Article | Published:

An Argonaute phosphorylation cycle promotes microRNA-mediated silencing

Nature volume 542, pages 197202 (09 February 2017) | Download Citation

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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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.

Author information

Affiliations

  1. Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    • Ryan J. Golden
    • , Tuo Li
    • , Juliane Braun
    • , Hema Manjunath
    • , Xiang Chen
    • , Tsung-Cheng Chang
    • , Florian Kopp
    • , Andres Ramirez-Martinez
    • , Vincent S. Tagliabracci
    • , Zhijian J. Chen
    •  & Joshua T. Mendell
  2. Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    • Ryan J. Golden
  3. Quantitative Biomedical Research Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    • Beibei Chen
    •  & Yang Xie
  4. Department of Clinical Sciences, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    • Beibei Chen
    •  & Yang Xie
  5. Department of Microbiology and Immunology, University of California San Francisco, San Francisco, California 94143, USA

    • Jiaxi Wu
  6. Eugene McDermott Center for Human Growth & Development, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    • Vanessa Schmid
  7. Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    • Zhijian J. Chen
    •  & Joshua T. Mendell
  8. Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    • Yang Xie
    •  & Joshua T. Mendell
  9. Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    • Joshua T. Mendell

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Joshua T. Mendell.

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

Supplementary information

PDF files

  1. 1.

    Supplementary Figure 1

    This file contains the gel source data.

Excel files

  1. 1.

    Supplementary Table 1.

    This file contains the simulated sgRNA enrichment in top 0.5% brightest cells.

  2. 2.

    Supplementary Table 2.

    This file contains the RIGER analysis of CRISPR-Cas9 screen in HCT116EGFP-miR19 cells.

  3. 3.

    Supplementary Table 3.

    This file contains the RIGER analysis of CRISPR-Cas9 screen in HCT116EGFP cells.

  4. 4.

    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.

  5. 5.

    Supplementary Table 5.

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

  6. 6.

    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.

  7. 7.

    Supplementary Table 7.

    This file contains the eCLIP results.

  8. 8.

    Supplementary Table 8.

    This file contains the oligonucleotides and peptides used in this study.

About this article

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DOI

https://doi.org/10.1038/nature21025

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