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.

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Figure 1: A genome-wide CRISPR–Cas9 screen reveals known and novel regulators of the miRNA pathway.
Figure 2: Loss of ANKRD52–PPP6C activity globally impairs miRNA-mediated silencing and results in AGO2 hyperphosphorylation.
Figure 3: AGO2 phosphorylation impairs mRNA target association.
Figure 4: A CRISPR–Cas9 suppressor screen reveals CSNK1A1 as the inhibitory AGO2 kinase.
Figure 5: Argonaute S824–S834 phosphorylation is required for fully efficient miRNA-mediated silencing and is triggered by target binding.
Figure 6: Expansion of the target repertoire bound by AGO25XA.

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Acknowledgements

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.

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Authors

Contributions

R.J.G. performed most experiments and B.C. performed most bioinformatics analyses. T.L., X.C., J.W., and R.J.G. performed mass spectrometry analyses. J.B. and H.M. generated plasmid constructs, cell lines, and performed CLIP validation experiments. V.S. provided technical assistance for sequencing sgRNA libraries. T.-C.C. performed qPCR analyses. F.K. assisted with CLIP experiments. A.R.-M. generated plasmid constructs. B.C., Y.X., and J.T.M. performed bioinformatics analyses. V.S.T. provided guidance for the in vitro kinase assays. R.J.G., J.T.M., B.C., Y.X., Z.J.C., and T.L. designed most experiments. R.J.G and J.T.M. wrote the manuscript.

Corresponding author

Correspondence to Joshua T. Mendell.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks S. Tavazoie, W. Wei and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Knockout of candidate miRNA regulators in HCT116EGFP cells.

Flow cytometry analysis of EGFP in HCT116EGFP cells after transduction with lentiCRISPR vectors targeting the indicated genes.

Extended Data Figure 2 BRD4, CTNNB1, and POU2F1 positively regulate miR-19 expression.

a, Model depicting how each gene may promote expression of the miR-17-92 cluster. bd, Western blot analysis confirming loss of expression of the indicated gene in HCT116 knockout clones. Asterisk indicates non-specific band. For each protein, all lanes came from the same blot but irrelevant lanes were removed. eg, qRT–PCR assays demonstrating reduced expression of MYC (e), pri-miR-17-92 (f), or mature miR-19a/b (g) in BRD4−/−, CTNNB1−/−, or POU2F1−/− cells. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 3 Western blot analysis confirms loss of protein expression in AGO2 and ANKRD52 HCT116 clonal knockout lines.

For both AGO2 (a) and ANKRD52 (b) western blots, all lanes came from the same blot but irrelevant lanes were removed. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 4 General impairment of miRNA-mediated silencing in ANKRD52–/– cells.

a, b, Flow cytometry analysis of EGFP expression in HCT116 cells stably expressing reporters for miR-16 (a) or miR-200 (b) after transduction with lentiCRISPR vectors targeting ANKRD52 or expressing a non-targeting sgRNA. c, qRT–PCR showing de-repression of established let-7 targets (DICER1 or HMGA2) in AGO2–/– or ANKRD52–/– cells. *P < 0.05, **P < 0.01, two-tailed Student’s t-test comparing AGO2–/– or ANKRD52–/– to parental. (n = 3 biological replicates, each assayed in triplicate.) d, qRT–PCR analysis of DICER1 and HMGA2 in non-transfected (NT) HCT116EGFP-miR-19 cells or after transfection with miR-19 antisense oligonucleotides (Anti-miR-19) confirms that these transcripts are not regulated by miR-19. Upregulation of the EGFP miR-19 reporter transcript served as a positive control in this experiment. (n = 3 biological replicates, each assayed in triplicate.) e, qRT–PCR was performed for the indicated miRNAs and expression levels were normalized to U6 snRNA (n = 2 biological replicates, each assayed in triplicate).

Extended Data Figure 5 The ANKRD52–PPP6C complex interacts with and dephosphorylates AGO proteins.

a, Co-immunoprecipitation of Flag–HA-AGO2 (FH-AGO2) with V5-ANKRD52 or V5-PPP6C with or without RNase A treatment. b, Phos-tag electrophoresis demonstrating AGO2 hyperphosphorylation in multiple ANKRD52/PPP6C-deficient cell lines. c, Phos-tag western blot analysis of Flag–HA-AGO1 (FH-AGO1) stably expressed in ANKRD52+/+ and ANKRD52−/− HCT116 cells. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 6 Identification of multiple definitively phosphorylated residues in the S824–S834 region of AGO2 by mass spectrometry.

a, Full-scan mass spectra zoomed to the region for the AGO2 815–837 peptide. The unphosphorylated and multiply phosphosphorylated precursor ions are shown in red. Peak labels indicate the mass-to-charge ratios and the charge state. The singly charged ion with grey label (top panel) does not correspond to peptides 815–837. Data at two close elution time points are shown for ANKRD52−/− to illustrate the unphosphorylated (0P), singly (1P), doubly (2P), and triply (3P) phosphorylated peptides. b, Quantification of the indicated endogenous AGO2 phosphopeptides relative to unphosphorylated peptide as determined by mass spectrometry. Labels 1P, 2P, or 3P respectively denote singly, doubly, or triply phosphorylated peptides spanning residues 815–837 of AGO2. Superscript indicates peptide charge state. ND, not detected. c, MS/MS spectra demonstrating phosphorylation of endogenous AGO2 at S824 in ANKRD52−/− cells. Red bars denote site-determining ions. d, e, MS/MS spectra demonstrating phosphorylation of FH-AGO2 (T830A) at S824 and S828 (d) or phosphorylation of FH-AGO2 (S824A/T830A) at S828 and S831(e) in ANKRD52−/− cells.

