Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1993 Nov 11;21(22):5130–5136. doi: 10.1093/nar/21.22.5130

Purification and characterization of human autoantibodies directed to specific regions on U1RNA; recognition of native U1RNP complexes.

R M Hoet 1, B Kastner 1, R Lührmann 1, W J van Venrooij 1
PMCID: PMC310627  PMID: 8255767

Abstract

Antibodies against naked U1RNA can be found in sera from patients with overlap syndromes of systemic lupus erythematosus (SLE) in addition to antibodies directed to the proteins of U1 ribonucleoproteins (U1RNP). We investigated the reactivity of these U1RNA specific autoantibodies with the native U1RNP particle both in vitro and inside the cell. For this purpose a method was developed to purify human autoantibodies directed to specific regions of U1RNA. The antibodies are specifically directed to either stemloop II or stemloop IV of U1RNA and do not crossreact with protein components of U1RNP. Both types of antibody are able to precipitate from cell extracts native U1snRNPs containing most, if not all, protein components. Immunofluorescence patterns indicate that the antigenic sites on the RNA, i.e. the stem of stemloop II and the loop of stemloop IV, are also available after fixation of the cells. Immunoelectron microscopy employing anti-stemloop IV antibodies and purified, complete U1snRNP particles showed that stemloop IV is located within the body of the U1RNP complex, which also comprises the Sm site and the common Sm proteins. The anti-U1RNA autoantibodies described in this paper recognize native U1RNP particles within the cell and can therefore be used as tools to study mechanisms involved in splicing of pre-mRNA.

