title: Spliceosome Sm proteins D1, D3, and B/B' are asymmetrically dimethylated at arginine residues in the nucleus.
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 omega-NG-monomethylarginine and symmetric omega-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 omega-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 omega-NG-monomethylarginine and symmetric omega-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 omega-NG,NG'-dimethylarginine and asymmetric omega-NG,NG-dimethylarginine when reactions were done in a Tris/HCl buffer, but only detected symmetric omega-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.
authors.affiliation: Department of Chemistry and Biochemistry, Molecular Biology Institute, UCLA, Los Angeles, CA 90095-1569, USA.
authors.forename: Tina Branscombe
authors.initials: TB
authors.lastname: Miranda
authors.forename: Permanan
authors.initials: P
authors.lastname: Khusial
authors.forename: Jeffry R
authors.initials: JR
authors.lastname: Cook
authors.forename: Jin-Hyung
authors.initials: JH
authors.lastname: Lee
authors.forename: Samuel I
authors.initials: SI
authors.lastname: Gunderson
authors.forename: Sidney
authors.initials: S
authors.lastname: Pestka
authors.forename: Gary W
authors.initials: GW
authors.lastname: Zieve
authors.forename: Steven
authors.initials: S
authors.lastname: Clarke
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002457:Cell Extracts / Q000737:chemistry; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry* / Q000378:metabolism*; D017381:Spliceosomes / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D002457:Cell Extracts; D017411:Ribonucleoproteins, Small Nuclear; D001120:Arginine