Abstract
N6-methyladenosine (m6A) is the most abundant internal mRNA nucleotide modification in mammals, regulating critical aspects of cell physiology and differentiation. The YTHDF proteins are the primary readers of m6A modifications and exert physiological functions of m6A in the cytosol. Elucidating the regulatory mechanisms of YTHDF proteins is critical to understanding m6A biology. Here we report a mechanism that protein post-translational modifications control the biological functions of the YTHDF proteins. We find that YTHDF1 and YTHDF3, but not YTHDF2, carry high levels of nutrient-sensing O-GlcNAc modifications. O-GlcNAcylation attenuates the translation-promoting function of YTHDF1 and YTHDF3 by blocking their interactions with proteins associated with mRNA translation. We further demonstrate that O-GlcNAc modifications on YTHDF1 and YTHDF3 regulate the assembly, stability and disassembly of stress granules to enable better recovery from stress. Therefore, our results discover an important regulatory pathway of YTHDF functions, adding an additional layer of complexity to the post-transcriptional regulation function of mRNA m6A.
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Data availability
The MS proteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository104 with the dataset identifier PXD037017. High-throughput sequencing data of CLIP and RiboLace can be accessed in the Gene Expression Omnibus under the accession number GSE216570. Previously published datasets that were used for reanalysis33,35,72. Swiss-Prot human database (release 10 December 2018) containing 26,448 entries from UniProt was used for proteomics analysis. Previously published data used for design of constructs and plasmids are available from RefSeq under the accession numbers NM_017798.4, NM_016258.3 and NM_152758.6. All other data supporting the findings of this study are available from the corresponding authors on reasonable request. Source data are provided with this paper.
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Acknowledgements
This study was supported by the National Key R&D Program of China (grant no. 2019YFA09006600 to S.L.), the National Natural Science Foundation of China (grant nos 22222705, 91953113 and 92253302 to S.L. and 22277080 to Y.G.) and the Startup Fund from Shenzhen Bay Laboratory (grant no. 21230102 to Y.G.). C.H. is a Howard Hughes Medical Institute Investigator. We thank the core facility of the Life Sciences Institute Zhejiang University and the Bioimaging Core of Shenzhen Bay Laboratory. We also thank X. He for editing and helpful discussions.
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S.L. and C.H. conceived the idea and supervised the study. Y.G. supervised the in vitro LLPS experiments. Y.C., R.W. and Z.Z. designed and conducted all experiments and also analysed the data together. L.L., Y.Z. and L.T. assisted with experiments and provided valuable discussion. G.S. performed the in vitro LLPS experiments. Y.Y. provided technical advice. S.L., C.H., Y.C., R.W. and Z.Z. wrote the paper. All authors commented on the final draft of the paper.
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Extended data
Extended Data Fig. 1 Post-translational modifications on YTHDFs identified by LC–MS/MS.
a) Filtered identified modifications in HEK293T were annotated on the sites (score > 100 for all modifications and diagnostic peak for O-GlcNAcylation). b, O-GlcNAcylation ratio of each site of YTHDF1 and YTHDF3 by LC–MS/MS analysis in HEK293T calculated with intensities (in blue) and MSMS counts (in orange) of the respective modified and unmodified peptide. (c) Estimated ratio of unmodified, single modified and double modified of YTHDF1 and YTHDF3 by the intensity ratio in (b). The estimated modification level is highly similar to the O-GlcNAcylation stoichiometry analysis in Fig. 1e. (d) O-GlcNAcylation stoichiometry analysis of Flag-tagged MYPT1, CSNK2A1 and CREB1 in HEK293T and HeLa. Arrowheads indicates the O-GlcNAcylation modified proteins. Flag* denoted samples were performed O-GlcNAcylation stoichiometry analysis and immunoblotting by anti-Flag. (e) Quantification result of O-GlcNAcylation level of MYPT1, CSNK2A1 and CREB1 in (d). Data are presented as mean ± s.d. (n = 3 biologically independent repeats). Significance was calculated using two-sided t-test.
