title: An Argonaute phosphorylation cycle promotes microRNA-mediated silencing.
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.
authors.affiliation: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.|Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Golden
authors.affiliation: Quantitative Biomedical Research Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.|Department of Clinical Sciences, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Beibei
authors.initials: B
authors.lastname: Chen
authors.affiliation: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Tuo
authors.initials: T
authors.lastname: Li
authors.affiliation: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Juliane
authors.initials: J
authors.lastname: Braun
authors.affiliation: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Hema
authors.initials: H
authors.lastname: Manjunath
authors.affiliation: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Xiang
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Microbiology and Immunology, University of California San Francisco, San Francisco, California 94143, USA.
authors.forename: Jiaxi
authors.initials: J
authors.lastname: Wu
authors.affiliation: Eugene McDermott Center for Human Growth &Development, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Schmid
authors.affiliation: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Tsung-Cheng
authors.initials: TC
authors.lastname: Chang
authors.affiliation: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Florian
authors.initials: F
authors.lastname: Kopp
authors.affiliation: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Andres
authors.initials: A
authors.lastname: Ramirez-Martinez
authors.affiliation: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Vincent S
authors.initials: VS
authors.lastname: Tagliabracci
authors.affiliation: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.|Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Zhijian J
authors.initials: ZJ
authors.lastname: Chen
authors.affiliation: Quantitative Biomedical Research Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.|Department of Clinical Sciences, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.|Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Xie
authors.affiliation: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.|Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.|Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.|Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Joshua T
authors.initials: JT
authors.lastname: Mendell
mesh_terms: D000595:Amino Acid Sequence; D060565:Argonaute Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D064113:CRISPR-Cas Systems / Q000235:genetics; D047390:Casein Kinase II / Q000378:metabolism; D020868:Gene Silencing; D045325:HCT116 Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D035683:MicroRNAs; D012333:RNA, Messenger; C570325:CSNK2A1 protein, human; D047390:Casein Kinase II; C000621177:ANKRD52 protein, human; D010749:Phosphoprotein Phosphatases; C106079:protein phosphatase 6