An Argonaute phosphorylation cycle promotes microRNA-mediated silencing

Ryan J. Golden, Beibei Chen, Tuo Li, Juliane Braun, Hema Manjunath, Xiang Chen, Jiaxi Wu, Vanessa Schmid, Tsung Cheng Chang, Florian Kopp, Andres Ramirez-Martinez, Vincent S. Tagliabracci, Zhijian J. Chen, Yang Xie, Joshua T. Mendell

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)197-202
Number of pages6
JournalNature
Volume542
Issue number7640
DOIs
StatePublished - Feb 9 2017
Externally publishedYes

ASJC Scopus subject areas

  • Medicine(all)
  • General

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