Fig. 2: OGT mediates O-GlcNAcylation of METTL3 on Thr186/Ser192/Ser193.

A Co-IP assay of the physical interaction between endogenous METTL3 and OGT in Huh-7 cells. B Flag-METTL3 and HA-OGT were transfected into Huh-7 cells. Cell extracts were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with the indicated antibodies. C Pull-down assays were performed to observe the direct interaction between METTL3 and OGT in vitro. Immunoblot analysis and Coomassie blue staining are shown. D Immunofluorescence staining of METTL3 and OGT in Huh-7 cells. Nuclei were counterstained with DAPI. Scale bar: 10 μm. E Schematic representation of the domain structure of METTL3. Full-length METTL3 consists of two domains, a C-terminal region (1–259 aa, ΔC) and the methyltransferase domain (260–580 aa, ΔN). F The interactions between HA-OGT and Flag-METTL3 (WT, ΔC, ΔN) were verified by Co-IP in HEK293 cells. G LC-MS identified Ser193 as the METTL3 O-GlcNAcylation site, which corresponded to O-GlcNAcylated METTL3 peptide AEQDLTTVTTFASSLASGLASSASEPAK. H Cross-species METTL3 sequence alignment. I HEK293 cells were transfected with Flag-tagged METTL3 (WT, T186A, S192A, S193A, or T186A/S192A/S193A). An sWGA pull-down assay was used to identify METTL3 O-GlcNAcylation sites. J, K Huh-7 cells were transfected with Flag-METTL3 WT, 3A mutant, or vector, followed by 25 μM TMG treatment for 12 h. sWGA pull-down (J) and IP (K) assays were used to identify O-GlcNAcylation sites.