Extended Data Fig. 2 Validation O-GlcNAcylation sites on YTHDF1 and Reversible O-GlcNAcylation sites of YTHDF3.
(a) O-GlcNAcylation stoichiometry analysis of WT-YTHDF1 and S157A mutant. The experiment was repeated twice with similar results. (b) O-GlcNAcylation stoichiometry analysis of WT-YTHDF1 and S196A mutant. The experiment was repeated twice with similar results. (c) O-GlcNAcylation stoichiometry analysis shows O-GlcNAcylation can be transferred in S196, S197 and S198, while S196 is the major O-GlcNAcylation site of YTHDF1. The experiment was repeated twice with similar results. (d) O-GlcNAcylation stoichiometry analysis of WT-YTHDF1 and S196/197/198 A mutant. The experiment was repeated twice with similar results. (e) O-GlcNAcylation stoichiometry analysis of WT-YTHDF3 and YTHDF3 with mutations on modification sites. The experiment was repeated twice with similar results. (f) Tandem mass spectrum of YTHDF3 peptide with T205 modified by O-GlcNAcylation. The spectrum of unmodified peptide was used for side-by-side comparison. The arrows pointed to the signature b–y ions in the modification spectrum. (g) Tandem mass spectrum of YTHDF3 peptide with S229 modified by O-GlcNAcylation. The spectrum of unmodified peptide was used for side-by-side comparison. The arrows pointed to the signature b–y ions in the modification spectrum. (h) O-GlcNAcylation stoichiometry analysis of YTHDF3 treated without and with 50 µM OSMI-1 or 10 µM Thiamet G for 5 h in HEK293T. The experiment was repeated twice with similar results. Arrowheads indicates the O-GlcNAcylation modified proteins. Flag* denotes samples were performed O-GlcNAcylation stoichiometry analysis and immunoblotting with anti-Flag.
Extended Data Fig. 3 The region of YTHDF1/3 modified by O-GlcNAcylation is essential for translation related protein binding but irrelevant for m6A binding.
(a, b) Pulldown assay of purified Flag-EGFP-tagged YTHDF1/3 by purified GST tagged EIF2S3, EIF3M and EIF4E. YTHDF1/3 can directly bind to these translation related proteins. The experiment was repeated twice with similar results. (c, d) Pulldown assay of purified his tagged EIF2S3 by purified Flag-EGFP-tagged YTHDF1/3 truncations (bottom) and the truncation scheme (up). Only the truncated YTHDF1/3 containing O-GlcNAcylation sites can directly bind to EIF2S3. (e, f) EMSA assay using vehicle, OGT or OGT-K852A treated EGFP–YTHDF1/3 and Cy3-m6A-RNA probe, (GGm6ACUC)10. Vehicle treated YTHDF1/3 contained no O-GlcNAcylation modification, OGT can modify YTHDF1/3 by O-GlcNAcylation and OGT-K852M mutant contains no catalytic activity. (g, h) Quantification analysis of shift RNA probe ratio in (e-f). Data are presented as mean ± s.d. (n = 3 biologically independent repeats). O-GlcNAcylation on the YTHDF1/3 contains no effect for the m6A RNA binding.
Extended Data Fig. 4 O-GlcNAcylation of YTHDF3 repressed target mRNA translation efficiency.
