Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 May;15(5):2800–2808. doi: 10.1128/mcb.15.5.2800

In vivo and in vitro arginine methylation of RNA-binding proteins.

Q Liu 1, G Dreyfuss 1
PMCID: PMC230511  PMID: 7739561

Abstract

Heterogenous nuclear ribonucleoproteins (hnRNPs) bind pre-mRNAs and facilitate their processing into mRNAs. Many of the hnRNPs undergo extensive posttranslational modifications including methylation on arginine residues. hnRNPs contain about 65% of the total NG,NG-dimethylarginine found in the cell nucleus. The role of this modification is not known. Here we identify the hnRNPs that are methylated in HeLa cells and demonstrate that most of the pre-mRNA-binding proteins receive this modification. Using recombinant human hnRNP A1 as a substrate, we have partially purified and characterized a protein-arginine N-methyltransferase specific for hnRNPs from HeLa cells. This methyltransferase can methylate the same subset of hnRNPs in vitro as are methylated in vivo. Furthermore, it can also methylate other RNA-binding proteins that contain the RGG motif RNA-binding domain. This activity is evolutionarily conserved from lower eukaryotes to mammals, suggesting that methylation has a significant role in the function of RNA-binding proteins.

Full Text

The Full Text of this article is available as a PDF (398.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson J. T., Wilson S. M., Datar K. V., Swanson M. S. NAB2: a yeast nuclear polyadenylated RNA-binding protein essential for cell viability. Mol Cell Biol. 1993 May;13(5):2730–2741. doi: 10.1128/mcb.13.5.2730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baldwin G. S., Carnegie P. R. Specific enzymic methylation of an arginine in the experimental allergic encephalomyelitis protein from human myelin. Science. 1971 Feb 12;171(3971):579–581. doi: 10.1126/science.171.3971.579. [DOI] [PubMed] [Google Scholar]
  3. Beyer A. L., Christensen M. E., Walker B. W., LeStourgeon W. M. Identification and characterization of the packaging proteins of core 40S hnRNP particles. Cell. 1977 May;11(1):127–138. doi: 10.1016/0092-8674(77)90323-3. [DOI] [PubMed] [Google Scholar]
  4. Birney E., Kumar S., Krainer A. R. Analysis of the RNA-recognition motif and RS and RGG domains: conservation in metazoan pre-mRNA splicing factors. Nucleic Acids Res. 1993 Dec 25;21(25):5803–5816. doi: 10.1093/nar/21.25.5803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boffa L. C., Karn J., Vidali G., Allfrey V. G. Distribution of NG, NG,-dimethylarginine in nuclear protein fractions. Biochem Biophys Res Commun. 1977 Feb 7;74(3):969–976. doi: 10.1016/0006-291x(77)91613-8. [DOI] [PubMed] [Google Scholar]
  6. Brostoff S., Eylar E. H. Localization of methylated arginine in the A1 protein from myelin. Proc Natl Acad Sci U S A. 1971 Apr;68(4):765–769. doi: 10.1073/pnas.68.4.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bugler B., Bourbon H., Lapeyre B., Wallace M. O., Chang J. H., Amalric F., Olson M. O. RNA binding fragments from nucleolin contain the ribonucleoprotein consensus sequence. J Biol Chem. 1987 Aug 15;262(23):10922–10925. [PubMed] [Google Scholar]
  8. Burd C. G., Dreyfuss G. Conserved structures and diversity of functions of RNA-binding proteins. Science. 1994 Jul 29;265(5172):615–621. doi: 10.1126/science.8036511. [DOI] [PubMed] [Google Scholar]
