Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

m6A-binding YTHDF proteins promote stress granule formation

Nature Chemical Biology volume 16, pages 955–963 (2020)Cite this article

Subjects

Abstract

Diverse RNAs and RNA-binding proteins form phase-separated, membraneless granules in cells under stress conditions. However, the role of the prevalent mRNA methylation, m6A, and its binding proteins in stress granule (SG) assembly remain unclear. Here, we show that m6A-modified mRNAs are enriched in SGs, and that m6A-binding YTHDF proteins are critical for SG formation. Depletion of YTHDF1/3 inhibits SG formation and recruitment of mRNAs to SGs. Both the N-terminal intrinsically disordered region and the C-terminal m6A-binding YTH domain of YTHDF proteins are important for SG formation. Super-resolution imaging further reveals that YTHDF proteins appear to be in a super-saturated state, forming clusters that often reside in the periphery of or at the junctions between SG core clusters, and potentially promote SG formation by reducing the activation energy barrier and critical size for SG condensate formation. Our results suggest a new function of the m6A-binding YTHDF proteins in regulating SG formation.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: m6A-modified mRNAs are enriched in stress granules in U2OS cells under oxidative stress.
Fig. 2: YTHDF proteins promote SG formation.
Fig. 3: Both the N-terminal intrinsically disordered region and the C-terminal YTH domain are important for YTHDF’s role in promoting SG formation.
Fig. 4: Inhibiting m6A-binding of YTHDF proteins partially impairs SG formation.
Fig. 5: YTHDF protein reduces the critical size and activation energy barrier for SG condensate formation.

Similar content being viewed by others

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Code availability

Custom Python and MATLAB codes for image acquisition and STORM analysis are available at https://github.com/ZhuangLab. Custom MATLAB codes for the two-color STORM data analysis, data fitting for the classical nucleation model and SG identification are available at https://github.com/yefu01/ythdf.

References

  1. Protter, D. S. & Parker, R. Principles and properties of stress granules. Trends Cell Biol. 26, 668–679 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Shin, Y. & Brangwynne, C. P. Liquid phase condensation in cell physiology and disease. Science 357, eaaf4382 (2017).

    Article  PubMed  CAS  Google Scholar 

  3. Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18, 285–298 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Kato, M. & McKnight, S. L. A solid-state conceptualization of information transfer from gene to message to protein. Annu. Rev. Biochem. 87, 351–390 (2018).

    Article  CAS  PubMed  Google Scholar 

  5. Ivanov, P., Kedersha, N. & Anderson, P. Stress granules and processing bodies in translational control. Cold Spring Harb. Perspect. Biol 11, a032813 (2018).

    Article  CAS  Google Scholar 

  6. Eliscovich, C. & Singer, R. H. RNP transport in cell biology: the long and winding road. Curr. Opin. Cell Biol. 45, 38–46 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Shukla, S. & Parker, R. Hypo- and hyper-assembly diseases of RNA–protein complexes. Trends Mol. Med. 22, 615–628 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Nedelsky, N. B. & Taylor, J. P. Bridging biophysics and neurology: aberrant phase transitions in neurodegenerative disease. Nat. Rev. Neurol. 15, 272–286 (2019).

    Article  PubMed  Google Scholar 

  9. Kato, M. et al. Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels. Cell 149, 753–767 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Han, T. W. et al. Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies. Cell 149, 768–779 (2012).

    Article  CAS  PubMed  Google Scholar 

  11. Van Treeck, B. & Parker, R. Emerging roles for intermolecular RNA–RNA interactions in RNP assemblies. Cell 174, 791–802 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Jain, S. et al. ATPase-modulated stress granules contain a diverse proteome and substructure. Cell 164, 487–498 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Dominissini, D. et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485, 201–206 (2012).

    Article  CAS  PubMed  Google Scholar 

  14. Meyer, K. D. et al. Comprehensive analysis of mRNA methylation reveals enrichment in 3’ UTRs and near stop codons. Cell 149, 1635–1646 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Fu, Y., Dominissini, D., Rechavi, G. & He, C. Gene expression regulation mediated through reversible m6A RNA methylation. Nat. Rev. Genet. 15, 293–306 (2014).

