The multifunctional
Ewing Sarcoma (
EWS) protein, a member of a large family of
RNA-binding proteins, is extensively asymmetrically dimethylated at
arginine residues within RGG consensus sequences. Using
recombinant proteins we examined whether
type I protein arginine methyltransferase (PRMT)1 or 3 is responsible for asymmetric dimethylations of the
EWS protein. After in vitro methylation of the
EWS protein by GST-PRMT1, we identified 27 dimethylated
arginine residues out of 30 potential methylation sites by mass spectrometry-based techniques (MALDI-TOF MS and MS/MS). Thus, PRMT1 recognizes most if not all methylation sites of the
EWS protein. With GST-PRMT3, however, only nine dimethylated arginines, located mainly in the C-terminal region of
EWS protein, could be assigned, indicating that structural determinants prevent complete methylation. In contrary to previous reports this study also revealed that
trypsin is able to cleave after methylated arginines. Pull-down experiments showed that endogenous
EWS protein binds efficiently to GST-PRMT1 but less to GST-PRMT3, which is in accordance to the in vitro methylation results. Furthermore, methylation of a
peptide containing different methylation sites revealed differences in the site selectivity as well as in the kinetic properties of GST-PRMT1 and GST-PRMT3. Kinetic differences due to an inhibition effect of the methylation inhibitor S-adenosyl-L-
homocysteine could be excluded by determining the corresponding K(i) values of the two
enzymes and the K(d) values for the methyl donor
S-adenosyl-L-methionine. The study demonstrates the strength of MS-based methods for a qualitative and quantitative analysis of enzymic
arginine methylation, a posttranslational modification that becomes more and more the object of investigations.