dimethylated at arginine residues in the nucleus

We report a novel modification of spliceosome proteins Sm D1, Sm D3, and Sm B/B 0. L292 mouse fibroblasts were labeled in vivo with [H]methionine. Sm D1, Sm D3, and Sm B/B 0 were purified from either nuclear extracts, cytosolic extracts or a cytosolic 6S complex by immunoprecipitation of the Sm protein-containing complexes and then separation by electrophoresis on a polyacrylamide gel containing urea. The isolated Sm D1, Sm D3 or Sm B/B 0 proteins were hydrolyzed to amino acids and the products were analyzed by high-resolution cation exchange chromatography. Sm D1, Sm D3, and Sm B/B 0 isolated from nuclear fractions were all found to contain x-N-monomethylarginine and symmetric x-N,N 0 -dimethylarginine, modifications that have been previously described. In addition, Sm D1, Sm D3, and Sm B/B 0 were also found to contain asymmetric x-N,N-dimethylarginine in these nuclear fractions. Analysis of Sm B/B 0 from cytosolic fractions and Sm B/B 0 and Sm D1 from cytosolic 6S complexes showed only the presence of x-N-monomethylarginine and symmetric x-N,N 0 -dimethylarginine. These results indicate that Sm D1, Sm D3, and Sm B/B 0 are asymmetrically dimethylated and that these modified proteins are located in the nucleus. In reactions in which Sm D1 or Sm D3 was methylated in vitro with a hemagglutinin-tagged PRMT5 purified from HeLa cells, we detected both symmetric xN,N 0 -dimethylarginine and asymmetric x-N,N-dimethylarginine when reactions were done in a Tris/HCl buffer, but only detected symmetric x-N,N 0 -dimethylarginine when a sodium phosphate buffer was used. These results suggest that the activity responsible for the formation of asymmetric dimethylated arginine residues in Sm proteins is either PRMT5 or a protein associated with it in the immunoprecipitated complex. 2004 Elsevier Inc. All rights reserved.