(a, b) Expression analysis of N-WT-YTHDF1/3-λ and N-YTHDF1/3-Mut-λ in of Fig. 4b,c and Extended Data Fig. 4f-g. (c) Translation efficiency analysis of N-WT-YTHDF1-λ containing no, either or both mutation on O-GlcNAcylation sites. Error bars, mean ± s.d., n = 3 biologically independent repeats. Significance was calculated using two-sided t-test. (d) Translation efficiency analysis of N-WT-YTHDF1-λ and λ treated with different concentration of OGT inhibitor OSMI-1 in HEK293T. Error bars, mean ± s.d., n = 3 biologically independent repeats. Significance was calculated using two-sided t-test. (e) Translation efficiency analysis of N-WT-YTHDF1-λ and λ treated with different concentration of OGA inhibitor Thiamet G in HeLa. Error bars, mean ± s.d., n = 3 biologically independent repeats. Significance was calculated using two-sided t-test. (f) Translation efficiency analysis of N-WT-YTHDF3-λ, N-YTHDF3-Mut-λ and λ in HEK293T. Error bars, mean ± s.d., n = 3 biologically independent repeats. Significance was calculated using two-sided t-test. (g) Translation efficiency analysis of N-WT-YTHDF3-λ, N-YTHDF3-Mut-λ and λ in HeLa. Error bars, mean ± s.d., n = 3 biologically independent repeats. Significance was calculated using two-sided t-test. (h) RNA abundance analysis of N-WT-YTHDF3-λ, N-YTHDF3-Mut-λ and λ in HEK293T by qPCR. Error bars, mean ± s.d., n = 3 biologically independent repeats. Significance was calculated using two-sided t-test. (i) Translation efficiency analysis of N-WT-YTHDF3-λ containing no, either or both mutation on O-GlcNAcylation sites. Error bars, mean ± s.d., n = 3 biologically independent repeats. Significance was calculated using two-sided t-test. (j) Comparison of fold change of label-free quantification of immunoprecipitated translation related proteins interacting with WT-YTHDF1/3 and YTHDF1/3-Mut in HEK293T and HeLa by LC–MS/MS. The red lines denoted the mean fold change of the proteins. Mean fold change in HEK293T is lower than in HeLa, which might be due to the low O-GlcNAcylation level of YTHDF1/3 in HeLa.
Extended Data Fig. 5 Co-localization of YTHDF1/3 with P-body.
Co-localization of mCherry-YTHDF1/EGFP–YTHDF3, m6A RNA and EDC4 (P-body marker) in HeLa shows YTHDF1/3 localized in P-body with m6A RNA. OE, overexpress. Scale bars: 10 μm. The experiment was repeated twice with similar results.
Extended Data Fig. 6 Cellular localization and expression level of YTHDF1/3.
(a, b) Immunoblotting of endogenous YTHDF1/3 in HEK293T, HeLa, OSMI-1 treated HEK293T and Thiamet G treated HeLa. OSMI-1: 50 µM, 5 hr; Thiamet G: 10 µM, 5 h. c, d) Quantification analysis of YTHDF1/3 expression level in (a-b). Error bars, mean ± s.d., n = 3 biologically independent repeats. Significance was calculated using two-sided t-test. The expression level of YTHDF1/3 in HeLa was lower than in HEK293T, but the inhibitors contained no effect on the expression level of YTHDF1/3. (e) Immunostaining of endogenous YTHDF1/3 in HEK293T, HeLa and Thiamet G treated HeLa. The localization of YTHDF1/3 shows not significantly different among cell lines. Scale bars: 10 μm. The experiment was repeated twice with similar results. (f) Immunostaining of Flag-tagged WT-YTHDF1/3 and YTHDF1/3-Mut in HEK293T and HeLa. The localization of YTHDF1/3 shows not significantly different among cell lines or between WT and Mut. Scale bars: 10 μm. The experiment was repeated twice with similar results.
Extended Data Fig. 7 Features of YTHDF1/3 PAR-CLIP data and mRNA-seq.
