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.
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We thank D. Bartel, C. Cepko, D. Sabatini, P. Sharp, D. Trono, T. Tuschl, and F. Zhang for plasmids; A. Guzman and R. Bruce in the McDermott Center Next Generation Sequencing Core; A. Mobley and the University of Texas Southwestern Flow Cytometry Core; H. Ball and the University of Texas Southwestern Protein Chemistry Technology Core; S. Johnson for assistance with software implementation; J. Cabrera for assistance with figure preparation; and K. O’Donnell for advice on the manuscript. This work was supported by grants from the Cancer Prevention and Research Institute of Texas (CPRIT) (R1008 and RP160249 to J.T.M., RP101251 to Y.X., RP120718 to Z.J.C., and RR150033 to V.S.T.) and the National Institutes of Health (R01CA120185 and R35CA197311 to J.T.M., R01CA152301 to Y.X., and R00DK099254 to V.S.T.). T.L. is supported by a fellowship from Cancer Research Institute. F.K. is supported by the Leopoldina Fellowship Program (LPDS 2014-12) from the German National Academy of Sciences Leopoldina. J.T.M. and V.S.T. are CPRIT Scholars in Cancer Research. J.T.M. and Z.J.C. are Investigators of the Howard Hughes Medical Institute.
Extended data figures
This file contains the simulated sgRNA enrichment in top 0.5% brightest cells.
This file contains the RIGER analysis of CRISPR-Cas9 screen in HCT116EGFP-miR19 cells.
This file contains the RIGER analysis of CRISPR-Cas9 screen in HCT116EGFP cells.
This file contains the RNA-seq results showing genes with altered expression in AGO2−/−, ANKRD52−/−, and CSNK1A1−/−; ANKRD52−/− HCT116 cells compared to wild-type HCT116 cells.
This file contains the RIGER analysis of CRISPR-Cas9 suppressor screen in ANKRD52−/− HCT116EGFP-miR19 cells.
This file contains the RNA-seq results showing genes with altered expression in AGO2−/− cells and AGO2−/− cells reconstituted with FH-AGO2WT or FH-AGO25XA compared to wild-type HCT116 cells.
This file contains the eCLIP results.
This file contains the oligonucleotides and peptides used in this study.
About this article
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