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. 1995 Dec 11;23(23):4864–4871. doi: 10.1093/nar/23.23.4864

Homodimerization of the human U1 snRNP-specific protein C.

J M Gunnewiek 1, Y van Aarssen 1, R Wassenaar 1, P Legrain 1, W J van Venrooij 1, R L Nelissen 1
PMCID: PMC307476  PMID: 8532530

Abstract

The U1 snRNP-specific protein C contains an N-terminal zinc finger-like CH motif which is required for the binding of the U1C protein to the U1 snRNP particle. Recently a similar motif was reported to be essential for in vivo homodimerization of the yeast splicing factor PRP9. In the present study we demonstrate that the human U1C protein is able to form homodimers as well. U1C homodimers are found when (i) the human U1C protein is expressed in Escherichia coli, (ii) immunoprecipitations with anti-U1C antibodies are performed on in vitro translated U1C, and when (iii) the yeast two hybrid system is used. Analyses of mutant U1C proteins in an in vitro dimerization assay and the yeast two hybrid system revealed that amino acids within the CH motif, i.e. between positions 22 and 30, are required for homodimerization.

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  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. Benezra R. An intermolecular disulfide bond stabilizes E2A homodimers and is required for DNA binding at physiological temperatures. Cell. 1994 Dec 16;79(6):1057–1067. doi: 10.1016/0092-8674(94)90036-1. [DOI] [PubMed] [Google Scholar]
  3. Boelens W., Scherly D., Jansen E. J., Kolen K., Mattaj I. W., van Venrooij W. J. Analysis of in vitro binding of U1-A protein mutants to U1 snRNA. Nucleic Acids Res. 1991 Sep 11;19(17):4611–4618. doi: 10.1093/nar/19.17.4611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Carlson J. D., Yarmush M. L. Antibody assisted protein refolding. Biotechnology (N Y) 1992 Jan;10(1):86–91. doi: 10.1038/nbt0192-86. [DOI] [PubMed] [Google Scholar]
  6. Chang T. H., Clark M. W., Lustig A. J., Cusick M. E., Abelson J. RNA11 protein is associated with the yeast spliceosome and is localized in the periphery of the cell nucleus. Mol Cell Biol. 1988 Jun;8(6):2379–2393. doi: 10.1128/mcb.8.6.2379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Feeney R. J., Sauterer R. A., Feeney J. L., Zieve G. W. Cytoplasmic assembly and nuclear accumulation of mature small nuclear ribonucleoprotein particles. J Biol Chem. 1989 Apr 5;264(10):5776–5783. [PubMed] [Google Scholar]
  8. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  9. Habets W. J., Hoet M. H., De Jong B. A., Van der Kemp A., Van Venrooij W. J. Mapping of B cell epitopes on small nuclear ribonucleoproteins that react with human autoantibodies as well as with experimentally-induced mouse monoclonal antibodies. J Immunol. 1989 Oct 15;143(8):2560–2566. [PubMed] [Google Scholar]
  10. Harper J. W., Adami G. R., Wei N., Keyomarsi K., Elledge S. J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993 Nov 19;75(4):805–816. doi: 10.1016/0092-8674(93)90499-g. [DOI] [PubMed] [Google Scholar]
  11. Heinrichs V., Bach M., Winkelmann G., Lührmann R. U1-specific protein C needed for efficient complex formation of U1 snRNP with a 5' splice site. Science. 1990 Jan 5;247(4938):69–72. doi: 10.1126/science.2136774. [DOI] [PubMed] [Google Scholar]
  12. Heinrichs V., Hackl W., Lührmann R. Direct binding of small nuclear ribonucleoprotein G to the Sm site of small nuclear RNA. Ultraviolet light cross-linking of protein G to the AAU stretch within the Sm site (AAUUUGUGG) of U1 small nuclear ribonucleoprotein reconstituted in vitro. J Mol Biol. 1992 Sep 5;227(1):15–28. doi: 10.1016/0022-2836(92)90678-d. [DOI] [PubMed] [Google Scholar]
