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m6A-binding YTHDF proteins promote stress granule formation

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.

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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.

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.

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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.

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

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Correspondence to Xiaowei Zhuang.

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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

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