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.
From: O-GlcNAcylation determines the translational regulation and phase separation of YTHDF proteins
(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.