    Article  CAS  PubMed  Google Scholar 

  16. Delaunay, S. & Frye, M. RNA modifications regulating cell fate in cancer. Nat. Cell Biol. 21, 552–559 (2019).

    Article  CAS  PubMed  Google Scholar 

  17. Wang, X. et al. N 6-methyladenosine modulates messenger RNA translation efficiency. Cell 161, 1388–1399 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Markmiller, S. et al. Context-dependent and disease-specific diversity in protein interactions within stress granules. Cell 172, 590–604 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Li, A. et al. Cytoplasmic m6A reader YTHDF3 promotes mRNA translation. Cell Res. 27, 444–447 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Shi, H. et al. YTHDF3 facilitates translation and decay of N 6-methyladenosine-modified RNA. Cell Res. 27, 315–328 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Tourriere, H. et al. The RasGAP-associated endoribonuclease G3BP assembles stress granules. J. Cell Biol. 160, 823–831 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kedersha, N. et al. G3BP-Caprin1-USP10 complexes mediate stress granule condensation and associate with 40S subunits. J. Cell Biol. 212, 845–860 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Edupuganti, R. R. et al. N 6-methyladenosine (m6A) recruits and repels proteins to regulate mRNA homeostasis. Nat. Struct. Mol. Biol. 24, 870–878 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Xiang, Y. et al. RNA m6A methylation regulates the ultraviolet-induced DNA damage response. Nature 543, 573–576 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Roundtree, I. A. et al. YTHDC1 mediates nuclear export of N 6-methyladenosine methylated mRNAs. Elife 6, e31311 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Wang, X. et al. N 6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505, 117–120 (2014).

    Article  PubMed  CAS  Google Scholar 

  27. Zheng, D. et al. Deadenylation is prerequisite for P-body formation and mRNA decay in mammalian cells. J. Cell Biol. 182, 89–101 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Khong, A. et al. The stress granule transcriptome reveals principles of mRNA accumulation in stress granules. Mol. Cell 68, 808–820 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Molinie, B. et al. m6A-LAIC-seq reveals the census and complexity of the m6A epitranscriptome. Nat. Methods 13, 692–698 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Femino, A. M., Fay, F. S., Fogarty, K. & Singer, R. H. Visualization of single RNA transcripts in situ. Science 280, 585–590 (1998).

    Article  CAS  PubMed  Google Scholar 

  31. Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–879 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Namkoong, S., Ho, A., Woo, Y. M., Kwak, H. & Lee, J. H. Systematic characterization of stress-induced RNA granulation. Mol. Cell 70, 175–187 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Hubstenberger, A. et al. P-body purification reveals the condensation of repressed mRNA regulons. Mol. Cell 68, 144–157 (2017).

    Article  CAS  PubMed  Google Scholar 

  34. Alberti, S., Gladfelter, A. & Mittag, T. Considerations and challenges in studying liquid–liquid phase separation and biomolecular condensates. Cell 176, 419–434 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Klausen, M. S. et al. NetSurfP-2.0: improved prediction of protein structural features by integrated deep learning. Proteins 87, 520–527 (2019).

    Article  CAS  PubMed  Google Scholar 

  36. Lancaster, A. K., Nutter-Upham, A., Lindquist, S. & King, O. D. PLAAC: a web and command-line application to identify proteins with prion-like amino acid composition. Bioinformatics 30, 2501–2502 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Peng, K., Radivojac, P., Vucetic, S., Dunker, A. K. & Obradovic, Z. Length-dependent prediction of protein intrinsic disorder. BMC Bioinformatics 7, 208 (2006).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Xu, C. et al. Structural basis for the discriminative recognition of N 6-methyladenosine RNA by the human YT521-B homology domain family of proteins. J. Biol. Chem. 290, 24902–24913 (2015).

    Article  CAS  PubMed  Google Scholar 

  39. Taslimi, A. et al. An optimized optogenetic clustering tool for probing protein interaction and function. Nat. Commun. 5, 4925 (2014).

    Article  CAS  PubMed  Google Scholar 

  40. Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Brangwynne, C. P. et al. Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science 324, 1729–1732 (2009).