  9. Carnegie P. R. Amino acid sequence of the encephalitogenic basic protein from human myelin. Biochem J. 1971 Jun;123(1):57–67. doi: 10.1042/bj1230057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Choi Y. D., Dreyfuss G. Monoclonal antibody characterization of the C proteins of heterogeneous nuclear ribonucleoprotein complexes in vertebrate cells. J Cell Biol. 1984 Dec;99(6):1997–1204. doi: 10.1083/jcb.99.6.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Christensen M. E., Beyer A. L., Walker B., Lestourgeon W. M. Identification of NG, NG-dimethylarginine in a nuclear protein from the lower eukaryote physarum polycephalum homologous to the major proteins of mammalian 40S ribonucleoprotein particles. Biochem Biophys Res Commun. 1977 Jan 24;74(2):621–629. doi: 10.1016/0006-291x(77)90348-5. [DOI] [PubMed] [Google Scholar]
  12. Christensen M. E., Fuxa K. P. The nucleolar protein, B-36, contains a glycine and dimethylarginine-rich sequence conserved in several other nuclear RNA-binding proteins. Biochem Biophys Res Commun. 1988 Sep 30;155(3):1278–1283. doi: 10.1016/s0006-291x(88)81279-8. [DOI] [PubMed] [Google Scholar]
  13. Cobianchi F., Calvio C., Stoppini M., Buvoli M., Riva S. Phosphorylation of human hnRNP protein A1 abrogates in vitro strand annealing activity. Nucleic Acids Res. 1993 Feb 25;21(4):949–955. doi: 10.1093/nar/21.4.949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cobianchi F., Karpel R. L., Williams K. R., Notario V., Wilson S. H. Mammalian heterogeneous nuclear ribonucleoprotein complex protein A1. Large-scale overproduction in Escherichia coli and cooperative binding to single-stranded nucleic acids. J Biol Chem. 1988 Jan 15;263(2):1063–1071. [PubMed] [Google Scholar]
  15. Desrosiers R., Tanguay R. M. Methylation of Drosophila histones at proline, lysine, and arginine residues during heat shock. J Biol Chem. 1988 Apr 5;263(10):4686–4692. [PubMed] [Google Scholar]
  16. Dreyfuss G., Choi Y. D., Adam S. A. Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies. Mol Cell Biol. 1984 Jun;4(6):1104–1114. doi: 10.1128/mcb.4.6.1104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dreyfuss G., Matunis M. J., Piñol-Roma S., Burd C. G. hnRNP proteins and the biogenesis of mRNA. Annu Rev Biochem. 1993;62:289–321. doi: 10.1146/annurev.bi.62.070193.001445. [DOI] [PubMed] [Google Scholar]
  18. Ghisolfi L., Joseph G., Amalric F., Erard M. The glycine-rich domain of nucleolin has an unusual supersecondary structure responsible for its RNA-helix-destabilizing properties. J Biol Chem. 1992 Feb 15;267(5):2955–2959. [PubMed] [Google Scholar]
  19. Ghosh S. K., Paik W. K., Kim S. Purification and molecular identification of two protein methylases I from calf brain. Myelin basic protein- and histone-specific enzyme. J Biol Chem. 1988 Dec 15;263(35):19024–19033. [PubMed] [Google Scholar]
  20. Girard J. P., Lehtonen H., Caizergues-Ferrer M., Amalric F., Tollervey D., Lapeyre B. GAR1 is an essential small nucleolar RNP protein required for pre-rRNA processing in yeast. EMBO J. 1992 Feb;11(2):673–682. doi: 10.1002/j.1460-2075.1992.tb05099.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Henríquez R., Blobel G., Aris J. P. Isolation and sequencing of NOP1. A yeast gene encoding a nucleolar protein homologous to a human autoimmune antigen. J Biol Chem. 1990 Feb 5;265(4):2209–2215. [PubMed] [Google Scholar]
  22. Holcomb E. R., Friedman D. L. Phosphorylation of the C-proteins of HeLa cell hnRNP particles. Involvement of a casein kinase II-type enzyme. J Biol Chem. 1984 Jan 10;259(1):31–40. [PubMed] [Google Scholar]
  23. Jong A. Y., Clark M. W., Gilbert M., Oehm A., Campbell J. L. Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins. Mol Cell Biol. 1987 Aug;7(8):2947–2955. doi: 10.1128/mcb.7.8.2947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Karn J., Vidali G., Boffa L. C., Allfrey V. G. Characterization of the non-histone nuclear proteins associated with rapidly labeled heterogeneous nuclear RNA. J Biol Chem. 1977 Oct 25;252(20):7307–7322. [PubMed] [Google Scholar]
