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Acetylation of intrinsically disordered regions regulates phase separation


Liquid–liquid phase separation (LLPS) of proteins containing intrinsically disordered regions (IDRs) has been proposed as a mechanism underlying the formation of membrane-less organelles. Tight regulation of IDR behavior is essential to ensure that LLPS only takes place when necessary. Here, we report that IDR acetylation/deacetylation regulates LLPS and assembly of stress granules (SGs), membrane-less organelles forming in response to stress. Acetylome analysis revealed that the RNA helicase DDX3X, an important component of SGs, is a novel substrate of the deacetylase HDAC6. The N-terminal IDR of DDX3X (IDR1) can undergo LLPS in vitro, and its acetylation at multiple lysine residues impairs the formation of liquid droplets. We also demonstrated that enhanced LLPS propensity through deacetylation of DDX3X-IDR1 by HDAC6 is necessary for SG maturation, but not initiation. Our analysis provides a mechanistic framework to understand how acetylation and deacetylation of IDRs regulate LLPS spatiotemporally, and impact membrane-less organelle formation in vivo.

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We are grateful to T. Hyman for use of the microscope with thermal stage on short notice and for comments on the manuscript, and R. Voit (German Cancer Research Center, Heidelberg) for HAT expression vectors. We thank L. Gelman and S. Bourke for help with microscopic analysis, H. Kohler for FACS analysis, J. Seebacher and V. Iesmantavicius for interpretation of mass spectrometry data, H. Gut for help with structure predictions, M.B. Stadler for acetylome-wide IDR analysis, J. Wilbertz for help with live-cell imaging, L. Giorgetti and Y. Zhan for help with mathematical modeling, W. Filipowicz and J. Chao for critical comments on the manuscript. We thank C. Schölz for valuable suggestions. We also thank L. Wang for advice on protein purification, G. Matthias and C. Cao for their helpful technical assistance, Y. Miyake for providing us with biological materials for experiments, R. Clerc for critical comments on the manuscript, and all the Matthias laboratory members for fruitful discussions. M. Saito is supported in part by a fellowship from the Nakajima Foundation. A.W. Fritsch is supported by the ELBE postdoctoral fellows program. The Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation (Grant agreement NNF14CC0001). This work was supported by the Novartis Research Foundation.

Author information

M.S. and P.M. designed the project; M.S. performed all experiments and interpreted the data for the manuscript under the supervision of P.M.; M.S. and D.H. performed mass spectrometry analysis; M.S. and J.E. performed microscopy and image analysis; M.S., A.W.F. and M.K. performed temperature-dependent microscopy measurements and their image analysis; M.S. and B.T.W. analyzed HDAC6 related acetylome data under the supervision of C.C.; M.S. and P.M. wrote the manuscript and all authors contributed to the final version.

Competing interests

The authors declare no competing interests.

Correspondence to Patrick Matthias.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–27

  2. Reporting Summary

  3. Supplementary Dataset 1

    Total DDX3X SG volume (related to Fig. 5)

  4. Supplementary Dataset 2

    DDX3X-interactome (related to Fig. 6)

  5. Supplementary Dataset 3

    Raw data used for mathematical modeling of SG growth (related to Fig. 6)

  6. Supplementary Video 1

    Fusion behavior of DDX3X-IDR1 droplet (related to Fig. 3)

  7. Supplementary Video 2

    DDX3X-IDR1 droplet disappearance by LLPS following a temperature increase (related to Fig. 3)

  8. Supplementary Video 3

    Liquid-like properties of mCherry-DDX3X SGs (related to Fig. 4)

  9. Supplementary Video 4

    SG formation of WT DDX3X and its mutants (related to Fig. 6)

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Further reading

Fig. 1: DDX3X-IDR1 is specifically deacetylated by HDAC6.
Fig. 2: Stress induces acetylation of DDX3X and other proteins.
Fig. 3: Acetylation of DDX3X-IDR1 impairs its droplet formation by LLPS in vitro.
Fig. 4: Acetyl-mimic/dead mutations alter DDX3X SG dynamics.
Fig. 5: Deacetylation of DDX3X is required for normal SG size.
Fig. 6: SG maturation is promoted by deacetylation of DDX3X.