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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 miRNA pathway is essential for development and homeostasis in diverse species1,2. miRNAs associate with Argonaute (AGO) proteins, which they guide to partially complementary sites in messenger RNAs (mRNAs)3, leading to reduced stability and translation of targeted messages4. miRNAs select targets primarily through base pairing of their seed regions, nucleotides 2-7. Consequently, the potential target repertoire for a given miRNA is vast. Multiple sequence features of bona fide target sites distinguish them from non-functional sites with seed complementarity5. Nevertheless, recent experiments have demonstrated that the functional pool of targets greatly exceeds the quantity of miRNAs in mammalian cells6,7. While the intrinsic sequence characteristics of miRNA binding sites strongly influence the kinetics of AGO:target interactions8,9, it is currently unknown whether additional, active mechanisms exist that influence mRNA binding to facilitate navigation of the extensive target landscape.
RNA interference (RNAi) screens have been used to dissect the miRNA pathway in invertebrates10,11. Analogous experiments in human cells, however, have been hindered by the fact that the RNAi and miRNA pathways share common molecular machinery. This limitation may be circumvented by recent advances in CRISPR-mediated genome editing, which offers a robust alternative for genetic loss-offunction...