An Argonaute phosphorylation cycle promotes microRNA-mediated silencing

doi:10.1038/nature21025

. 2017 Feb 9;542(7640):197-202.

doi: 10.1038/nature21025. Epub 2017 Jan 23.

An Argonaute phosphorylation cycle promotes microRNA-mediated silencing

Ryan J Golden^{1

2}, Beibei Chen^{3

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 Chen^{1

7}, Yang Xie^{3

4

8}, Joshua T Mendell^{1

7

8

9}

Affiliations

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

PMID: 28114302
PMCID: PMC5302127
DOI: 10.1038/nature21025

An Argonaute phosphorylation cycle promotes microRNA-mediated silencing

Ryan J Golden et al. Nature. 2017.

. 2017 Feb 9;542(7640):197-202.

doi: 10.1038/nature21025. Epub 2017 Jan 23.

Authors

Affiliations

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

PMID: 28114302
PMCID: PMC5302127
DOI: 10.1038/nature21025

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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Figures

**Extended Data Figure 1. Knockout of candidate miRNA regulators in HCT116^EGFP cells**
Flow cytometry analysis of EGFP in HCT116^EGFP 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. **b, c, d,** 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. **e, f, g,** 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 Figure 1.

**Extended Data Figure 3. Western blot analysis confirms loss of protein expression in *AGO2* (a) and *ANKRD52* (b) HCT116 clonal knockout lines**
For each protein, all lanes came from the same blot but irrelevant lanes were removed. For gel source data, see Supplementary Figure 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) HCT116^EGFP-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 Figure 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 peptide 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. 1P, 2P, or 3P respectively denotes 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 (upper, middle, lower), 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 Figure 3a (N = 4 biological replicates, each assayed in triplicate). For gel source data, see Supplementary Figure 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 HCT116^EGFP-miR19 (a) or HCT116^EGFP cells (b) after transduction with lentiCRISPR vectors targeting the indicated genes. c, Flow cytometry demonstrating EGFP expression in HCT116^EGFP-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 Figure 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 Figure 1.

**Extended Data Figure 10. Generation and eCLIP analysis of *AGO2*^−/− cells reconstituted with AGO2^WT or AGO2^5XA**
a, Western blot showing equivalent expression of FH-AGO2^WT and FH-AGO2^5XA at physiologic levels. b, Distribution of AGO2 binding sites determined by eCLIP. For gel source data, see Supplementary Figure 1. 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-AGO2^5XA to FH-AGO2^WT. n.s., not significant. d, FH-AGO2^WT CLIP coverage (normalized total number of reads in clusters in a given 3' UTR divided by FPKM) of genes whose AGO2-mediated repression is or is not rescued by FH-AGO2^5XA. e, 8mer, 7mer, or 6mer binding sites for active miRNAs in HCT116 were identified within FH-AGO2^WT/FH-AGO2^5XA-common CLIP clusters or FH-AGO2^5XA-unique CLIP clusters in 3' UTRs. CDF plots show CLIP coverage for each class of site (normalized number of crosslinking events within 10 nucleotides of each site). n.s., Kolmogorov-Smirnov (KS) test not significant. f, CDF plot showing the fold-change in CLIP coverage comparing FH-AGO2^5XA to FH-AGO2^WT for transcripts with long half-lives (top quartile) *vs.* 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.

**Fig. 1. A genome-wide CRISPR-Cas9 screen reveals known and novel regulators of the miRNA pathway**
a, Design of CRISPR-Cas9 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 HCT116^EGFP-miR19 (top) or HCT116^EGFP 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 HCT116^EGFP-miR19 cells after transduction with lentiCRISPR vectors.

**Fig. 2. Loss of ANKRD52-PPP6C activity globally impairs miRNA-mediated silencing and results in AGO2 hyperphosphorylation**
a, Cumulative distribution function (CDF) plot demonstrating that genes upregulated in *AGO2*^−/− HCT116 cells (Supplementary Table 4; FDR ≤ 0.05) are similarly upregulated in *ANKRD52*^−/− cells. Kolmogorov-Smirnov (KS) p value shown for this and all subsequent CDF plots. **b, c,** Phos-tag electrophoresis demonstrating enhanced AGO2 phosphorylation in ANKRD52/PPP6C-deficient HCT116 cells (b) and sensitivity of AGO2 phosphorylation to lambda protein phosphatase (λPP) (c). d, Evolutionary conservation of AGO2 S824-S834. Putative phosphorylation sites shown in red. e, Phos-tag analysis of FH-AGO2 mutants stably expressed in *ANKRD52*^−/− cells. f, EGFP expression in *ANKRD52*^−/− HCT116^EGFP-miR19 cells expressing the indicated FH-AGO2 constructs. For gel source data, see Supplementary Figure 1.

