The guide RNA sequence dictates the slicing kinetics and conformational dynamics of the Argonaute silencing complex

Abstract

SUMMARYThe RNA-induced silencing complex (RISC), which powers RNA interference (RNAi), consists of a guide RNA and an Argonaute protein that slices target RNAs complementary to the guide. We find that for different guide-RNA sequences, slicing rates of perfectly paired, bound targets can be surprisingly different (>250-fold range), and that faster slicing confers greater knockdown efficacy in cells. Four sequence determinants underlie much of this variation in slicing rates. Analyses of these determinants implicate a structural distortion at guide nucleotides 6–7 in promoting slicing, and indicate that, in contrast to the prevailing model, slicing is primarily limited by the rate at which the enzyme−substrate complex reaches a slicing-competent conformation. Moreover, different guide sequences have an unanticipated, 600-fold range in 3′-mismatch tolerance, which is attributable to the GC-content of their central regions. Together, our analyses identify sequence determinants of RISC activity and provide biochemical, conformational, and functional rationale for their action.