Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1989 Dec 1;8(12):3853–3860. doi: 10.1002/j.1460-2075.1989.tb08563.x

Cloned human snRNP proteins B and B' differ only in their carboxy-terminal part.

A van Dam 1, I Winkel 1, J Zijlstra-Baalbergen 1, R Smeenk 1, H T Cuypers 1
PMCID: PMC402073  PMID: 2531083

Abstract

Antibodies to the snRNP proteins B' and B are frequently observed in autoimmune diseases. We analyzed different types of cDNAs encoding these proteins. One type of cDNA encoded a protein whose predicted mol. wt is 24.6 kd, whereas another type encoded a protein with a predicted mol wt of 23.7 kd. When translated in vitro from cDNA transcripts, the apparent mol. wts of these proteins on SDS-polyacrylamide gel were 29.5 and 28.5 kd respectively. The main difference between these two types of cDNAs proved to be the presence of an additional sequence of 146 nucleotides in the 3' part of the open reading frame of the clone encoding the shorter protein. This insert contains a termination codon in frame, at 9 nucleotides downstream from the 5' end of the insert. The additional sequence revealed at the 3' end a consensus sequence of vertebrates for intron-exon junctions. We demonstrated the presence of mRNAs corresponding with both types of cDNA in human cells. We hypothesize that the B' and B protein are derived from one pre-mRNA by alternative splicing, and show that they differ only at the carboxy terminus, where a proline rich motive is repeated once more in B'. A comparison with amino acid sequences of other cloned snRNP proteins is included.