(a) Immunoblotting of Ythdf1/3 knockdown HEK293T and HeLa rescued by YTHDF1 or YTHDF3. Labelled transfection conditions were used for YTHDF1/3 PAR-CLIP and RNA-seq. The experiment was performed once. (b) FPKM of YTHDFs in HEK293T (b) and HeLa (c). Ctrl, Ythdf1/3 knockdown. WT, Ythdf1/3 knockdown rescued by WT-YTHDF1/3. Mut, Ythdf1/3 knockdown rescued by YTHDF1/3-Mut. Cumulative distribution log2-fold changes of mRNA input between Ythdf1/3 knockdown and Ythdf1/3 knockdown rescued by WT-YTHDF1/3 (d, f) or YTHDF1/3-Mut (e, g) in HEK293T (d, e) and HeLa (f, g) for non-target (grey) and YTHDF1/3 target (red). Distribution with boxplot and p values were calculated using two-sided t-test. Boxplot bounds depict quartile 1, median and quartile 3, with whiskers at 1.5× interquartile range. n = 58813 genes in total. The experiment was performed with 3 independent replicates. (h) Translation efficiency analysis of N-YTHDF1-λ and N-YTHDF3-λ treated with 1 or 9 g/L glucose in HeLa and the F-luc/R-luc was normalized by respective λ group. Error bars, mean ± s.d., n = 3 biologically independent repeats. Significance was calculated using two-sided t-test. (i) Distribution of translation efficiency of mRNA in HeLa cultured with no glucose or high glucose medium. n = 4655 genes and the experiment was performed with 3 independent replicates. (j) Distribution of log2-fold changes of translation efficiency of YTHDF1 target mRNAs in HeLa cultured with no glucose or high glucose medium. n = 1095 and 2268 genes. The experiment was performed with 3 independent replicates. (k) Distribution of log2-fold changes of translation efficiency of mRNAs containing different m6A numbers in HeLa cultured with no glucose or high glucose medium. n = 1694, 600, 1054 and 1306 genes. The experiment was performed with 3 independent replicates. Distribution with boxplot and p values were calculated using two-sided t-test. Boxplot bounds depict quartile 1, median and quartile 3, with whiskers at 1.5× interquartile range and outlier points.
Extended Data Fig. 8 Dynamic O-GlcNAcylation of YTHDF1/3 regulated m6A mRNA translation efficiency in a cell cycle-dependent manner.
(a) Detailed description of Fig. 6a. mRNAs in YTHDF1 PAR-CLIP and IP dataset were enriched by YTHDF1 PAR-CLIP or YTHDF1 IP in HeLa. mRNAs in YTHDFs iCLIP dataset were enriched by YTHDF1/2/3 iCLIP in HeLa. Translation efficiency in dataset was calculated by bulk RNA-seq and ribosome profiling seq results. Three datasets were combined to analyse the translation efficiency of m6A containing mRNAs. (b) Distribution of log2-fold changes of translation efficiency of mRNAs containing different m6A numbers between M and S phase. The dataset of m6A-modified mRNA in HeLa cells is generated by iCLIP. n = 4,384 genes in total. Boxplot bounds depict quartile 1, median and quartile 3, with whiskers at 1.5× interquartile range and outlier points. The analysis was performed using previously reported dataset (X. Wang et al.33, S. Zaccara et al.35 and J. Park et al.72) with 3 independent replicates. (c) O-GlcNAcylation stoichiometry analysis for YTHDF1 in HeLa at different cell cycle stages by double thymidine release. YTHDF1* denotes samples were performed O-GlcNAcylation stoichiometry analysis and immunoblotting by YTHDF1 antibodies. Arrowheads indicates the O-GlcNAcylation modified proteins. Quantification analysis was shown in Fig. 5g. (d) Immunoblotting of HeLa synchronized to M phase by nocodazole and released for 14 h. The experiment was repeated twice with similar results. (e) Immunoblotting for the cell cycle marker proteins in (f). H3pS10, M phase. Cyclin A2, late G1-S-G2. p-Cyclin E1-T395, G1. Cyclin B1, G2-M. The experiment was repeated twice with similar results. (f) Immunostaining of endogenous YTHDF1 in HeLa released from nocodazole for 4 (M), 7 (early G1) or 11 h (late G1). The localization of YTHDF1 shows not significantly different though different cell cycles. Scale bars: 10 μm. The experiment was repeated twice with similar results.
Extended Data Fig. 9 O-GlcNAcylation of YTHDF1 increased its dynamic nature in stress granules.
