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. 1988 Jun;81(6):1716–1724. doi: 10.1172/JCI113511

The U2 small nuclear ribonucleoprotein particle as an autoantigen. Analysis with sera from patients with overlap syndromes.

J Craft 1, T Mimori 1, T L Olsen 1, J A Hardin 1
PMCID: PMC442616  PMID: 2968364

Abstract

We identified eight patients whose sera contained autoantibodies to the U2 small nuclear ribonucleoprotein (snRNP), an RNA protein particle involved in the splicing of newly transcribed messenger RNA. Each of these patients had an overlap syndrome that included features of either systemic lupus erythematosus (SLE), scleroderma, and/or polymyositis. We then used these sera to characterize the autoantigenic polypeptides of the U1 and U2 snRNP particles. In immunoblots, all sera contained antibodies to the B" polypeptide of the U2 snRNP. A subset of these sera that more effectively immunoprecipitated the native U2 particle contained an additional antibody system that recognized the A' polypeptide of this snRNP. Antibodies eluted from the B" protein bound the A polypeptide of the U1 snRNP and vice versa. Moreover, antibodies to the B" polypeptide were accompanied by antibodies to the 68K and C polypeptides of the U1 snRNP. Finally, the A' and B" polypeptides remained physically associated after the U2 particle was cleaved with RNase. Thus these sera contain multiple autoantibody systems that, at one level, target two physically associated antigenic polypeptides of the U2 particle and, at another, target two snRNP particles which are associated during the splicing of premessenger RNA. These linked autoantibody sets provide further evidence that intact macromolecular structures are targeted by the immune response in SLE and related diseases.

