Protein arginine methyltransferase 6 is a novel substrate of protein arginine methyltransferase 1

Figure 6 Methylation by protein arginine methyltransferase 1 may regulate the enzymatic activity of protein arginine methyltransferase 6. A: Radioactive methyltransferase assay was performed to show activity of protein arginine methyltransferase (PRMT) 6 wild type and PRMT6 mutants on histone H3 (1-31). 0.6 M PRMT6 mutant and 30 µM 14C-S-adenosyl methionine (SAM) were incubated with 0 to 200 µM of H3 (1-31). After the enzyme was added, the reaction was continuously incubated for 60 min and then quenched by spreading the mixture onto P81 filter paper. Following washing and drying the paper, liquid scintillation was performed to quantify the methylated products. The data were fit to the Michaelis-Menten curve; B: Radioactive methyltransferase assay was performed to show histone H3 methylation level change as PRMT1 concentration change. Different concentration of PRMT1 was incubated with 30 µM 14C-SAM and 1 µM PRMT6 for 1 h, histone H3 was added and incubated for another 1 h. The systems were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and the phosphor image was captured after 72 h; C: Histone H3 methylation of HEK293T cell lysate was detected by western blot using anti-H3R2me2a antibody. HEK293T cell was transfected with HA-PRMT1 or control and cell lysate was extracted; D: Radioactive methyltransferase assay was performed to show histone H4 methylation level change as PRMT6 concentration change. Different concentration of PRMT6 was incubated with PRMT1 and 30 µM 14C-SAM, then 1 µM histone H4 was added and incubated for another 1 h. The systems were separated by 12% SDS-PAGE gel and the phosphor image was captured after 72 h. PRMT: Protein arginine methyltransferase.