Fig. 4: O-GlcNAcylation stabilizes METTL3 by decreasing its ubiquitination.

A Immunoblotting of METTL3. Huh-7 cells were treated with 25 μM TMG for 12 h. Then, 100 μM CHX was added to block protein synthesis for the indicated times. The half-life of METTL3 was quantified in three independent immunoblotting experiments. METTL3 levels at 0 h were arbitrarily set to 100%. B Half-life and quantitative analysis of METTL3. Huh-7 cells were infected with OGT shRNA lentivirus and treated with 100 μM CHX for the indicated times. METTL3 levels were measured by immunoblotting (n = 3). C Half-life of Flag-METTL3 and quantitative analysis in Huh-7 cells. Huh-7 cells were transfected with Flag-METTL3-WT or -3A and treated with 100 μM CHX for the indicated times. Cell lysates were immunoblotted with anti-Flag. Data are representative of three independent experiments. Data in (A–C) were analyzed using an unpaired Student’s t-test. *P < 0.05, **P < 0.01. D, E Hepatoma cells were co-transfected with His-Ub and Flag-METTL3 (WT or 3A) and treated or untreated with TMG. Cells were treated with 20 μM MG132 for 8 h, and cell lysates were subjected to IP. Input and IP proteins were immunoblotted with indicated antibodies. FBXW7 increases the poly-ubiquitination level of METTL3 in Huh-7 (F) and PCL/PRF/5 cells (G). H Huh-7 cells were co-transfected Flag-METTL3 (WT or 3A) and HA-FBXW7, cell lysates were subjected to Co-IP to observe their interactions. I USP5 decreases the poly-ubiquitination level of METTL3 in Huh-7 cells.