Acellular METTL3 Methylation Assay

METTL3-including complexes were immunoprecipitated from cellular lysate obtained after sonication and the use of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) buffer (40 mM HEPES [pH 7.4], 120 mM NaCl, 1% CHAPS, and 1 mM EDTA, supplemented with protease and phosphatase inhibitors). IPs were performed using the Catch and Release v2.0 reversible IP system (Merck, France) and anti-METTL3 (Abcam, France). Immunoglobulin G (IgG) (Abcam, France) was used as a control. Elutions from IP were performed using the non-denaturing elution buffer according to the manufacturer’s instructions. Then, 30 μL of elution was used in the METTL3 enzymatic assay. The METTL3 enzymatic assay was conducted in reaction buffer (20 mM Tris [pH 7.5], 1 mM DTT, 0.01% Triton X-100, and 40 U/100 mL of RNaseOUT buffer). The reaction mixture contained unmethylated mimic miR-200b-3p with biotin tag and S-adenosyl methionine (SAM). Enzymatic assay reactions were incubated overnight at room temperature on a shaker. After streptavidin isolation, the presence of N6-adenosine methylation was determined by a dot blot. Dots were then incubated with anti-m6A and anti-adenosine (as a loading control) antibodies overnight. For signal detection, secondary horseradish peroxidase (HRP) antibodies were used and signal was detected on ChemiDoc MP (Bio-Rad, France).