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Selected References

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  1. Billings P. B., Hoch S. O. Characterization of U small nuclear RNA-associated proteins. J Biol Chem. 1984 Oct 25;259(20):12850–12856. [PubMed] [Google Scholar]
  2. Black D. L., Chabot B., Steitz J. A. U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing. Cell. 1985 Oct;42(3):737–750. doi: 10.1016/0092-8674(85)90270-3. [DOI] [PubMed] [Google Scholar]
  3. Bohan A., Peter J. B. Polymyositis and dermatomyositis (first of two parts). N Engl J Med. 1975 Feb 13;292(7):344–347. doi: 10.1056/NEJM197502132920706. [DOI] [PubMed] [Google Scholar]
  4. Bringmann P., Lührmann R. Purification of the individual snRNPs U1, U2, U5 and U4/U6 from HeLa cells and characterization of their protein constituents. EMBO J. 1986 Dec 20;5(13):3509–3516. doi: 10.1002/j.1460-2075.1986.tb04676.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bringmann P., Rinke J., Appel B., Reuter R., Lührmann R. Purification of snRNPs U1, U2, U4, U5 and U6 with 2,2,7-trimethylguanosine-specific antibody and definition of their constituent proteins reacting with anti-Sm and anti-(U1)RNP antisera. EMBO J. 1983;2(7):1129–1135. doi: 10.1002/j.1460-2075.1983.tb01557.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chambers J. C., Keene J. D. Isolation and analysis of cDNA clones expressing human lupus La antigen. Proc Natl Acad Sci U S A. 1985 Apr;82(7):2115–2119. doi: 10.1073/pnas.82.7.2115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Conner G. E., Nelson D., Wisniewolski R., Lahita R. G., Blobel G., Kunkel H. G. Protein antigens of the RNA-protein complexes detected by anti-SM and anti-RNP antibodies found in serum of patients with systemic lupus erythematosus and related disorders. J Exp Med. 1982 Nov 1;156(5):1475–1485. doi: 10.1084/jem.156.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Habets W. J., Sillekens P. T., Hoet M. H., Schalken J. A., Roebroek A. J., Leunissen J. A., van de Ven W. J., van Venrooij W. J. Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B" antigen. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2421–2425. doi: 10.1073/pnas.84.8.2421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Habets W. J., de Rooij D. J., Hoet M. H., van de Putte L. B., van Venrooij W. J. Quantitation of anti-RNP and anti-Sm antibodies in MCTD and SLE patients by immunoblotting. Clin Exp Immunol. 1985 Feb;59(2):457–466. [PMC free article] [PubMed] [Google Scholar]
  11. Habets W. J., de Rooij D. J., Salden M. H., Verhagen A. P., van Eekelen C. A., van de Putte L. B., van Venrooij W. J. Antibodies against distinct nuclear matrix proteins are characteristic for mixed connective tissue disease. Clin Exp Immunol. 1983 Oct;54(1):265–276. [PMC free article] [PubMed] [Google Scholar]
  12. Habets W., Hoet M., Bringmann P., Lührmann R., van Venrooij W. Autoantibodies to ribonucleoprotein particles containing U2 small nuclear RNA. EMBO J. 1985 Jun;4(6):1545–1550. doi: 10.1002/j.1460-2075.1985.tb03815.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hardin J. A. The lupus autoantigens and the pathogenesis of systemic lupus erythematosus. Arthritis Rheum. 1986 Apr;29(4):457–460. doi: 10.1002/art.1780290401. [DOI] [PubMed] [Google Scholar]
  14. Hardin J. A., Thomas J. O. Antibodies to histones in systemic lupus erythematosus: localization of prominent autoantigens on histones H1 and H2B. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7410–7414. doi: 10.1073/pnas.80.24.7410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hendrick J. P., Wolin S. L., Rinke J., Lerner M. R., Steitz J. A. Ro small cytoplasmic ribonucleoproteins are a subclass of La ribonucleoproteins: further characterization of the Ro and La small ribonucleoproteins from uninfected mammalian cells. Mol Cell Biol. 1981 Dec;1(12):1138–1149. doi: 10.1128/mcb.1.12.1138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hinterberger M., Pettersson I., Steitz J. A. Isolation of small nuclear ribonucleoproteins containing U1, U2, U4, U5, and U6 RNAs. J Biol Chem. 1983 Feb 25;258(4):2604–2613. [PubMed] [Google Scholar]
  17. Kieber-Emmons T., Kohler H. Evolutionary origin of autoreactive determinants (autogens). Proc Natl Acad Sci U S A. 1986 Apr;83(8):2521–2525. doi: 10.1073/pnas.83.8.2521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kinlaw C. S., Dusing-Swartz S. K., Berget S. M. Human U1 and U2 small nuclear ribonucleoproteins contain common and unique polypeptides. Mol Cell Biol. 1982 Oct;2(10):1159–1166. doi: 10.1128/mcb.2.10.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kinlaw C. S., Robberson B. L., Berget S. M. Fractionation and characterization of human small nuclear ribonucleoproteins containing U1 and U2 RNAs. J Biol Chem. 1983 Jun 10;258(11):7181–7189. [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Liautard J. P., Sri-Widada J., Brunel C., Jeanteur P. Structural organization of ribonucleoproteins containing small nuclear RNAs from HeLa cells. Proteins interact closely with a similar structural domain of U1, U2, U4 and U5 small nuclear RNAs. J Mol Biol. 1982 Dec 15;162(3):623–643. doi: 10.1016/0022-2836(82)90392-8. [DOI] [PubMed] [Google Scholar]
  23. Mattaj I. W., De Robertis E. M. Nuclear segregation of U2 snRNA requires binding of specific snRNP proteins. Cell. 1985 Jan;40(1):111–118. doi: 10.1016/0092-8674(85)90314-9. [DOI] [PubMed] [Google Scholar]
  24. Mattaj I. W., Habets W. J., van Venrooij W. J. Monospecific antibodies reveal details of U2 snRNP structure and interaction between U1 and U2 snRNPs. EMBO J. 1986 May;5(5):997–1002. doi: 10.1002/j.1460-2075.1986.tb04314.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Matter L., Schopfer K., Wilhelm J. A., Nyffenegger T., Parisot R. F., De Robertis E. M. Molecular characterization of ribonucleoprotein antigens bound by antinuclear antibodies. A diagnostic evaluation. Arthritis Rheum. 1982 Nov;25(11):1278–1283. doi: 10.1002/art.1780251102. [DOI] [PubMed] [Google Scholar]
  26. Mattioli M., Reichlin M. Physical association of two nuclear antigens and mutual occurrence of their antibodies: the relationship of the SM and RNAprotein (MO) systems in SLE sera. J Immunol. 1973 May;110(5):1318–1324. [PubMed] [Google Scholar]
