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. 1992 Sep;90(3):1061–1066. doi: 10.1172/JCI115921

Purification and partial sequencing of the nuclear autoantigen RA33 shows that it is indistinguishable from the A2 protein of the heterogeneous nuclear ribonucleoprotein complex.

G Steiner 1, K Hartmuth 1, K Skriner 1, I Maurer-Fogy 1, A Sinski 1, E Thalmann 1, W Hassfeld 1, A Barta 1, J S Smolen 1
PMCID: PMC329965  PMID: 1522214

Abstract

RA33 is a nuclear autoantigen with an apparent molecular mass of 33 kD. Autoantibodies against RA33 are found in about 30% of sera from RA patients, but only occasionally in sera from patients with other connective tissue diseases. To characterize RA33, the antigen was purified from HeLa cell nuclear extracts to more than 90% homogeneity by affinity chromatography on heparin-Sepharose and by chromatofocusing. Sequence analysis of five tryptic peptides revealed that their sequences matched corresponding sequences of the A2 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex. Furthermore, RA33 was shown to be present in the 40S hnRNP complex and to behave indistinguishably from A2 in binding to single stranded DNA. In summary, these data strongly indicate that RA33 and A2 are the same protein, and thus identify on a molecular level a new autoantigen.

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Selected References

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

  1. Alspaugh M. A., Henle G., Lennette E. T., Henle W. Elevated levels of antibodies to Epstein-Barr virus antigens in sera and synovial fluids of patients with rheumatoid arthritis. J Clin Invest. 1981 Apr;67(4):1134–1140. doi: 10.1172/JCI110127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bandziulis R. J., Swanson M. S., Dreyfuss G. RNA-binding proteins as developmental regulators. Genes Dev. 1989 Apr;3(4):431–437. doi: 10.1101/gad.3.4.431. [DOI] [PubMed] [Google Scholar]
  3. Barnett S. F., Northington S. J., LeStourgeon W. M. Isolation and in vitro assembly of nuclear ribonucleoprotein particles and purification of core particle proteins. Methods Enzymol. 1990;181:293–307. doi: 10.1016/0076-6879(90)81130-m. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Burd C. G., Swanson M. S., Görlach M., Dreyfuss G. Primary structures of the heterogeneous nuclear ribonucleoprotein A2, B1, and C2 proteins: a diversity of RNA binding proteins is generated by small peptide inserts. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9788–9792. doi: 10.1073/pnas.86.24.9788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buvoli M., Cobianchi F., Bestagno M. G., Mangiarotti A., Bassi M. T., Biamonti G., Riva S. Alternative splicing in the human gene for the core protein A1 generates another hnRNP protein. EMBO J. 1990 Apr;9(4):1229–1235. doi: 10.1002/j.1460-2075.1990.tb08230.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Buvoli M., Cobianchi F., Biamonti G., Riva S. Recombinant hnRNP protein A1 and its N-terminal domain show preferential affinity for oligodeoxynucleotides homologous to intron/exon acceptor sites. Nucleic Acids Res. 1990 Nov 25;18(22):6595–6600. doi: 10.1093/nar/18.22.6595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Choi Y. D., Dreyfuss G. Isolation of the heterogeneous nuclear RNA-ribonucleoprotein complex (hnRNP): a unique supramolecular assembly. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7471–7475. doi: 10.1073/pnas.81.23.7471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Choi Y. D., Grabowski P. J., Sharp P. A., Dreyfuss G. Heterogeneous nuclear ribonucleoproteins: role in RNA splicing. Science. 1986 Mar 28;231(4745):1534–1539. doi: 10.1126/science.3952495. [DOI] [PubMed] [Google Scholar]
  10. Cobianchi F., SenGupta D. N., Zmudzka B. Z., Wilson S. H. Structure of rodent helix-destabilizing protein revealed by cDNA cloning. J Biol Chem. 1986 Mar 15;261(8):3536–3543. [PubMed] [Google Scholar]
  11. Economidis I. V., Pederson T. Structure of nuclear ribonucleoprotein: heterogeneous nuclear RNA is complexed with a major sextet of proteins in vivo. Proc Natl Acad Sci U S A. 1983 Mar;80(6):1599–1602. doi: 10.1073/pnas.80.6.1599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fritzler M. J., Ali R., Tan E. M. Antibodies from patients with mixed connective tissue disease react with heterogeneous nuclear ribonucleoprotein or ribonucleic acid (hnRNP/RNA) of the nuclear matrix. J Immunol. 1984 Mar;132(3):1216–1222. [PubMed] [Google Scholar]
