Single-Molecule Analysis of the Target Cleavage Reaction by the Drosophila RNAi Enzyme Complex

Small interfering RNAs (siRNAs) direct cleavage of complementary target RNAs via an RNA-induced silencing complex (RISC) that contains Argonatute2 protein at its core. However, what happens after target cleavage remains unclear. Here we analyzed the cleavage reaction by Drosophila Argonaute2-RISC using single-molecule imaging and revealed a series of intermediate states in target recognition, cleavage, and product release. Our data suggest that, after cleavage, RISC generally releases the 50 cleavage fragment from the guide 30 supplementary region first and then the 30 fragment from the seed region, highlighting the reinforcement of the seed pairing in RISC. However, this order can be reversed by extreme stabilization of the 30 supplementary region or mismatches in the seed region. Therefore, the release order of the two cleavage fragments is influenced by the stability in each region, in contrast to the unidirectional base pairing propagation from the seed to the 30 supplementary region upon target recognition.

小干扰RNA（siRNAs）通过一种以Argonaute2蛋白为核心的RNA诱导沉默复合体（RISC）引导互补靶RNA的切割。然而，靶标切割之后发生了什么仍不清楚。在此我们利用单分子成像技术分析了果蝇Argonaute2 - RISC的切割反应，并揭示了在靶标识别、切割和产物释放过程中的一系列中间状态。我们的数据表明，切割之后，RISC通常首先从引导链3′辅助区域释放5′切割片段，然后从种子区域释放3′片段，这凸显了RISC中种子配对的加强。然而，3′辅助区域的极度稳定或种子区域的错配可使这种顺序逆转。因此，与靶标识别时从种子区域到3′辅助区域的单向碱基配对延伸相反，两个切割片段的释放顺序受各个区域稳定性的影响。