    Article  CAS  PubMed  Google Scholar 

  42. Narayanan, A. et al. A first order phase transition mechanism underlies protein aggregation in mammalian cells. Elife 8, e39695 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Anders, M. et al. Dynamic m6A methylation facilitates mRNA triaging to stress granules. Life Sci. Alliance 1, e201800113 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Kedersha, N. & Anderson, P. Mammalian stress granules and processing bodies. Methods Enzymol. 431, 61–81 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. Wang, J. et al. Binding to m6A RNA promotes YTHDF2-mediated phase separation. Protein Cell 11, 304–307 (2020).

    Article  PubMed  Google Scholar 

  46. Wang, J. et al. A molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins. Cell 174, 688–699 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Gao, Y. et al. Multivalent m6A motifs promote phase separation of YTHDF proteins. Cell Res. 29, 767–769 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Ries, R. J. et al. m6A enhances the phase separation potential of mRNA. Nature 571, 424–428 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Buchan, J. R., Kolaitis, R. M., Taylor, J. P. & Parker, R. Eukaryotic stress granules are cleared by autophagy and Cdc48/VCP function. Cell 153, 1461–1474 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Wheeler, J. R., Matheny, T., Jain, S., Abrisch, R. & Parker, R. Distinct stages in stress granule assembly and disassembly. Elife 5, e18413 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  51. Sarkar, S. & Hopper, A. K. tRNA nuclear export in Saccharomyces cerevisiae: in situ hybridization analysis. Mol. Biol. Cell 9, 3041–3055 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Khatter, H., Myasnikov, A. G., Natchiar, S. K. & Klaholz, B. P. Structure of the human 80S ribosome. Nature 520, 640–645 (2015).

    Article  CAS  PubMed  Google Scholar 

  53. Natchiar, S. K., Myasnikov, A. G., Kratzat, H., Hazemann, I. & Klaholz, B. P. Visualization of chemical modifications in the human 80S ribosome structure. Nature 551, 472–477 (2017).

    Article  CAS  PubMed  Google Scholar 

  54. Richter, K. N. et al. Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy. EMBO J. 37, 139–159 (2018).

    Article  CAS  PubMed  Google Scholar 

  55. Wang, W., Li, G. W., Chen, C., Xie, X. S. & Zhuang, X. Chromosome organization by a nucleoid-associated protein in live bacteria. Science 333, 1445–1449 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Otsu, N. Threshold selection method from gray-level histograms. IEEE Trans. Syst. Man Cybern. 9, 62–66 (1979).

    Article  Google Scholar 

  58. Slezov, V. V. Kinetics of First‐Order Phase Transitions (Wiley, 2009).

  59. Karthika, S., Radhakrishnan, T. K. & Kalaichelvi, P. A review of classical and nonclassical nucleation theories. Cryst. Growth Des. 16, 6663–6681 (2016).

    Article  CAS  Google Scholar 

  60. Shin, Y. et al. Liquid nuclear condensates mechanically sense and restructure the genome. Cell 175, 1481–1491 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank members of Zhuang Lab for help, especially R. Zhou and B. Han for assistance with the two-color STORM set-up and data analysis and G. Wang and M. Thanawala for help with data analysis. We thank K. Xu for help with the script for two-color STORM data analysis. We thank P. Anderson and N. Kedersha for helpful discussions and Y. Shi (Harvard Medical School) for providing the U2OS-METTL3-KO cell line. This work was in part supported by NIH (to X.Z.). X.Z. is an HHMI investigator.

Author information

Authors and Affiliations

  1. Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA

    Ye Fu & Xiaowei Zhuang

  2. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Ye Fu & Xiaowei Zhuang

  3. Department of Physics, Harvard University, Cambridge, MA, USA

    Xiaowei Zhuang

Authors
  1. Ye Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaowei Zhuang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.F. and X.Z. designed the experiments. Y.F. performed experiments and analyzed data. Y.F. and X.Z. wrote the paper.

Corresponding author

Correspondence to Xiaowei Zhuang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Fu, Y., Zhuang, X. m6A-binding YTHDF proteins promote stress granule formation. Nat Chem Biol 16, 955–963 (2020). https://doi.org/10.1038/s41589-020-0524-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-020-0524-y

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing