Human Argonaute 2 Has Diverse Reaction Pathways on Target RNAs

Argonaute is a key enzyme of various RNA silencing pathways. We use single-molecule fluorescence measurements to characterize the reaction mechanisms of the core-RISC (RNA-induced silencing complex) composed of human Argonaute 2 and a small RNA. We found that target binding of core-RISC starts at the seed region, resulting in four distinct reaction pathways: target cleavage, transient binding, stable binding, and Argonaute unloading. The target cleavage requires extensive sequence complementarity and dramatically accelerates core-RISC recycling. The stable binding of core-RISC is efficiently established with the seed match only, providing a potential explanation for the seed-match rule of miRNA (microRNA) target selection. Target cleavage on perfect-match targets sensitively depends on RNA sequences, providing an insight into designing more efficient siRNAs (small interfering RNAs). [Display omitted] •Reaction pathways of core-RISC and their kinetic parameters are characterized•Argonaute dramatically accelerates core-RISC recycling after target cleavage•Seed-match is sufficient for stable core-RISC binding on target RNAs•Target cleavage efficiency of core-RISC is sensitive to RNA sequences By using single-molecule fluorescence measurements, Jo et al. show that target binding of core-RISC starts at the seed region, resulting in four distinct reaction pathways: target cleavage, transient binding, stable binding, and Argonaute unloading. Especially, the stable binding of core-RISC provides a potential explanation for the seed-match rule of miRNA.