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 and Joshua T. Mendell ()
Additional contact information
Ryan J. Golden: University of Texas Southwestern Medical Center
Beibei Chen: Quantitative Biomedical Research Center, University of Texas Southwestern Medical Center
Tuo Li: University of Texas Southwestern Medical Center
Juliane Braun: University of Texas Southwestern Medical Center
Hema Manjunath: University of Texas Southwestern Medical Center
Xiang Chen: University of Texas Southwestern Medical Center
Jiaxi Wu: University of California San Francisco
Vanessa Schmid: Eugene McDermott Center for Human Growth & Development, University of Texas Southwestern Medical Center
Tsung-Cheng Chang: University of Texas Southwestern Medical Center
Florian Kopp: University of Texas Southwestern Medical Center
Andres Ramirez-Martinez: University of Texas Southwestern Medical Center
Vincent S. Tagliabracci: University of Texas Southwestern Medical Center
Zhijian J. Chen: University of Texas Southwestern Medical Center
Yang Xie: Quantitative Biomedical Research Center, University of Texas Southwestern Medical Center
Joshua T. Mendell: University of Texas Southwestern Medical Center

Nature, 2017, vol. 542, issue 7640, 197-202

Abstract: 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.

Date: 2017
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DOI: 10.1038/nature21025

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