2017-Golden-An Argonaute phosphorylation cycle.pdf

9 f e b r u a r y 2 0 1 7|V O L 5 4 2|N a T u r e|1 9 7arTicLedoi:10.1038/nature21025An Argonaute phosphorylation cycle promotes microRNA-mediated silencingryan J.Golden1,2,beibei chen3,4,Tuo Li1,Juliane braun1,Hema Manjunath1,Xiang chen1,Jiaxi Wu5,Vanessa Schmid6,Tsung-cheng chang1,florian Kopp1,andres ramirez-Martinez1,Vincent S.Tagliabracci1,Zhijian J.chen1,7,yang Xie3,4,8&Joshua T.Mendell1,7,8,9The miRNA pathway is essential for development and homeostasis in diverse species1,2.miRNAs associate with Argonaute(AGO)proteins,which they guide to partially complementary sites in messenger RNAs(mRNAs)3,leading to reduced stability and translation of targeted messages4.miRNAs select targets primarily through base pairing of their seed regions,nucleotides 27.Consequently,the potential target repertoire for a given miRNA is vast.Multiple sequence features of bona fide target sites distinguish them from non-functional sites with seed complementarity5.Nevertheless,recent experiments have demon-strated that the functional pool of targets greatly exceeds the quantity of miRNAs in mammalian cells6,7.While the intrinsic sequence char-acteristics of miRNA binding sites strongly influence the kinetics of AGO:target interactions8,9,it is currently unknown whether additional,active mechanisms exist that influence mRNA binding to facilitate nav-igation of the extensive target landscape.RNA interference(RNAi)screens have been used to dissect the miRNA pathway in invertebrates10,11.Analogous experiments in human cells,however,have been hindered by the fact that the RNAi and miRNA pathways share common molecular machinery.This lim-itation may be circumvented by recent advances in CRISPR-mediated genome editing,which offers a robust alternative for genetic loss-of-function screens in human cells12,13.Here we describe the application of CRISPRCas9 screening to identify novel regulators of miRNA-me-diated silencing.These experiments reveal that the ANKRD52PPP6C phosphatase complex performs a critical function in the miRNA path-way by dephosphorylating a set of highly conserved amino acids in AGO2.A secondary genome-wide screen revealed CSNK1A1 as the kinase that phosphorylates AGO2 on these sites.This AGO2 phospho-rylation cycle is triggered by target engagement and negatively regulates target association,yet is essential to maintain the global efficiency of miRNA-mediated silencing.Transcriptome-wide AGO2 binding studies show that S824S834 phosphorylation remodels the target pool bound by AGO2 at steady-state.These data reveal a previously unrecognized mechanism that regulates AGO:target interactions to promote miRNA-mediated repression.CRISPRCas9 screen for miRNA regulatorsTo apply CRISPRCas9 screening to interrogate the miRNA pathway,we first generated a cell line expressing a fluorescent reporter of miRNA activity.Enhanced green fluorescent protein(EGFP)transcripts with or without a 3 untranslated region(UTR)harbouring eight imper-fectly complementary binding sites for miR-19,an abundant miRNA,were expressed in the stably diploid cell line HCT116(ref.14)(Fig.1a).The miR-19 reporter line(HCT116EGFP-miR19)but not the control line lacking miR-19 sites(HCT116EGFP),exhibited robust de-repression of EGFP upon infection with a CRISPR lentivirus targeting the essential miRNA biogenesis factor DROSHA or after transfection with an anti-sense miR-19 inhibitor(Fig.1b).A genome-wide CRISPRCas9 screen was performed by infecting HCT116EGFP-miR19 and HCT116EGFP cells with a lentiviral library tar-geting over 19,000 human genes and 1,864 miRNAs12,15.After 14 days of growth,the brightest 0.5%of cells,representing those with defi-cient miRNA-mediated silencing,were collected(Fig.1a).Simulations demonstrated that collection of cells in this gate could theoretically yield 150-fold enrichment of highly effective single-guide RNAs(sgR-NAs)that target essential genes in the miRNA pathway while still allow-ing significant enrichment of partly effective guides that incompletely impair miRNA-mediated silencing(Supplementary Table 1).sgRNA 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(S824S834),followed by rapid dephosphorylation by the ANKRD52PPP6C 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.