  25. Kiledjian M., Dreyfuss G. Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box. EMBO J. 1992 Jul;11(7):2655–2664. doi: 10.1002/j.1460-2075.1992.tb05331.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kumar A., Casas-Finet J. R., Luneau C. J., Karpel R. L., Merrill B. M., Williams K. R., Wilson S. H. Mammalian heterogeneous nuclear ribonucleoprotein A1. Nucleic acid binding properties of the COOH-terminal domain. J Biol Chem. 1990 Oct 5;265(28):17094–17100. [PubMed] [Google Scholar]
  27. Kumar A., Williams K. R., Szer W. Purification and domain structure of core hnRNP proteins A1 and A2 and their relationship to single-stranded DNA-binding proteins. J Biol Chem. 1986 Aug 25;261(24):11266–11273. [PubMed] [Google Scholar]
  28. Kumar A., Wilson S. H. Studies of the strand-annealing activity of mammalian hnRNP complex protein A1. Biochemistry. 1990 Dec 4;29(48):10717–10722. doi: 10.1021/bi00500a001. [DOI] [PubMed] [Google Scholar]
  29. Käufer N. F., Simanis V., Nurse P. Fission yeast Schizosaccharomyces pombe correctly excises a mammalian RNA transcript intervening sequence. Nature. 1985 Nov 7;318(6041):78–80. doi: 10.1038/318078a0. [DOI] [PubMed] [Google Scholar]
  30. Lapeyre B., Amalric F., Ghaffari S. H., Rao S. V., Dumbar T. S., Olson M. O. Protein and cDNA sequence of a glycine-rich, dimethylarginine-containing region located near the carboxyl-terminal end of nucleolin (C23 and 100 kDa). J Biol Chem. 1986 Jul 15;261(20):9167–9173. [PubMed] [Google Scholar]
  31. Lapeyre B., Bourbon H., Amalric F. Nucleolin, the major nucleolar protein of growing eukaryotic cells: an unusual protein structure revealed by the nucleotide sequence. Proc Natl Acad Sci U S A. 1987 Mar;84(6):1472–1476. doi: 10.1073/pnas.84.6.1472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  33. Lischwe M. A., Cook R. G., Ahn Y. S., Yeoman L. C., Busch H. Clustering of glycine and NG,NG-dimethylarginine in nucleolar protein C23. Biochemistry. 1985 Oct 22;24(22):6025–6028. doi: 10.1021/bi00343a001. [DOI] [PubMed] [Google Scholar]
  34. Lischwe M. A., Ochs R. L., Reddy R., Cook R. G., Yeoman L. C., Tan E. M., Reichlin M., Busch H. Purification and partial characterization of a nucleolar scleroderma antigen (Mr = 34,000; pI, 8.5) rich in NG,NG-dimethylarginine. J Biol Chem. 1985 Nov 15;260(26):14304–14310. [PubMed] [Google Scholar]
  35. Lothstein L., Arenstorf H. P., Chung S. Y., Walker B. W., Wooley J. C., LeStourgeon W. M. General organization of protein in HeLa 40S nuclear ribonucleoprotein particles. J Cell Biol. 1985 May;100(5):1570–1581. doi: 10.1083/jcb.100.5.1570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Matunis E. L., Matunis M. J., Dreyfuss G. Characterization of the major hnRNP proteins from Drosophila melanogaster. J Cell Biol. 1992 Jan;116(2):257–269. doi: 10.1083/jcb.116.2.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Matunis M. J., Matunis E. L., Dreyfuss G. Isolation of hnRNP complexes from Drosophila melanogaster. J Cell Biol. 1992 Jan;116(2):245–255. doi: 10.1083/jcb.116.2.245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Matunis M. J., Michael W. M., Dreyfuss G. Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein. Mol Cell Biol. 1992 Jan;12(1):164–171. doi: 10.1128/mcb.12.1.164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Merrill B. M., LoPresti M. B., Stone K. L., Williams K. R. High pressure liquid chromatography purification of UP1 and UP2, two related single-stranded nucleic acid-binding proteins from calf thymus. J Biol Chem. 1986 Jan 15;261(2):878–883. [PubMed] [Google Scholar]
