Beyond the seed: structural basis for supplementary mircoRNA targeting

microRNAs (miRNA) guide Argonaute proteins to mRNAs targeted for repression. Target recognition occurs primarily through the miRNA seed region, composed of guide (g) nucleotides g2-g8. However, nucleotides beyond the seed are also important for some known miRNA-target interactions. Here, we report the structure of human Argonaute2 (Ago2) engaged with a target RNA recognized through both miRNA seed and (g13-g16) regions. Ago2 creates a supplementary that accommodates up to 5 miRNA-target base pairs. Seed and chambers are adjacent to each other, and can be bridged by an unstructured target loop of 1-15 nucleotides. Opening of the chamber may be constrained by tension in the miRNA 39 tail as increases in miRNA length stabilize interactions. Contrary to previous reports, we demonstrate optimal interactions can increase target affinity u003e20-fold. These results provide a mechanism for extended miRNA-targeting, suggest a function for 39 isomiRs in tuning miRNA targeting specificity, and indicate that interactions may contribute more to target recognition than is widely appreciated.