Full text

PDF
3853

Images in this article

3854Image
on p.3854
3854Image
on p.3854
3857Image
on p.3857
3857Image
on p.3857

Selected References

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

  1. Agris C. H., Nemeroff M. E., Krug R. M. A block in mammalian splicing occurring after formation of large complexes containing U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins. Mol Cell Biol. 1989 Jan;9(1):259–267. doi: 10.1128/mcb.9.1.259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Earnshaw W. C., Sullivan K. F., Machlin P. S., Cooke C. A., Kaiser D. A., Pollard T. D., Rothfield N. F., Cleveland D. W. Molecular cloning of cDNA for CENP-B, the major human centromere autoantigen. J Cell Biol. 1987 Apr;104(4):817–829. doi: 10.1083/jcb.104.4.817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fisher D. E., Conner G. E., Reeves W. H., Blobel G., Kunkel H. G. Synthesis and assembly of human small nuclear ribonucleoproteins generated by cell-free translation. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6356–6360. doi: 10.1073/pnas.80.20.6356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fisher D. E., Conner G. E., Reeves W. H., Wisniewolski R., Blobel G. Small nuclear ribonucleoprotein particle assembly in vivo: demonstration of a 6S RNA-free core precursor and posttranslational modification. Cell. 1985 Oct;42(3):751–758. doi: 10.1016/0092-8674(85)90271-5. [DOI] [PubMed] [Google Scholar]
  7. Francoeur A. M., Peebles C. L., Heckman K. J., Lee J. C., Tan E. M. Identification of ribosomal protein autoantigens. J Immunol. 1985 Oct;135(4):2378–2384. [PubMed] [Google Scholar]
  8. Frendewey D., Krämer A., Keller W. Different small nuclear ribonucleoprotein particles are involved in different steps of splicing complex formation. Cold Spring Harb Symp Quant Biol. 1987;52:287–298. doi: 10.1101/sqb.1987.052.01.034. [DOI] [PubMed] [Google Scholar]
  9. Fresco L. D., Kurilla M. G., Keene J. D. Rapid inhibition of processing and assembly of small nuclear ribonucleoproteins after infection with vesicular stomatitis virus. Mol Cell Biol. 1987 Mar;7(3):1148–1155. doi: 10.1128/mcb.7.3.1148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Garoff H., Frischauf A. M., Simons K., Lehrach H., Delius H. The capsid protein of Semliki Forest virus has clusters of basic amino acids and prolines in its amino-terminal region. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6376–6380. doi: 10.1073/pnas.77.11.6376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gelpi C., Rodriguez-Sanchez J. L., Martinez M. A., Craft J., Hardin J. A. Murine graft vs host disease. A model for study of mechanisms that generate autoantibodies to ribonucleoproteins. J Immunol. 1988 Jun 15;140(12):4160–4166. [PubMed] [Google Scholar]
  12. Grabowski P. J., Seiler S. R., Sharp P. A. A multicomponent complex is involved in the splicing of messenger RNA precursors. Cell. 1985 Aug;42(1):345–353. doi: 10.1016/s0092-8674(85)80130-6. [DOI] [PubMed] [Google Scholar]
  13. Habets W. J., Berden J. H., Hoch S. O., Van Venrooij W. J. Further characterization and subcellular localization of Sm and U1 ribonucleoprotein antigens. Eur J Immunol. 1985 Oct;15(10):992–997. doi: 10.1002/eji.1830151006. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Hamm J., Kazmaier M., Mattaj I. W. In vitro assembly of U1 snRNPs. EMBO J. 1987 Nov;6(11):3479–3485. doi: 10.1002/j.1460-2075.1987.tb02672.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hamm J., van Santen V. L., Spritz R. A., Mattaj I. W. Loop I of U1 small nuclear RNA is the only essential RNA sequence for binding of specific U1 small nuclear ribonucleoprotein particle proteins. Mol Cell Biol. 1988 Nov;8(11):4787–4791. doi: 10.1128/mcb.8.11.4787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Konarska M. M., Sharp P. A. Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes. Cell. 1987 Jun 19;49(6):763–774. doi: 10.1016/0092-8674(87)90614-3. [DOI] [PubMed] [Google Scholar]
  19. Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
  20. Kruijer W., van Schaik F. M., Sussenbach J. S. Structure and organization of the gene coding for the DNA binding protein of adenovirus type 5. Nucleic Acids Res. 1981 Sep 25;9(18):4439–4457. doi: 10.1093/nar/9.18.4439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Leff S. E., Rosenfeld M. G., Evans R. M. Complex transcriptional units: diversity in gene expression by alternative RNA processing. Annu Rev Biochem. 1986;55:1091–1117. doi: 10.1146/annurev.bi.55.070186.005303. [DOI] [PubMed] [Google Scholar]
  22. Maddison P. J., Skinner R. P., Vlachoyiannopoulos P., Brennand D. M., Hough D. Antibodies to nRNP, Sm, Ro(SSA) and La(SSB) detected by ELISA: their specificity and inter-relations in connective tissue disease sera. Clin Exp Immunol. 1985 Nov;62(2):337–345. [PMC free article] [PubMed] [Google Scholar]
  23. McAllister G., Amara S. G., Lerner M. R. Tissue-specific expression and cDNA cloning of small nuclear ribonucleoprotein-associated polypeptide N. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5296–5300. doi: 10.1073/pnas.85.14.5296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McAllister G., Roby-Shemkovitz A., Amara S. G., Lerner M. R. cDNA sequence of the rat U snRNP-associated protein N: description of a potential Sm epitope. EMBO J. 1989 Apr;8(4):1177–1181. doi: 10.1002/j.1460-2075.1989.tb03489.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Ohosone Y., Mimori T., Griffith A., Akizuki M., Homma M., Craft J., Hardin J. A. Molecular cloning of cDNA encoding Sm autoantigen: derivation of a cDNA for a B polypeptide of the U series of small nuclear ribonucleoprotein particles. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4249–4253. doi: 10.1073/pnas.86.11.4249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Padgett R. A., Grabowski P. J., Konarska M. M., Seiler S., Sharp P. A. Splicing of messenger RNA precursors. Annu Rev Biochem. 1986;55:1119–1150. doi: 10.1146/annurev.bi.55.070186.005351. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Reuter R., Rothe S., Lührmann R. Molecular relationships between U snRNP proteins as investigated by rabbit antisera and peptide mapping. Nucleic Acids Res. 1987 May 26;15(10):4021–4034. doi: 10.1093/nar/15.10.4021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rokeach L. A., Haselby J. A., Hoch S. O. Molecular cloning of a cDNA encoding the human Sm-D autoantigen. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4832–4836. doi: 10.1073/pnas.85.13.4832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rokeach L. A., Jannatipour M., Haselby J. A., Hoch S. O. Primary structure of a human small nuclear ribonucleoprotein polypeptide as deduced by cDNA analysis. J Biol Chem. 1989 Mar 25;264(9):5024–5030. [PubMed] [Google Scholar]
  33. Schmauss C., McAllister G., Ohosone Y., Hardin J. A., Lerner M. R. A comparison of snRNP-associated Sm-autoantigens: human N, rat N and human B/B'. Nucleic Acids Res. 1989 Feb 25;17(4):1733–1743. doi: 10.1093/nar/17.4.1733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sillekens P. T., Beijer R. P., Habets W. J., van Venrooij W. J. Human U1 snRNP-specific C protein: complete cDNA and protein sequence and identification of a multigene family in mammals. Nucleic Acids Res. 1988 Sep 12;16(17):8307–8321. doi: 10.1093/nar/16.17.8307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sillekens P. T., Beijer R. P., Habets W. J., van Verooij W. J. Molecular cloning of the cDNA for the human U2 snRNA-specific A' protein. Nucleic Acids Res. 1989 Mar 11;17(5):1893–1906. doi: 10.1093/nar/17.5.1893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Spritz R. A., Strunk K., Surowy C. S., Hoch S. O., Barton D. E., Francke U. The human U1-70K snRNP protein: cDNA cloning, chromosomal localization, expression, alternative splicing and RNA-binding. Nucleic Acids Res. 1987 Dec 23;15(24):10373–10391. doi: 10.1093/nar/15.24.10373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stanford D. R., Rohleder A., Neiswanger K., Wieben E. D. DNA sequence of a human Sm autoimmune antigen. The multigene family contains a processed pseudogene. J Biol Chem. 1987 Jul 25;262(21):9931–9934. [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  42. 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]
  43. Westgeest A. A., Bons J. C., Van den Brink H. G., Aarden L. A., Smeenk R. J. An improved incubation apparatus for Western blots used for the detection of antinuclear antibodies. J Immunol Methods. 1986 Dec 24;95(2):283–288. doi: 10.1016/0022-1759(86)90417-5. [DOI] [PubMed] [Google Scholar]
  44. Wieben E. D., Madore S. J., Pederson T. Protein binding sites are conserved in U1 small nuclear RNA from insects and mammals. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1217–1220. doi: 10.1073/pnas.80.5.1217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Williams D. G., Stocks M. R., Smith P. R., Maini R. N. Murine lupus monoclonal antibodies define five epitopes on two different Sm polypeptides. Immunology. 1986 Jul;58(3):495–500. [PMC free article] [PubMed] [Google Scholar]
  46. Yamamoto K., Miura H., Moroi Y., Yoshinoya S., Goto M., Nishioka K., Miyamoto T. Isolation and characterization of a complementary DNA expressing human U1 small nuclear ribonucleoprotein C polypeptide. J Immunol. 1988 Jan 1;140(1):311–317. [PubMed] [Google Scholar]
  47. Zillmann M., Zapp M. L., Berget S. M. Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles. Mol Cell Biol. 1988 Feb;8(2):814–821. doi: 10.1128/mcb.8.2.814. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES