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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The data that support the findings of this study are available from the corresponding author upon reasonable request.
Protter, D. S. & Parker, R. Principles and properties of stress granules. Trends Cell Biol. 26, 668–679 (2016).
Shin, Y. & Brangwynne, C. P. Liquid phase condensation in cell physiology and disease. Science 357, eaaf4382 (2017).
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).
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).
Ivanov, P., Kedersha, N. & Anderson, P. Stress granules and processing bodies in translational control. Cold Spring Harb. Perspect. Biol 11, a032813 (2018).
Eliscovich, C. & Singer, R. H. RNP transport in cell biology: the long and winding road. Curr. Opin. Cell Biol. 45, 38–46 (2017).
Shukla, S. & Parker, R. Hypo- and hyper-assembly diseases of RNA–protein complexes. Trends Mol. Med. 22, 615–628 (2016).
Nedelsky, N. B. & Taylor, J. P. Bridging biophysics and neurology: aberrant phase transitions in neurodegenerative disease. Nat. Rev. Neurol. 15, 272–286 (2019).
Kato, M. et al. Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels. Cell 149, 753–767 (2012).
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).
Van Treeck, B. & Parker, R. Emerging roles for intermolecular RNA–RNA interactions in RNP assemblies. Cell 174, 791–802 (2018).
Jain, S. et al. ATPase-modulated stress granules contain a diverse proteome and substructure. Cell 164, 487–498 (2016).
Dominissini, D. et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485, 201–206 (2012).
Meyer, K. D. et al. Comprehensive analysis of mRNA methylation reveals enrichment in 3’ UTRs and near stop codons. Cell 149, 1635–1646 (2012).
Fu, Y., Dominissini, D., Rechavi, G. & He, C. Gene expression regulation mediated through reversible m6A RNA methylation. Nat. Rev. Genet. 15, 293–306 (2014).
Delaunay, S. & Frye, M. RNA modifications regulating cell fate in cancer. Nat. Cell Biol. 21, 552–559 (2019).
Wang, X. et al. N 6-methyladenosine modulates messenger RNA translation efficiency. Cell 161, 1388–1399 (2015).
Markmiller, S. et al. Context-dependent and disease-specific diversity in protein interactions within stress granules. Cell 172, 590–604 (2018).
Li, A. et al. Cytoplasmic m6A reader YTHDF3 promotes mRNA translation. Cell Res. 27, 444–447 (2017).
Shi, H. et al. YTHDF3 facilitates translation and decay of N 6-methyladenosine-modified RNA. Cell Res. 27, 315–328 (2017).
Tourriere, H. et al. The RasGAP-associated endoribonuclease G3BP assembles stress granules. J. Cell Biol. 160, 823–831 (2003).
Kedersha, N. et al. G3BP-Caprin1-USP10 complexes mediate stress granule condensation and associate with 40S subunits. J. Cell Biol. 212, 845–860 (2016).
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).
Xiang, Y. et al. RNA m6A methylation regulates the ultraviolet-induced DNA damage response. Nature 543, 573–576 (2017).
Roundtree, I. A. et al. YTHDC1 mediates nuclear export of N 6-methyladenosine methylated mRNAs. Elife 6, e31311 (2017).
Wang, X. et al. N 6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505, 117–120 (2014).
Zheng, D. et al. Deadenylation is prerequisite for P-body formation and mRNA decay in mammalian cells. J. Cell Biol. 182, 89–101 (2008).
Khong, A. et al. The stress granule transcriptome reveals principles of mRNA accumulation in stress granules. Mol. Cell 68, 808–820 (2017).
Molinie, B. et al. m6A-LAIC-seq reveals the census and complexity of the m6A epitranscriptome. Nat. Methods 13, 692–698 (2016).
Femino, A. M., Fay, F. S., Fogarty, K. & Singer, R. H. Visualization of single RNA transcripts in situ. Science 280, 585–590 (1998).
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).
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).
Hubstenberger, A. et al. P-body purification reveals the condensation of repressed mRNA regulons. Mol. Cell 68, 144–157 (2017).
Alberti, S., Gladfelter, A. & Mittag, T. Considerations and challenges in studying liquid–liquid phase separation and biomolecular condensates. Cell 176, 419–434 (2019).
Klausen, M. S. et al. NetSurfP-2.0: improved prediction of protein structural features by integrated deep learning. Proteins 87, 520–527 (2019).
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).
Peng, K., Radivojac, P., Vucetic, S., Dunker, A. K. & Obradovic, Z. Length-dependent prediction of protein intrinsic disorder. BMC Bioinformatics 7, 208 (2006).
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).
Taslimi, A. et al. An optimized optogenetic clustering tool for probing protein interaction and function. Nat. Commun. 5, 4925 (2014).
Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).
Brangwynne, C. P. et al. Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science 324, 1729–1732 (2009).
Narayanan, A. et al. A first order phase transition mechanism underlies protein aggregation in mammalian cells. Elife 8, e39695 (2019).
Anders, M. et al. Dynamic m6A methylation facilitates mRNA triaging to stress granules. Life Sci. Alliance 1, e201800113 (2018).
Kedersha, N. & Anderson, P. Mammalian stress granules and processing bodies. Methods Enzymol. 431, 61–81 (2007).
Wang, J. et al. Binding to m6A RNA promotes YTHDF2-mediated phase separation. Protein Cell 11, 304–307 (2020).
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).
Gao, Y. et al. Multivalent m6A motifs promote phase separation of YTHDF proteins. Cell Res. 29, 767–769 (2019).
Ries, R. J. et al. m6A enhances the phase separation potential of mRNA. Nature 571, 424–428 (2019).
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).
Wheeler, J. R., Matheny, T., Jain, S., Abrisch, R. & Parker, R. Distinct stages in stress granule assembly and disassembly. Elife 5, e18413 (2016).
Sarkar, S. & Hopper, A. K. tRNA nuclear export in Saccharomyces cerevisiae: in situ hybridization analysis. Mol. Biol. Cell 9, 3041–3055 (1998).
Khatter, H., Myasnikov, A. G., Natchiar, S. K. & Klaholz, B. P. Structure of the human 80S ribosome. Nature 520, 640–645 (2015).
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).
Richter, K. N. et al. Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy. EMBO J. 37, 139–159 (2018).
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).
Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).
Otsu, N. Threshold selection method from gray-level histograms. IEEE Trans. Syst. Man Cybern. 9, 62–66 (1979).
Slezov, V. V. Kinetics of First‐Order Phase Transitions (Wiley, 2009).
Karthika, S., Radhakrishnan, T. K. & Kalaichelvi, P. A review of classical and nonclassical nucleation theories. Cryst. Growth Des. 16, 6663–6681 (2016).
Shin, Y. et al. Liquid nuclear condensates mechanically sense and restructure the genome. Cell 175, 1481–1491 (2018).
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.
The authors declare no competing interests.
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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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