Nature. 2017 Jan 23. doi: 10.1038/nature21025. [Epub ahead of print]

An Argonaute phosphorylation cycle promotes microRNA-mediated silencing.

Golden RJ^1,², Chen B^3,⁴, Li T¹, Braun J¹, Manjunath H¹, Chen X¹, Wu J⁵, Schmid V⁶, Chang TC¹, Kopp F¹, Ramirez-Martinez A¹, Tagliabracci VS¹, Chen ZJ^1,⁷, Xie Y^3,^4,⁸, Mendell JT^1,^7,^8,⁹.

Author information

¹Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
²Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
³Quantitative Biomedical Research Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
⁴Department of Clinical Sciences, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
⁵Department of Microbiology and Immunology, University of California San Francisco, San Francisco, California 94143, USA.
⁶Eugene McDermott Center for Human Growth &Development, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
⁷Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
⁸Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
⁹Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

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.