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.

Source data