Extended Data Figure 7 Phosphomimetic mutants of FH-AGO2 do not exhibit reduced miRNA association.

a, Western blots showing expression of the indicated FH-AGO2 mutants. Within each panel (top, middle, bottom), all lanes came from the same blot but irrelevant lanes were removed. b, miRNA association of wild-type or mutant FH-AGO2 assessed as described in Fig. 3a (n = 4 biological replicates, each assayed in triplicate). For gel source data, see Supplementary Fig. 1.

Extended Data Figure 8 Analysis of serine/threonine kinases identified in the CRISPR–Cas9 suppressor screen.

a, b, Flow cytometry demonstrating EGFP expression in HCT116EGFP-miR19 (a) or HCT116EGFP cells (b) after transduction with lentiCRISPR vectors targeting the indicated genes. c, Flow cytometry demonstrating EGFP expression in HCT116EGFP-miR19 cells treated with the indicated dose of rapamycin. NT, not treated. d, Phos-tag western blot analysis of AGO2 in ANKRD52−/− cells after treatment with rapamycin. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 9 Functional characterization of CSNK1A1 and AGO2 target binding mutants.

a, Western blot analysis confirms loss of CSNK1A1 expression in HCT116 ANKRD52−/−;CSNK1A1−/− clonal knockout cells. All lanes came from the same blot but irrelevant lanes were removed. b, miR-19 expression normalized to U6 expression, assessed by qRT–PCR, in cells of the indicated genotypes (n = 4 biological replicates, each assayed in triplicate). c, Co-immunoprecipitation of V5-CSNK1A1 with FH-AGO2, with or without RNase A treatment. d, miRNA association of FH-AGO2 assessed as in Fig. 3e (n = 4 biological replicates, each assayed in triplicate). *P < 0.05 comparing mutant to wild-type AGO2, two-tailed Student’s t-test. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 10 Generation and eCLIP analysis of AGO2–/– cells reconstituted with AGO2WT or AGO25XA.

a, Western blot showing equivalent expression of FH-AGO2WT and FH-AGO25XA at physiological levels. For gel source data, see Supplementary Fig. 1. b, Distribution of AGO2 binding sites determined by eCLIP. c, Validation of targets identified by eCLIP using FH-AGO2 pull-down assays performed in reconstituted AGO2−/− cells. Experiment was performed as in Fig. 3a except anti-Flag antibody was used for immunoprecipitation (n = 3 biological replicates, each assayed in triplicate). *P < 0.05, **P < 0.01, one-tailed Student’s t-test comparing FH-AGO25XA with FH-AGO2WT. NS, not significant. d, FH-AGO2WT CLIP coverage (normalized total number of reads in clusters in a given 3′ UTR divided by the FPKM) of genes whose AGO2-mediated repression is or is not rescued by FH-AGO25XA. e, The 8-, 7-, or 6-nucleotide binding sites for active miRNAs in HCT116 were identified within FH-AGO2WT/FH-AGO25XA-common CLIP clusters or FH-AGO25XA-unique CLIP clusters in 3′ UTRs. CDF plots show CLIP coverage for each class of site (normalized number of crosslinking events within ten nucleotides of each site). NS, not significant, assessed by Kolmogorov–Smirnov test. f, CDF plot showing the fold change in CLIP coverage comparing FH-AGO25XA to FH-AGO2WT for transcripts with long half-lives (top quartile) versus those with short half-lives (bottom quartile). g, Summary of the newly defined AGO2 phosphorylation cycle. Target engagement triggers the hierarchical, multi-site phosphorylation of AGO2 by CSNK1A1, which inhibits target binding. The ANKRD52–PPP6C phosphatase complex dephosphorylates these residues, allowing AGO2 to engage new targets. Continual phosphorylation/de-phosphorylation of AGO2 through this cycle is necessary to maintain the global efficiency of miRNA-mediated silencing.

Supplementary information

Supplementary Figure 1

This file contains the gel source data. (PDF 1155 kb)

Supplementary Table 1.

This file contains the simulated sgRNA enrichment in top 0.5% brightest cells. (XLSX 9 kb)

Supplementary Table 2.

This file contains the RIGER analysis of CRISPR-Cas9 screen in HCT116EGFP-miR19 cells. (XLSX 2290 kb)

Supplementary Table 3.

This file contains the RIGER analysis of CRISPR-Cas9 screen in HCT116EGFP cells. (XLSX 2291 kb)

Supplementary Table 4.

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. (XLSX 160 kb)

Supplementary Table 5.

This file contains the RIGER analysis of CRISPR-Cas9 suppressor screen in ANKRD52−/− HCT116EGFP-miR19 cells. (XLSX 2096 kb)

Supplementary Table 6.

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. (XLSX 886 kb)

Supplementary Table 7.

This file contains the eCLIP results. (XLSX 271 kb)

Supplementary Table 8.

This file contains the oligonucleotides and peptides used in this study. (XLSX 18 kb)

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Golden, R., Chen, B., Li, T. et al. An Argonaute phosphorylation cycle promotes microRNA-mediated silencing. Nature 542, 197–202 (2017). https://doi.org/10.1038/nature21025

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