A unifying model for microRNA-guided silencing of messenger RNAs

Silencing by the miRNA-guided RNA induced silencing complex (miRISC) is dependent on Ago2-chaperoned base pairing between the miRNA 5′ seed (5′S) and a complementary sequence in the 3′ untranslated region of an mRNA. Prevailing mechanistic understanding posits that initial 5′S pairing can further allow functional base pair expansion into the 3′ non-seed (3′NS), while functionally distinct non-canonical pairing was reported between only the 3′NS and the mRNA coding sequence. We developed single-molecule kinetics through equilibrium Poisson sampling (SiMKEPS) to measure highly precise binding and dissociation rate constants of varying-length target sequences to 5′S and 3′NS in a paradigmatic miRISC isolated from human cells, revealing distinct stable states of miRISC with mutually exclusive 5′S and 3′NS pairing. Our data suggest conformational rearrangements of the Ago2-bound miRNA that regulate alternative 5′S- and 3′NS-driven target recognition. The resulting model reconciles previously disparate observations and deepens our acumen for successfully marshaling RNA silencing therapies.