  27. Mimori T., Hardin J. A., Steitz J. A. Characterization of the DNA-binding protein antigen Ku recognized by autoantibodies from patients with rheumatic disorders. J Biol Chem. 1986 Feb 15;261(5):2274–2278. [PubMed] [Google Scholar]
  28. Mimori T., Hinterberger M., Pettersson I., Steitz J. A. Autoantibodies to the U2 small nuclear ribonucleoprotein in a patient with scleroderma-polymyositis overlap syndrome. J Biol Chem. 1984 Jan 10;259(1):560–565. [PubMed] [Google Scholar]
  29. 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]
  30. Pettersson I., Hinterberger M., Mimori T., Gottlieb E., Steitz J. A. The structure of mammalian small nuclear ribonucleoproteins. Identification of multiple protein components reactive with anti-(U1)ribonucleoprotein and anti-Sm autoantibodies. J Biol Chem. 1984 May 10;259(9):5907–5914. [PubMed] [Google Scholar]
  31. Pettersson I., Wang G., Smith E. I., Wigzell H., Hedfors E., Horn J., Sharp G. C. The use of immunoblotting and immunoprecipitation of (U) small nuclear ribonucleoproteins in the analysis of sera of patients with mixed connective tissue disease and systemic lupus erythematosus. A cross-sectional, longitudinal study. Arthritis Rheum. 1986 Aug;29(8):986–996. doi: 10.1002/art.1780290807. [DOI] [PubMed] [Google Scholar]
  32. Preliminary criteria for the classification of systemic sclerosis (scleroderma). Subcommittee for scleroderma criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. Arthritis Rheum. 1980 May;23(5):581–590. doi: 10.1002/art.1780230510. [DOI] [PubMed] [Google Scholar]
  33. Reeves W. H., Fisher D. E., Lahita R. G., Kunkel H. G. Autoimmune sera reactive with Sm antigen contain high levels of RNP-like antibodies. J Clin Invest. 1985 Feb;75(2):580–587. doi: 10.1172/JCI111734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Reeves W. H., Fisher D. E., Wisniewolski R., Gottlieb A. B., Chiorazzi N. Psoriasis and Raynaud's phenomenon associated with autoantibodies to U1 and U2 small nuclear ribonucleoproteins. N Engl J Med. 1986 Jul 10;315(2):105–111. doi: 10.1056/NEJM198607103150207. [DOI] [PubMed] [Google Scholar]
  35. Reuter R., Lehner C. F., Nigg E. A., Lührmann R. A monoclonal antibody specific for snRNPs U1 and U2. FEBS Lett. 1986 May 26;201(1):25–30. doi: 10.1016/0014-5793(86)80564-6. [DOI] [PubMed] [Google Scholar]
  36. Sharp G. C., Irvin W. S., LaRoque R. L., Velez C., Daly V., Kaiser A. D., Holman H. R. Association of autoantibodies to different nuclear antigens with clinical patterns of rheumatic disease and responsiveness to therapy. J Clin Invest. 1971 Feb;50(2):350–359. doi: 10.1172/JCI106502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sharp G. C., Irvin W. S., May C. M., Holman H. R., McDuffie F. C., Hess E. V., Schmid F. R. Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease, systematic lupus erythematosus and other rheumatic diseases. N Engl J Med. 1976 Nov 18;295(21):1149–1154. doi: 10.1056/NEJM197611182952101. [DOI] [PubMed] [Google Scholar]
  38. Sharp G. C., Irvin W. S., Tan E. M., Gould R. G., Holman H. R. Mixed connective tissue disease--an apparently distinct rheumatic disease syndrome associated with a specific antibody to an extractable nuclear antigen (ENA). Am J Med. 1972 Feb;52(2):148–159. doi: 10.1016/0002-9343(72)90064-2. [DOI] [PubMed] [Google Scholar]
  39. Sharp P. A. Splicing of messenger RNA precursors. Science. 1987 Feb 13;235(4790):766–771. doi: 10.1126/science.3544217. [DOI] [PubMed] [Google Scholar]
  40. Shero J. H., Bordwell B., Rothfield N. F., Earnshaw W. C. High titers of autoantibodies to topoisomerase I (Scl-70) in sera from scleroderma patients. Science. 1986 Feb 14;231(4739):737–740. doi: 10.1126/science.3003910. [DOI] [PubMed] [Google Scholar]
  41. Smith D. E., Fisher P. A. Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J Cell Biol. 1984 Jul;99(1 Pt 1):20–28. doi: 10.1083/jcb.99.1.20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Takano M., Agris P. F., Sharp G. C. Purification and biochemical characterization of nuclear ribonucleoprotein antigen using purified antibody from serum of a patient with mixed connective tissue disease. J Clin Invest. 1980 Jun;65(6):1449–1456. doi: 10.1172/JCI109809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Takano M., Golden S. S., Sharp G. C., Agris P. F. Molecular relationships between two nuclear antigens, ribonucleoprotein and Sm: purification of active antigens and their biochemical characterization. Biochemistry. 1981 Oct 13;20(21):5929–5936. doi: 10.1021/bi00524a001. [DOI] [PubMed] [Google Scholar]
  44. Tan E. M. Autoantibodies to nuclear antigens (ANA): their immunobiology and medicine. Adv Immunol. 1982;33:167–240. doi: 10.1016/s0065-2776(08)60836-6. [DOI] [PubMed] [Google Scholar]
  45. Tan E. M., Cohen A. S., Fries J. F., Masi A. T., McShane D. J., Rothfield N. F., Schaller J. G., Talal N., Winchester R. J. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1982 Nov;25(11):1271–1277. doi: 10.1002/art.1780251101. [DOI] [PubMed] [Google Scholar]
  46. Theissen H., Etzerodt M., Reuter R., Schneider C., Lottspeich F., Argos P., Lührmann R., Philipson L. Cloning of the human cDNA for the U1 RNA-associated 70K protein. EMBO J. 1986 Dec 1;5(12):3209–3217. doi: 10.1002/j.1460-2075.1986.tb04631.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. White P. J., Gardner W. D., Hoch S. O. Identification of the immunogenically active components of the Sm and RNP antigens. Proc Natl Acad Sci U S A. 1981 Jan;78(1):626–630. doi: 10.1073/pnas.78.1.626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wieben E. D., Rohleder A. M., Nenninger J. M., Pederson T. cDNA cloning of a human autoimmune nuclear ribonucleoprotein antigen. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7914–7918. doi: 10.1073/pnas.82.23.7914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wooley J. C., Zukerberg L. R., Chung S. Y. Polypeptide components of human small nuclear ribonucleoproteins. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5208–5212. doi: 10.1073/pnas.80.17.5208. [DOI] [PMC free article] [PubMed] [Google Scholar]

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