Spliceosome Sm proteins D1, D3, and B/B′ are asymmetrically dimethylated at arginine residues in the nucleus

• Published in: Biochemical and biophysical research communications 2004-10, Vol.323 (2), p.382-387
• Main Authors: Miranda, Tina Branscombe, Khusial, Permanan, Cook, Jeffry R., Lee, Jin-Hyung, Gunderson, Samuel I., Pestka, Sidney, Zieve, Gary W., Clarke, Steven
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Arginine - chemistry; Arginine - metabolism; Binding Sites; Cell Extracts - chemistry; Cell Line; Cell Nucleus - chemistry; Cell Nucleus - metabolism; Fibroblasts - metabolism; Fibroblasts - ultrastructure; HeLa Cells; Humans; Methylation; Methyltransferase; Mice; Molecular Sequence Data; Protein Binding; Protein post-translational modification; Ribonucleoproteins, Small Nuclear - chemistry; Ribonucleoproteins, Small Nuclear - metabolism; Spliceosome; Spliceosomes - chemistry; Spliceosomes - metabolism; Splicing; Structure-Activity Relationship
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2004.08.107

We report a novel modification of spliceosome proteins Sm D1, Sm D3, and Sm B/B′. L292 mouse fibroblasts were labeled in vivo with [3H]methionine. Sm D1, Sm D3, and Sm B/B′ 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′ 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′ isolated from nuclear fractions were all found to contain ω-NG-monomethylarginine and symmetric ω-NG,NG′-dimethylarginine, modifications that have been previously described. In addition, Sm D1, Sm D3, and Sm B/B′ were also found to contain asymmetric ω-NG,NG-dimethylarginine in these nuclear fractions. Analysis of Sm B/B′ from cytosolic fractions and Sm B/B′ and Sm D1 from cytosolic 6S complexes showed only the presence of ω-NG-monomethylarginine and symmetric ω-NG,NG′-dimethylarginine. These results indicate that Sm D1, Sm D3, and Sm B/B′ 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 ω-NG,NG′-dimethylarginine and asymmetric ω-NG,NG-dimethylarginine when reactions were done in a Tris/HCl buffer, but only detected symmetric ω-NG,NG′-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.