The biochemical basis for the cooperative action of microRNAs

In cells, closely spaced microRNA (miRNA) target sites within a messenger RNA (mRNA) can act cooperatively, leading to more repression of the target mRNA than expected by independent action at each site. Using purified miRNA-Argonaute (AGO2) complexes, synthetic target RNAs, and a purified domain of TNRC6B (GW182 in flies) that is able to simultaneously bind multiple AGO proteins, we examined both the occupancies and binding affinities of miRNA-AGO2 complexes and target RNAs with either one site or two cooperatively spaced sites. On their own, miRNA-AGO2 complexes displayed little if any cooperative binding to dual sites. In contrast, in the presence of the AGO-binding region of TNRC6B, we observed strong cooperative binding to dual sites, with almost no singly bound target RNAs and substantially increased binding affinities and Hill coefficients. Cooperative binding was retained when the two sites were for two different miRNAs or when the two sites were bound to miRNAs loaded into two different AGO paralogs, AGO1 and AGO2. The improved binding affinity was attributable primarily to a reduced rate of dissociation between miRNA-AGO complexes and their dual-site targets. Thus, the multivalent binding of TNRC6 enables cooperative binding of miRNA-AGO complexes to target RNAs, thereby explaining the basis of cooperative action.