An Argonaute phosphorylation cycle promotes microRNA-mediated silencing

MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). Here we use clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity in human cells to identify new regulators of the miRNA pathway. By using iterative rounds of screening, we reveal a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824–S834), followed by rapid dephosphorylation by the ANKRD52–PPP6C phosphatase complex. Although genetic and biochemical studies demonstrate that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 reveals a pronounced expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per-target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing. The application of genome-wide CRISPR–Cas9 screening coupled with a fluorescent reporter to interrogate the microRNA pathway reveals that continual transient phosphorylation of Argonaute 2 is required to maintain the global efficiency of microRNA-mediated repression. Argonaute phosphorylation regulates microRNAs The small RNA regulators of gene expression, microRNAs (miRNAs), are loaded into an effector protein, Argonaute 2 (AGO2), before interacting with target messenger RNAs (mRNAs). Joshua Mendell and colleagues find that when this interaction occurs, CSNK1A1 phosphorylates several conserved sites on AGO2. This modification is quickly reversed through the activity of ANKRD52–PPP6C phosphatase. While phosphorylation of AGO2 inhibits the miRNA–mRNA interaction, a mutant AGO2 that cannot be phosphorylated also displays dysregulation of miRNA-dependent silencing. This finding is attributed to the loss of specificity of miRNA binding, suggesting that the role of the phosphorylation cycle is to enhance the preferential interaction of miRNAs with their target.