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.2017 Macmillan Publishers Limited,part of Springer Nature.All rights reserved.ArticlereSeArcH1 9 8|N a T u r e|V O L 5 4 2|9 f e b r u a r y 2 0 1 7representation in the sorted and unsorted cells was enumerated by high-throughput sequencing and the RNAi Gene Enrichment Ranking(RIGER)algorithm16 was used to identify genes targeted by multiple enriched sgRNAs,representing high-confidence hits(Supplementary Tables 2 and 3).A large number of established components of the miRNA pathway and miR-19 itself were identified as significant hits in HCT116EGFP-miR19 but not HCT116EGFP cells(Fig.1c,d),establishing the sensitivity of this approach.We noted two classes of highly ranked hits without a previously defined role in the miRNA pathway:transcriptional regulators(BRD4,CTNNB1,and POU2F1)and interacting components of the serine/threonine protein phosphatase 6(PPP6)complex(ANKRD52 and PPP6C)17.Loss of function of any of these genes measurably de-repressed EGFP in HCT116EGFP-miR19 but not HCT116EGFP cells(Fig.1e and Extended Data Fig.1).CTNNB1 and BRD4 promote MYC expression18,19,a known positive regulator of transcription of the miR-19 host transcript(pri-miR-17-92)20.Accordingly,analysis of MYC,pri-miR-17-92,and mature miR-19 levels in CTNNB1/,BRD4/,and POU2F1/cells provided evidence that CTNNB1 and BRD4 indirectly regulate transcription of miR-19 through MYC(Extended Data Fig.2).POU2F1,in contrast,promotes pri-miR-17-92 transcription through an MYC-independent mechanism that may include interaction with a binding site in the promoter,as proposed previously21.ANKRD52 and PPP6C dephosphorylate AGO2The identification of ANKRD52 and PPP6C as significant hits sug-gested that phosphorylation regulates the activity of an essential miRNA pathway component.Confirming a general impairment of miRNA-mediated silencing in cells deficient for this phosphatase com-plex,RNA sequencing(RNA-seq)demonstrated that genes upregu-lated in AGO2/cells were similarly upregulated in ANKRD52/cells(Fig.2a,Extended Data Fig.3 and Supplementary Table 4).Additional miRNA reporter constructs and endogenous let-7 targets2224 were also de-repressed by ANKRD52 knockout(Extended Data Fig.4ad).The steady-state abundance of representative miRNAs was not decreased(Extended Data Fig.4e),indicating that the ANKRD52PPP6C com-plex does not globally regulate miRNA biogenesis.Owing to their central role in miRNA-mediated silencing,we hypothesized that Argonaute proteins may be dephosphorylated by ANKRD52PPP6C.AGO2 and ANKRD52PPP6C interacted in an RNA-independent manner(Extended Data Fig.5a).Phos-tag electrophoresis,a sensitive method for detection of phosphorylated proteins25,revealed that AGO2 migrated as a doublet,with dramatic enhancement of the slowly migrating form in ANKRD52-or PPP6C-deficient cells(Fig.2b).Phosphatase treatment confirmed that the more slowly migrating species corresponded to phosphorylated AGO2(p-AGO2;Fig.2c).Deficiency of ANKRD52 or PPP6C in multiple human cell lines similarly led to accumulation of phospho-rylated AGO2(Extended Data Fig.5b).We also observed enhanced phosphorylation of AGO1 in ANKRD52/cells(Extended Data Fig.5c),suggesting broader regulation of Argonaute proteins by the ANKRD52PPP6C complex.Mass spectrometry was used to identify the relevant phosphores-idue(s)in endogenous AGO2.Enhanced phosphorylation within a region of the PIWI domain containing four highly conserved serine residues and a single poorly conserved threonine residue(S824S834)was detected in ANKRD52/cells,while the previously reported phosphorylation of S387 was not increased26(Fig.2d and Extended Data Fig.6a,b).Triply phosphorylated peptides spanning S824S834 were detected,with definitive detection of p-S824(Extended Data Fig.6c).Although close spacing prevented the assignment of addi-tional phosphorylations to specific residues,mass spectrometry using AGO2 alanine mutants allowed definitive identification of phospho-rylation at S828 and S831(Extended Data Fig.6d,e).Confirming these results,mutating all five serine/threonine residues in this region to alanine(5XA)completely abolished the Phos-tag p-AGO2 band(Fig.2e).Interestingly,a single S828A mutation also fully abolished the AGO2 mobility shift,suggesting that phosphorylation of this residue may be necessary to trigger hierarchical phosphorylation of additional amino acids within this region.Importantly,expression of AGO25XA or AGO2S828A,but not wild-type AGO2,rescued repression of the miR-19 EGFP reporter in ANKRD52/cells(Fig.2f),demon-strating that the defect in miRNA-mediated silencing caused by loss of ANKRD52 was specifically due to hyperphosphorylation of AGO2 at S824S834.CTNNB1DDX6BRD4AGO2DICER1DROSHAmiR-1306(DGCR8)miR-19b-1miR-92a-1ANKRD52DGCR8PPP6CPOU2F1XPO5XRN1012345605,00010,00015,00020,000GeneAGO2miR-1306(DGCR8)DICER1DROSHADDX6DGCR8XPO5ANKRD52BRD4CTNNB1miR-19b-1PPP6CPOU2F1XRN1miR-92a-1ANKRD52PPP6CPOU2F1CTNNB1DROSHA sgRNA EGFP sgRNA Target sgRNA1 Target sgRNA2 Control sgRNAControl sgRNAmiR-19 inhibitorUntransfectedGFP fuorescenceGFP fuorescenceParental HCT116BRD48 miR-19Brightest0.5%Identify enrichedsgRNAsNormalized cell countNormalized cell countHCT116EGFP-miR19HCT116EGFP sgRNA librarylog10(RIGER P value)012345605,00010,00015,00020,000Genelog10(RIGER P value)LTRsgRNACas9LTRGFPA(n)A(n)A(n)A(n)A(n)A(n)GFPGFPGFPA(n)GFPGFPGFPabcdeDROSHADICER1m7GAAAnAGO2DDX6DGCR8XRN1m7GAAAnXPO5Pri-miR-17-92Figure 1|A genome-wide CRISPRCas9 screen reveals known and novel regulators of the miRNA pathway.a,Design of CRISPRCas9 screen.b,Validation of reporter cell lines.EGFP fluorescence after introduction of lentiCRISPR vectors(top)or antisense miR-19 inhibitors(bottom).c,RIGER analysis of screening results in HCT116EGFP-miR19(top)or HCT116EGFP cells(bottom).Red dots,known components of the miRNA pathway;blue dots,putative novel regulators.d,Components of the miRNA pathway identified as significant hits.e,EGFP expression in HCT116EGFP-miR19 cells after transduction with lentiCRISPR vectors.2017 Macmillan Publishers Limited,part of Springer Nature.All rights reserved.Article reSeArcH9 f e b r u a r y 2 0 1 7|V O L 5 4 2|N a T u r e|1 9 9Phosphorylation inhibits target bindingAGO2 S824S834 is within a structurally unresolved loop of the PIWI domain that is located in the vicinity of the miRNA:target interface27,28.We therefore hypothesized that phosphorylation in this region may reduce miRNA and/or target association.Immunopurification of endogenous AGO2 from ANKRD52+/+or ANKRD52/cells demon-strated equivalent miRNA association(Fig.3a).In contrast,AGO2 target association was significantly reduced in ANKRD52/cells,as determined by assessing AGO2 binding to the miR-19 EGFP reporter transcript and two established targets of miR-16 or let-7(Fig.3b)22,23,29.To confirm these findings,AGO2:miRNA complexes were captured with an RNA oligonucleotide that mimics a target of miR-21,an abun-dant miRNA in HCT116 cells30,31(Fig.3c).Whereas unphosphorylated AGO2 was efficiently recovered using this approach,binding of phos-phorylated AGO2 to the synthetic target was dramatically decreased(Fig.3d).Importantly,both phosphorylated and unphosphorylated forms of AGO2 were efficiently recovered by immunoprecipitation using an anti-AGO2 antibody,demonstrating that the relevant phos-phoresidues were stable under these conditions.A series of phosphomimetic mutants were generated to identify the specific phosphoresidues that impair AGO2 target association(Extended Data Fig.7a).As expected,none of the mutations measurably decreased miRNA association(Extended Data Fig.7b).In contrast,target association was significantly impaired by mutation of all five serine and threonine residues in the S824S834 region to glutamic acid(5XE)and,more importantly,individual mutations or combinations of mutations that mimic definitive phosphorylation sites documented by mass spectrometry(S831E,S828E/S831E,and S824E/S828E/S831E)(Fig.3e).Notably,mutation of S828 or all serines and threonines in this region to alanine(S828A or 5XA,respec-tively)did not inhibit target interaction.Consistent with an isolated effect of S824S834 phosphorylation on target binding,tethering AGO2WT,AGO25XE,or AGO25XA to a luciferase transcript using the N peptide-boxB system32 resulted in equivalent repression(Fig.3f).Taken together,these findings establish that phosphorylation of S824S834 potently and specifically inhibits AGO2:target interactions.CSNK1A1 is the inhibitory AGO2 kinaseWe next sought to identify the kinase that initiates this inhib