Full text

PDF
5135

Images in this article

5132Image
on p.5132
5132Image
on p.5132
5133Image
on p.5133
5134Image
on p.5134

Selected References

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

  1. Bach M., Krol A., Lührmann R. Structure-probing of U1 snRNPs gradually depleted of the U1-specific proteins A, C and 70k. Evidence that A interacts differentially with developmentally regulated mouse U1 snRNA variants. Nucleic Acids Res. 1990 Feb 11;18(3):449–457. doi: 10.1093/nar/18.3.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boelens W. C., Jansen E. J., van Venrooij W. J., Stripecke R., Mattaj I. W., Gunderson S. I. The human U1 snRNP-specific U1A protein inhibits polyadenylation of its own pre-mRNA. Cell. 1993 Mar 26;72(6):881–892. doi: 10.1016/0092-8674(93)90577-d. [DOI] [PubMed] [Google Scholar]
  3. Deutscher S. L., Keene J. D. A sequence-specific conformational epitope on U1 RNA is recognized by a unique autoantibody. Proc Natl Acad Sci U S A. 1988 May;85(10):3299–3303. doi: 10.1073/pnas.85.10.3299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Hamm J., Dathan N. A., Scherly D., Mattaj I. W. Multiple domains of U1 snRNA, including U1 specific protein binding sites, are required for splicing. EMBO J. 1990 Apr;9(4):1237–1244. doi: 10.1002/j.1460-2075.1990.tb08231.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Hoet R. M., De Weerd P., Gunnewiek J. K., Koornneef I., Van Venrooij W. J. Epitope regions on U1 small nuclear RNA recognized by anti-U1RNA-specific autoantibodies. J Clin Invest. 1992 Nov;90(5):1753–1762. doi: 10.1172/JCI116049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hoet R. M., Koornneef I., de Rooij D. J., van de Putte L. B., van Venrooij W. J. Changes in anti-U1 RNA antibody levels correlate with disease activity in patients with systemic lupus erythematosus overlap syndrome. Arthritis Rheum. 1992 Oct;35(10):1202–1210. doi: 10.1002/art.1780351013. [DOI] [PubMed] [Google Scholar]
  9. Hoet R. M., van Venrooij W. J. B-cell epitopes of RNA autoantigens. Mol Biol Rep. 1992 Jun;16(3):199–205. doi: 10.1007/BF00464708. [DOI] [PubMed] [Google Scholar]
  10. Kastner B., Kornstädt U., Bach M., Lührmann R. Structure of the small nuclear RNP particle U1: identification of the two structural protuberances with RNP-antigens A and 70K. J Cell Biol. 1992 Feb;116(4):839–849. doi: 10.1083/jcb.116.4.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kastner B., Lührmann R. Electron microscopy of U1 small nuclear ribonucleoprotein particles: shape of the particle and position of the 5' RNA terminus. EMBO J. 1989 Jan;8(1):277–286. doi: 10.1002/j.1460-2075.1989.tb03374.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Krol A., Westhof E., Bach M., Lührmann R., Ebel J. P., Carbon P. Solution structure of human U1 snRNA. Derivation of a possible three-dimensional model. Nucleic Acids Res. 1990 Jul 11;18(13):3803–3811. doi: 10.1093/nar/18.13.3803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lührmann R., Kastner B., Bach M. Structure of spliceosomal snRNPs and their role in pre-mRNA splicing. Biochim Biophys Acta. 1990 Nov 30;1087(3):265–292. doi: 10.1016/0167-4781(90)90001-i. [DOI] [PubMed] [Google Scholar]
  14. Scherly D., Boelens W., Dathan N. A., van Venrooij W. J., Mattaj I. W. Major determinants of the specificity of interaction between small nuclear ribonucleoproteins U1A and U2B'' and their cognate RNAs. Nature. 1990 Jun 7;345(6275):502–506. doi: 10.1038/345502a0. [DOI] [PubMed] [Google Scholar]
  15. Sillekens P. T., Habets W. J., Beijer R. P., van Venrooij W. J. cDNA cloning of the human U1 snRNA-associated A protein: extensive homology between U1 and U2 snRNP-specific proteins. EMBO J. 1987 Dec 1;6(12):3841–3848. doi: 10.1002/j.1460-2075.1987.tb02721.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Slobbe R., Van Esch B., Kveder T., Van Venrooij W. J. The use of adenovirus-infected HeLa cells for the detection of low titer autoantibodies. J Immunol Methods. 1991 Apr 25;138(2):237–244. doi: 10.1016/0022-1759(91)90172-c. [DOI] [PubMed] [Google Scholar]
  17. Surowy C. S., van Santen V. L., Scheib-Wixted S. M., Spritz R. A. Direct, sequence-specific binding of the human U1-70K ribonucleoprotein antigen protein to loop I of U1 small nuclear RNA. Mol Cell Biol. 1989 Oct;9(10):4179–4186. doi: 10.1128/mcb.9.10.4179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Tan E. M. Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. Adv Immunol. 1989;44:93–151. doi: 10.1016/s0065-2776(08)60641-0. [DOI] [PubMed] [Google Scholar]
  19. Tsai D. E., Keene J. D. In vitro selection of RNA epitopes using autoimmune patient serum. J Immunol. 1993 Feb 1;150(3):1137–1145. [PubMed] [Google Scholar]
  20. Tsai D. E., Kenan D. J., Keene J. D. In vitro selection of an RNA epitope immunologically cross-reactive with a peptide. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):8864–8868. doi: 10.1073/pnas.89.19.8864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Verheijen R., de Jong B. A., Oberyé E. H., van Venrooij W. J. Molecular cloning of a major CENP-B epitope and its use for the detection of anticentromere autoantibodies. Mol Biol Rep. 1992 Feb;16(1):49–59. doi: 10.1007/BF00788753. [DOI] [PubMed] [Google Scholar]
  23. de Rooij D. J., Habets W. J., van de Putte L. B., Hoet M. H., Verbeek A. L., van Venrooij W. J. Use of recombinant RNP peptides 70K and A in an ELISA for measurement of antibodies in mixed connective tissue disease: a longitudinal follow up of 18 patients. Ann Rheum Dis. 1990 Jun;49(6):391–395. doi: 10.1136/ard.49.6.391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. van Venrooij W. J., Hoet R., Castrop J., Hageman B., Mattaj I. W., van de Putte L. B. Anti-(U1) small nuclear RNA antibodies in anti-small nuclear ribonucleoprotein sera from patients with connective tissue diseases. J Clin Invest. 1990 Dec;86(6):2154–2160. doi: 10.1172/JCI114954. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES