Exposure on cell surface and extensive arginine methylation of ewing sarcoma (EWS) protein

In contrast to the knowledge regarding the function of chimeric Ewing sarco ma (EWS) fusion proteins that arise from chromosomal translocation, the cel lular function of the RNA binding EWS protein is poorly characterized. EWS protein had been found mainly in the nucleus. In this report we show that E WS protein is not only found in the nucleus and cytosol but also on cell. s urfaces. After cell-surface biotinylation, isoelectric focusing of membrane fraction, avidin-agarose extraction of biotinylated proteins, and SDS-poly acrylamide gel electrophoresis, EWS protein was identified by matrix-assist ed laser desorption ionization and nanoelectro-spray tandem mass spectromet ry of in-gel-digested peptides. These analyses revealed that the protein, h aving repeated RGG motifs, is extensively asymmetrically dimethylated on ar ginine residues, the sites of which have been mapped by mass spectrometric methods. Out of a total of 30 Arg-Gly sequences, 29 arginines were found to be at least partially methylated. The Arg-Gly-Gly sequence was present in 21 of the 29 methylation sites, and in contrast to other methylated protein s, only 11 (38%) methylated arginine residues were found in the Gly-Arg-Gly sequence. The presence of Gly on the C-terminal side of the arginine resid ue seems to be a prerequisite for recognition by a protein-arginine N-methy ltransferase (PRMT) catalyzing this asymmetric dimethylation reaction. One monomethylarginine and no symmetrically methylated arginine residue was fou nd. The present findings imply that RNA-binding EWS protein shuttles from t he nucleus to the cell surface in a methylated form, the role of which is d iscussed.