  40. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  41. Munroe S. H., Dong X. F. Heterogeneous nuclear ribonucleoprotein A1 catalyzes RNA.RNA annealing. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):895–899. doi: 10.1073/pnas.89.3.895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Nadler S. G., Merrill B. M., Roberts W. J., Keating K. M., Lisbin M. J., Barnett S. F., Wilson S. H., Williams K. R. Interactions of the A1 heterogeneous nuclear ribonucleoprotein and its proteolytic derivative, UP1, with RNA and DNA: evidence for multiple RNA binding domains and salt-dependent binding mode transitions. Biochemistry. 1991 Mar 19;30(11):2968–2976. doi: 10.1021/bi00225a034. [DOI] [PubMed] [Google Scholar]
  43. Najbauer J., Johnson B. A., Young A. L., Aswad D. W. Peptides with sequences similar to glycine, arginine-rich motifs in proteins interacting with RNA are efficiently recognized by methyltransferase(s) modifying arginine in numerous proteins. J Biol Chem. 1993 May 15;268(14):10501–10509. [PubMed] [Google Scholar]
  44. O'Farrell P. Z., Goodman H. M., O'Farrell P. H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. doi: 10.1016/0092-8674(77)90176-3. [DOI] [PubMed] [Google Scholar]
  45. Park K. S., Frost B., Tuck M., Ho L. L., Kim S., Paik W. K. Enzymatic methylation of in vitro synthesized apocytochrome c enhances its transport into mitochondria. J Biol Chem. 1987 Oct 25;262(30):14702–14708. [PubMed] [Google Scholar]
  46. Pederson T. Proteins associated with heterogeneous nuclear RNA in eukaryotic cells. J Mol Biol. 1974 Feb 25;83(2):163–183. doi: 10.1016/0022-2836(74)90386-6. [DOI] [PubMed] [Google Scholar]
  47. Piñol-Roma S., Choi Y. D., Matunis M. J., Dreyfuss G. Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins. Genes Dev. 1988 Feb;2(2):215–227. doi: 10.1101/gad.2.2.215. [DOI] [PubMed] [Google Scholar]
  48. Piñol-Roma S., Dreyfuss G. Cell cycle-regulated phosphorylation of the pre-mRNA-binding (heterogeneous nuclear ribonucleoprotein) C proteins. Mol Cell Biol. 1993 Sep;13(9):5762–5770. doi: 10.1128/mcb.13.9.5762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Piñol-Roma S., Dreyfuss G. Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm. Nature. 1992 Feb 20;355(6362):730–732. doi: 10.1038/355730a0. [DOI] [PubMed] [Google Scholar]
  50. Piñol-Roma S., Dreyfuss G. hnRNP proteins: localization and transport between the nucleus and the cytoplasm. Trends Cell Biol. 1993 May;3(5):151–155. doi: 10.1016/0962-8924(93)90135-n. [DOI] [PubMed] [Google Scholar]
  51. Pontius B. W., Berg P. Renaturation of complementary DNA strands mediated by purified mammalian heterogeneous nuclear ribonucleoprotein A1 protein: implications for a mechanism for rapid molecular assembly. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8403–8407. doi: 10.1073/pnas.87.21.8403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Portman D. S., Dreyfuss G. RNA annealing activities in HeLa nuclei. EMBO J. 1994 Jan 1;13(1):213–221. doi: 10.1002/j.1460-2075.1994.tb06251.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Rajpurohit R., Lee S. O., Park J. O., Paik W. K., Kim S. Enzymatic methylation of recombinant heterogeneous nuclear RNP protein A1. Dual substrate specificity for S-adenosylmethionine:histone-arginine N-methyltransferase. J Biol Chem. 1994 Jan 14;269(2):1075–1082. [PubMed] [Google Scholar]
  54. Rajpurohit R., Paik W. K., Kim S. Enzymatic methylation of heterogeneous nuclear ribonucleoprotein in isolated liver nuclei. Biochim Biophys Acta. 1992 Jul 31;1122(2):183–188. doi: 10.1016/0167-4838(92)90322-5. [DOI] [PubMed] [Google Scholar]