  13. Gelpi C., Rodriguez-Sanchez J. L., Hardin J. A. Purification of hnRNP from HeLa cells with a monoclonal antibody and its application in ELISA: detection of autoantibodies. Clin Exp Immunol. 1988 Feb;71(2):281–288. [PMC free article] [PubMed] [Google Scholar]
  14. Hannestad K., Stollar B. D. Certain rheumatoid factors react with nucleosomes. Nature. 1978 Oct 19;275(5681):671–673. doi: 10.1038/275671a0. [DOI] [PubMed] [Google Scholar]
  15. Harris E. D., Jr Rheumatoid arthritis. Pathophysiology and implications for therapy. N Engl J Med. 1990 May 3;322(18):1277–1289. doi: 10.1056/NEJM199005033221805. [DOI] [PubMed] [Google Scholar]
  16. Hassfeld W., Steiner G., Hartmuth K., Kolarz G., Scherak O., Graninger W., Thumb N., Smolen J. S. Demonstration of a new antinuclear antibody (anti-RA33) that is highly specific for rheumatoid arthritis. Arthritis Rheum. 1989 Dec;32(12):1515–1520. doi: 10.1002/anr.1780321204. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Lerner M. R., Steitz J. A. Antibodies to small nuclear RNAs complexed with proteins are produced by patients with systemic lupus erythematosus. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5495–5499. doi: 10.1073/pnas.76.11.5495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Leser G. P., Escara-Wilke J., Martin T. E. Monoclonal antibodies to heterogeneous nuclear RNA-protein complexes. The core proteins comprise a conserved group of related polypeptides. J Biol Chem. 1984 Feb 10;259(3):1827–1833. [PubMed] [Google Scholar]
  20. Maniatis T., Reed R. The role of small nuclear ribonucleoprotein particles in pre-mRNA splicing. Nature. 1987 Feb 19;325(6106):673–678. doi: 10.1038/325673a0. [DOI] [PubMed] [Google Scholar]
  21. Mayrand S. H., Pederson T. Crosslinking of hnRNP proteins to pre-mRNA requires U1 and U2 snRNPs. Nucleic Acids Res. 1990 Jun 11;18(11):3307–3318. doi: 10.1093/nar/18.11.3307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Montecucco C., Caporali R., Negri C., de Gennaro F., Cerino A., Bestagno M., Cobianchi F., Astaldi-Ricotti G. C. Antibodies from patients with rheumatoid arthritis and systemic lupus erythematosus recognize different epitopes of a single heterogeneous nuclear RNP core protein. Possible role of cross-reacting antikeratin antibodies. Arthritis Rheum. 1990 Feb;33(2):180–186. doi: 10.1002/art.1780330205. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Sierakowska H., Szer W., Furdon P. J., Kole R. Antibodies to hnRNP core proteins inhibit in vitro splicing of human beta-globin pre-mRNA. Nucleic Acids Res. 1986 Jul 11;14(13):5241–5254. doi: 10.1093/nar/14.13.5241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Simon M. M., Prester M., Kramer M. D., Fruth U. An inhibitor specific for the mouse T-cell associated serine proteinase 1 (TSP-1) inhibits the cytolytic potential of cytoplasmic granules but not of intact cytolytic T cells. J Cell Biochem. 1989 May;40(1):1–13. doi: 10.1002/jcb.240400102. [DOI] [PubMed] [Google Scholar]
  26. Swanson M. S., Dreyfuss G. Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities. Mol Cell Biol. 1988 May;8(5):2237–2241. doi: 10.1128/mcb.8.5.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Swanson M. S., Dreyfuss G. RNA binding specificity of hnRNP proteins: a subset bind to the 3' end of introns. EMBO J. 1988 Nov;7(11):3519–3529. doi: 10.1002/j.1460-2075.1988.tb03228.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Swanson M. S., Nakagawa T. Y., LeVan K., Dreyfuss G. Primary structure of human nuclear ribonucleoprotein particle C proteins: conservation of sequence and domain structures in heterogeneous nuclear RNA, mRNA, and pre-rRNA-binding proteins. Mol Cell Biol. 1987 May;7(5):1731–1739. doi: 10.1128/mcb.7.5.1731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tan E. M., Chan E. K., Sullivan K. F., Rubin R. L. Antinuclear antibodies (ANAs): diagnostically specific immune markers and clues toward the understanding of systemic autoimmunity. Clin Immunol Immunopathol. 1988 May;47(2):121–141. doi: 10.1016/0090-1229(88)90066-9. [DOI] [PubMed] [Google Scholar]
  30. Van Venrooij W. J., Sillekens P. T. Small nuclear RNA associated proteins: autoantigens in connective tissue diseases. Clin Exp Rheumatol. 1989 Nov-Dec;7(6):635–645. [PubMed] [Google Scholar]
  31. Venables P. Epstein-Barr virus infection and autoimmunity in rheumatoid arthritis. Ann Rheum Dis. 1988 Apr;47(4):265–269. doi: 10.1136/ard.47.4.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]

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