Journal: Nature communications
Article Title: PRMT9 is a Type II methyltransferase that methylates the splicing factor SAP145
Figure Lengend Snippet: PRMT9 catalyzes symmetrical dimethylation of SAP145 at Arginine 508 ( a ) PRMT9 methylates SAP145 fragment F3 (a.a. 401–550). The in vitro methylation was performed by incubating either wild type or enzymatic mutant recombinant HA-PRMT9 (purified from Sf21 cells) with GST or GST-tag SAP145 fragments (F1–F4, as described in Fig. 3a and b ). The loading of PRMT9 was detected by western blotting using αHA antibody. ( b ) PRMT9 symmetrically dimethylates SAP145 as detected by amino acid analysis. Amino acid analysis of in vitro methylation products from wild type and enzymatic mutant GFP-PRMT9 as enzymes and GST-SAP145 (401–550) fragment as substrate. Black dashed line indicates elution of nonradiolabeled standards. The radioactive peaks elute 1–2 min before the nonradiolabeled standards due to a tritium isotope effect 39 . ( c ) PRMT9 symmetrically dimethylates SAP145 as detected by thin layer chromatography (TLC). SDMA fractions from cation-exchange chromatography of the in vitro 3 H-methylation reaction were separated using thin layer chromatography (TLC). Fractions were spotted on a cellulose plate along with the addition of 5 nmol MMA, 15 nmol ADMA, and 5 nmol SDMA internal standards. Individual standards were also spotted in adjacent lanes to determine the migration distance of each methylated arginine derivative. The origin is indicated by fraction 2. The solvent front was run near the end of the plate to fraction 32. The plate was air-dried and each lane was subsequently sliced in 5 mm fractions and counted for three 30-min counting cycles. The radioactive peaks elute 1–2 min before the nonradiolabeled standards due to a tritium isotope effect 39 . This experiment was repeated 3 times and similar migration patterns were observed. ( d ) PRMT9 methylates SAP145 at R508 in vitro . The in vitro methylation assay was performed by incubating recombinant HA-PRMT9 with a series of Arg to Lys (R to K) mutants of SAP145 fragment F3 (see Fig. 3a and b for description) for 1 h at 30° C. After exposure at −80 °C for 3 days, the membrane was stained with Coomassie blue to check the protein loading. Arrows indicate the positions of the substrates and stars indicate the positions of the recombinant HA-PRMT9.
Article Snippet: To prepare samples for high resolution cation-exchange chromatography, the hydrolyzed samples were resuspended in 50 μl of water and mixed with sodium citrate buffer (0.2 M in Na+, pH 2.2) and standards including 1 μmol each of ω-MMA (acetate salt, Sigma, M7033), SDMA (di(p-hydroxyazobenzene)-p′-sulfonate salt, Sigma, D0390) and ADMA (hydrochloride salt, Sigma, D4268).
Techniques: In Vitro, Methylation, Mutagenesis, Recombinant, Purification, Western Blot, Thin Layer Chromatography, Chromatography, Migration, Staining