Immunostaining of EGFP–WT-YTHDF1 and EGFP–YTHDF1-Mut treated with or without OSMI-1 (a) or Thiamet G (b) in HEK293T (a) and HeLa (b). Scale bars: 10 μm. (c, d) Quantification result of SGs per cell of (a-b), n = 30 cells per condition from 3 independent experiments. Boxplot bounds depict quartile 1, median and quartile 3, with whiskers at 1.5× interquartile range. Significance was calculated using two-sided t-test. (e) Immunoblotting of expression control of (a). (f) Immunoblotting of expression control of (b). (g) Validation of Ythdf1/3 knockdown efficiency in HeLa by immunoblotting. The experiment was repeated twice with similar results. (h) Immunostaining of G3BP1 and YTHDF1 in parental and Ythdf1/3 knockdown HeLa treated with 0.6 M sorbitol stress. Scale bars: 10 μm. (i) Quantification result of SGs per cell of (h), n = 60 cells per condition from 3 independent experiments. Error bar, mean ± s.d. Significance was calculated using two-sided t-test. (j) Quantification result of fraction of YTHDF1 out of SGs of Fig. 6e, n = 69, 55 cells for WT and Mut group from 3 independent experiments. Error bar, mean ± s.d. Significance is calculated using two-sided t-test. (k) Quantification result of fraction of G3BP1 in SGs of Fig. 6e, n = 42, 37 cells for WT and Mut group from 3 independent experiments. Error bar, mean ± s.d. Significance was calculated using two-sided t-test. (l-n) FRAP analysis of EGFP–WT-YTHDF1 and EGFP–YTHDF1-Mut under 0.2 M NaCl stress in HEK293T. Scale bars: 1 μm. Error bar, mean ± s.d., n = 3 SGs per condition from 3 independent experiments. Significance was calculated using two-sided t-test. The white circles represent the photobleaching region. (o, p) O-GlcNAcylation stoichiometry analysis for YTHDF1 in HEK293T under sorbitol stress treatment and recovery. Arrowheads indicates the O-GlcNAcylation modified proteins. The experiment was performed once. YTHDF1* denotes samples were performed O-GlcNAcylation stoichiometry analysis and immunoblotting by YTHDF1 antibodies.
Extended Data Fig. 10 O-GlcNAcylation of YTHDF1 increased the dynamic nature of stress granules.
a–e, FRAP analysis of mCherry-WT-YTHDF1/YTHDF1-Mut and EGFP-G3BP1 treated with 0.6 M sorbitol stress in Ythdf1/3 knockdown HeLa. YTHDF1-Mut shows lower and slower recovery in fluorescence than WT-YTHDF1 both in YTHDF1 itself and SGs (G3BP1 as marker). Scale bars: 1 μm. Error bar, mean ± s.d., n = 3 SGs per condition from 3 independent experiments. Significance was calculated using two-sided t-test. The white circles represent the photobleaching region. (f, g) Time lapse imaging of mCherry-WT-YTHDF1/YTHDF1-Mut and EGFP-G3BP1 released from 0.6 M sorbitol stress in living Ythdf1/3 knockdown HeLa cells. Scale bars: 10 μm. Error bar, mean ± s.d., n = 7, 6 cells for WT and Mut group from 3 independent experiments. Significance was calculated using two-sided t-test. (h–j) FRAP analysis of Cy3-m6A-RNA probe, BFP-G3BP1 and EGFP–YTHDF1 or O-GlcNAcylated EGFP–YTHDF1 in the droplets in vitro. Scale bars: 5 μm. Error bar, mean ± s.d., n = 3 droplets per condition from 3 independent experiments. Significance was calculated using a two-sided t-test. OG denoted O-GlcNAcylated form by OGT reaction. The white circles represent the photobleaching region.
Supplementary information
Supplementary Data 1
Post-translational modifications identified on YTHDFs.
Supplementary Data 2
Interaction proteins of YTHDF1 and YTHDF3 by IP-MS.
Supplementary Data 3
CLIP peaks of the wild type and mutant.
Supplementary Data 4
RiboLace reads.
Supplementary Data 5
Translation efficiency analysis of m6A mRNAs in different glucose concentrations.
Supplementary Data 6
Translation efficiency analysis of cell cycle by reported RNA-seq datasets in cell cycles.
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Chen, Y., Wan, R., Zou, Z. et al. O-GlcNAcylation determines the translational regulation and phase separation of YTHDF proteins. Nat Cell Biol 25, 1676–1690 (2023). https://doi.org/10.1038/s41556-023-01258-x
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DOI: https://doi.org/10.1038/s41556-023-01258-x
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