**Fig. 3. AGO2 phosphorylation impairs mRNA target association**
a, Measurement of AGO2-associated miRNA by qRT-PCR. N = 2 biological replicates each assayed in triplicate. Error bars indicate SD for this and all subsequent qRT-PCR data. b, AGO2-target association assessed as described in (a). *p < 0.05, two-tailed student’s t test comparing *ANKRD52*^−/− to WT. **c, d,** Schematic (c) and phos-tag western blot (d) of AGO2:miRNA target capture experiment. e, *EGFP* target association of FH-AGO2 assessed as described in (a). *p < 0.05; **p < 0.001, two-tailed student’s t test (N = 4 biological replicates). f, AGO2 tethering experiments demonstrating repression of luciferase-BoxB (pGL3-BoxB) by λN-AGO2 (WT), λN-AGO2 (5XA), and λN-AGO2 (5XE), but not by untethered AGO2 or λN alone. A luciferase transcript lacking BoxB sites (pGL3-Control) served as a negative control. *p < 0.05; ** p < 0.01, two-tailed student’s t test (N = 3 biological replicates). For gel source data, see Supplementary Figure 1.

**Fig. 4. A CRISPR-Cas9 suppressor screen reveals CSNK1A1 as the inhibitory AGO2 kinase**
a, Design of CRISPR-Cas9 screen to identify *ANKRD52*^−/− suppressors. b, RIGER analysis with serine/threonine kinases highlighted. c, EGFP expression in *ANKRD52*^−/− HCT116^EGFP-miR19 cells transduced with lentiCRISPR vectors targeting *CSNK1A1*. d, Abrogated phosphorylation of AGO2 in *ANKRD52*^−/−; *CSNK1A1*^−/− cells. e, Relative association of miR-19a, miR-19b and *EGFP* target mRNA with AGO2 assessed as described in Fig. 3a (N = 3 biological replicates). f, *In vitro* phosphorylation of FH-AGO2 by CSNK1A1 with or without pre-treatment with λPP. g, In *vitro* CSNK1A1-mediated phosphorylation of the indicated peptides. For gel source data, see Supplementary Figure 1.

**Fig. 5. Argonaute S824-S834 phosphorylation is required for fully efficient miRNA-mediated silencing and is triggered by target binding**
a, CDF plot demonstrating that genes upregulated in *AGO2*^−/− HCT116 cells (Supplementary Table 4; FDR ≤ 0.05) are similarly upregulated in *CSNK1A1*^−/−*; ANKRD52*^−/− cells. b, CDF plot showing partially rescued repression of genes upregulated in *AGO2*^−/− cells (Supplementary Table 6; FDR ≤ 0.05) reconstituted with FH-AGO2^5XA compared to cells reconstituted with FH-AGO2^WT. c, *EGFP* target association of FH-AGO2 assessed as in Fig. 3e (N = 4 biological replicates, each assayed in triplicate). **d, e,** Phosphorylation of AGO2 mutants in *ANKRD52*^−/− cells (d) or endogenous AGO2 after transfection of *ciRS-7* constructs and the indicated miRNAs (e). For gel source data, see Supplementary Figure 1.

**Fig. 6. Expansion of the target repertoire bound by AGO2^5XA**
a, Number of AGO2 binding sites determined by eCLIP. b, Unique and common genes bound by FH-AGO2^WT and FH-AGO2^5XA. c, Binding sites for active miRNAs in HCT116 were identified within FH-AGO2^WT CLIP clusters in 3' UTRs. CDF plots show CLIP coverage for each class of site (normalized number of crosslinking events within 10 nucleotides of each site divided by FPKM of the transcript) in FH-AGO2^WT and FH-AGO2^5XA datasets.

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References

1. Mendell JT, Olson EN. MicroRNAs in stress signaling and human disease. Cell. 2012;148:1172–1187. - PMC - PubMed
1. Vidigal JA, Ventura A. The biological functions of miRNAs: lessons from in vivo studies. Trends Cell Biol. 2015;25:137–147. - PMC - PubMed
1. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136:215–233. - PMC - PubMed
1. Jonas S, Izaurralde E. Towards a molecular understanding of microRNA-mediated gene silencing. Nat Rev Genet. 2015;16:421–433. - PubMed
1. Grimson A, et al. MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell. 2007;27:91–105. - PMC - PubMed

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[1] Mendell JT, Olson EN. MicroRNAs in stress signaling and human disease. Cell. 2012;148:1172–1187. - PMC - PubMed

[2] Mendell JT, Olson EN. MicroRNAs in stress signaling and human disease. Cell. 2012;148:1172–1187. - PMC - PubMed

[3] Vidigal JA, Ventura A. The biological functions of miRNAs: lessons from in vivo studies. Trends Cell Biol. 2015;25:137–147. - PMC - PubMed

[4] Vidigal JA, Ventura A. The biological functions of miRNAs: lessons from in vivo studies. Trends Cell Biol. 2015;25:137–147. - PMC - PubMed

[5] Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136:215–233. - PMC - PubMed

[6] Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136:215–233. - PMC - PubMed

[7] Jonas S, Izaurralde E. Towards a molecular understanding of microRNA-mediated gene silencing. Nat Rev Genet. 2015;16:421–433. - PubMed

[8] Jonas S, Izaurralde E. Towards a molecular understanding of microRNA-mediated gene silencing. Nat Rev Genet. 2015;16:421–433. - PubMed

[9] Grimson A, et al. MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell. 2007;27:91–105. - PMC - PubMed

[10] Grimson A, et al. MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell. 2007;27:91–105. - PMC - PubMed

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An Argonaute phosphorylation cycle promotes microRNA-mediated silencing

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An Argonaute phosphorylation cycle promotes microRNA-mediated silencing

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