  55. Rawal N., Rajpurohit R., Paik W. K., Kim S. Purification and characterization of S-adenosylmethionine-protein-arginine N-methyltransferase from rat liver. Biochem J. 1994 Jun 1;300(Pt 2):483–489. doi: 10.1042/bj3000483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Reporterer M., Corbin J. L. N G ,N G ,-dimethylarginine in myosin during muscle development. Biochem Biophys Res Commun. 1971 May 7;43(3):644–650. doi: 10.1016/0006-291x(71)90663-2. [DOI] [PubMed] [Google Scholar]
  57. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  58. Siomi H., Matunis M. J., Michael W. M., Dreyfuss G. The pre-mRNA binding K protein contains a novel evolutionarily conserved motif. Nucleic Acids Res. 1993 Mar 11;21(5):1193–1198. doi: 10.1093/nar/21.5.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Siomi H., Siomi M. C., Nussbaum R. L., Dreyfuss G. The protein product of the fragile X gene, FMR1, has characteristics of an RNA-binding protein. Cell. 1993 Jul 30;74(2):291–298. doi: 10.1016/0092-8674(93)90420-u. [DOI] [PubMed] [Google Scholar]
  60. Soulard M., Della Valle V., Siomi M. C., Piñol-Roma S., Codogno P., Bauvy C., Bellini M., Lacroix J. C., Monod G., Dreyfuss G. hnRNP G: sequence and characterization of a glycosylated RNA-binding protein. Nucleic Acids Res. 1993 Sep 11;21(18):4210–4217. doi: 10.1093/nar/21.18.4210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Suzuki K., Olvera J., Wool I. G. Primary structure of rat ribosomal protein S2. A ribosomal protein with arginine-glycine tandem repeats and RGGF motifs that are associated with nucleolar localization and binding to ribonucleic acids. J Biol Chem. 1991 Oct 25;266(30):20007–20010. [PubMed] [Google Scholar]
  62. Tao J., Frankel A. D. Specific binding of arginine to TAR RNA. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2723–2726. doi: 10.1073/pnas.89.7.2723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wang C., Lazarides E., O'Connor C. M., Clarke S. Methylation of chicken fibroblast heat shock proteins at lysyl and arginyl residues. J Biol Chem. 1982 Jul 25;257(14):8356–8362. [PubMed] [Google Scholar]
  64. Wang C., Lin J. M., Lazarides E. Methylations of 70,000-Da heat shock proteins in 3T3 cells: alterations by arsenite treatment, by different stages of growth and by virus transformation. Arch Biochem Biophys. 1992 Aug 15;297(1):169–175. doi: 10.1016/0003-9861(92)90656-h. [DOI] [PubMed] [Google Scholar]
  65. Weng Z., Thomas S. M., Rickles R. J., Taylor J. A., Brauer A. W., Seidel-Dugan C., Michael W. M., Dreyfuss G., Brugge J. S. Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains. Mol Cell Biol. 1994 Jul;14(7):4509–4521. doi: 10.1128/mcb.14.7.4509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Wilk H. E., Werr H., Friedrich D., Kiltz H. H., Schäfer K. P. The core proteins of 35S hnRNP complexes. Characterization of nine different species. Eur J Biochem. 1985 Jan 2;146(1):71–81. doi: 10.1111/j.1432-1033.1985.tb08621.x. [DOI] [PubMed] [Google Scholar]
  67. Williams K. R., Stone K. L., LoPresti M. B., Merrill B. M., Planck S. R. Amino acid sequence of the UP1 calf thymus helix-destabilizing protein and its homology to an analogous protein from mouse myeloma. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5666–5670. doi: 10.1073/pnas.82.17.5666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Wong G., Müller O., Clark R., Conroy L., Moran M. F., Polakis P., McCormick F. Molecular cloning and nucleic acid binding properties of the GAP-associated tyrosine phosphoprotein p62. Cell. 1992 May 1;69(3):551–558. doi: 10.1016/0092-8674(92)90455-l. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES