the endonuclease Ago2.
Argonaute（AGO）蛋白构成 RNA 诱导沉默复合物（RISCs）的核心，并介导小 RNA 指导的基因沉默。在 RNAi 中，短干扰 RNA（siRNA）引导 RISCs 与互补的靶 RNA 结合，导致内切酶 Ago2 的切割。然而，非催化 AGO 蛋白也有助于 RNAi，但不能切割靶 RNA，并且经常产生脱靶效应。在这里，我们表明合成的 siRNA 双链与所有 AGO 蛋白相互作用，但只有功能性 RISC 才能迅速组装在 Ago2 周围。通过稳定 siRNA 双链，我们表明非催化的 AGO 蛋白 Ago1、-3 和 -4 可以被选择性阻断，并且不能形成功能性 RISCs。此外，稳定的 siRNA 更有效地形成 Ago2-RISC，从而导致沉默活性增加。我们的数据为设计具有选择性激活内切酶 Ago2 的 siRNA 提供了新的参数。
