Regulating the regulator: Phosphorylation-mediated regulation of the RNAi effector protein Argonaute

Argonaute (Ago) proteins play a central role in post-transcriptional gene regulation through RNA interference (RNAi). In this process, Ago binds to a small interfering RNA (siRNA) or a microRNA (miRNA) and uses it as a guide to target messenger RNAs containing regions of complementarity and down-regulates production of their corresponding proteins. It was previously shown that the kinase CK1α phosphorylates a cluster of residues in the eukaryotic insertion (EI) of Ago, leading to the alleviation of miRNA-mediated repression through an undetermined mechanism. We show that binding of miRNA-loaded human Argonaute-2 (hAgo2) to target RNA with complementarity to the seed and 3’ supplemental regions of the miRNA primes the EI for hierarchical phosphorylation by CK1α. The added negative charges electrostatically promote target release, freeing hAgo2 to seek out additional targets once it is dephosphorylated. The high conservation of potential phosphosites in the EI suggests that such a regulatory strategy may be a shared mechanism for regulating miRNA-mediated repression