  13. Janknecht R., de Martynoff G., Lou J., Hipskind R. A., Nordheim A., Stunnenberg H. G. Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8972–8976. doi: 10.1073/pnas.88.20.8972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Legrain P., Chapon C., Galisson F. Interactions between PRP9 and SPP91 splicing factors identify a protein complex required in prespliceosome assembly. Genes Dev. 1993 Jul;7(7B):1390–1399. doi: 10.1101/gad.7.7b.1390. [DOI] [PubMed] [Google Scholar]
  16. Legrain P., Choulika A. The molecular characterization of PRP6 and PRP9 yeast genes reveals a new cysteine/histidine motif common to several splicing factors. EMBO J. 1990 Sep;9(9):2775–2781. doi: 10.1002/j.1460-2075.1990.tb07465.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Legrain P., Dokhelar M. C., Transy C. Detection of protein-protein interactions using different vectors in the two-hybrid system. Nucleic Acids Res. 1994 Aug 11;22(15):3241–3242. doi: 10.1093/nar/22.15.3241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lehmeier T., Foulaki K., Lührmann R. Evidence for three distinct D proteins, which react differentially with anti-Sm autoantibodies, in the cores of the major snRNPs U1, U2, U4/U6 and U5. Nucleic Acids Res. 1990 Nov 25;18(22):6475–6484. doi: 10.1093/nar/18.22.6475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Mount S. M., Pettersson I., Hinterberger M., Karmas A., Steitz J. A. The U1 small nuclear RNA-protein complex selectively binds a 5' splice site in vitro. Cell. 1983 Jun;33(2):509–518. doi: 10.1016/0092-8674(83)90432-4. [DOI] [PubMed] [Google Scholar]
  21. Nelissen R. L., Heinrichs V., Habets W. J., Simons F., Lührmann R., van Venrooij W. J. Zinc finger-like structure in U1-specific protein C is essential for specific binding to U1 snRNP. Nucleic Acids Res. 1991 Feb 11;19(3):449–454. doi: 10.1093/nar/19.3.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nelissen R. L., Will C. L., van Venrooij W. J., Lührmann R. The association of the U1-specific 70K and C proteins with U1 snRNPs is mediated in part by common U snRNP proteins. EMBO J. 1994 Sep 1;13(17):4113–4125. doi: 10.1002/j.1460-2075.1994.tb06729.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  24. Scherly D., Boelens W., van Venrooij W. J., Dathan N. A., Hamm J., Mattaj I. W. Identification of the RNA binding segment of human U1 A protein and definition of its binding site on U1 snRNA. EMBO J. 1989 Dec 20;8(13):4163–4170. doi: 10.1002/j.1460-2075.1989.tb08601.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schmitt J., Hess H., Stunnenberg H. G. Affinity purification of histidine-tagged proteins. Mol Biol Rep. 1993 Oct;18(3):223–230. doi: 10.1007/BF01674434. [DOI] [PubMed] [Google Scholar]
  26. Sharp P. A. Split genes and RNA splicing. Cell. 1994 Jun 17;77(6):805–815. doi: 10.1016/0092-8674(94)90130-9. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  29. Studier F. W. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J Mol Biol. 1991 May 5;219(1):37–44. doi: 10.1016/0022-2836(91)90855-z. [DOI] [PubMed] [Google Scholar]
  30. Tatei K., Takemura K., Tanaka H., Masaki T., Ohshima Y. Recognition of 5' and 3' splice site sequences in pre-mRNA studied with a filter binding technique. J Biol Chem. 1987 Aug 25;262(24):11667–11674. [PubMed] [Google Scholar]
  31. Tazi J., Kornstädt U., Rossi F., Jeanteur P., Cathala G., Brunel C., Lührmann R. Thiophosphorylation of U1-70K protein inhibits pre-mRNA splicing. Nature. 1993 May 20;363(6426):283–286. doi: 10.1038/363283a0. [DOI] [PubMed] [Google Scholar]
  32. Will C. L., Behrens S. E., Lührmann R. Protein composition of mammalian spliceosomal snRNPs. Mol Biol Rep. 1993 Aug;18(2):121–126. doi: 10.1007/BF00986766. [DOI] [PubMed] [Google Scholar]

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