Spliceosome Sm proteins D1, D3, and B/B' are asymmetrically dimethylated at arginine residues in the nucleus |
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Authors: | Miranda Tina Branscombe Khusial Permanan Cook Jeffry R Lee Jin-Hyung Gunderson Samuel I Pestka Sidney Zieve Gary W Clarke Steven |
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Institution: | a Department of Chemistry and Biochemistry, Molecular Biology Institute, UCLA, Los Angeles, CA 90095-1569, USA b Department of Pathology, SUNY Stony Brook, Stony Brook, NY 11794-8691, USA c Department of Molecular Genetics, Microbiology, and Immunology, Robert Wood Johnson Medical School-UMDNJ, Piscataway, NJ 08854, USA d Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854, USA |
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Abstract: | 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. |
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Keywords: | Protein post-translational modification Methylation Methyltransferase Spliceosome Splicing |
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