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. 1994 Sep 1;13(17):4113–4125. doi: 10.1002/j.1460-2075.1994.tb06729.x

The association of the U1-specific 70K and C proteins with U1 snRNPs is mediated in part by common U snRNP proteins.

R L Nelissen 1, C L Will 1, W J van Venrooij 1, R Lührmann 1
PMCID: PMC395334  PMID: 8076607

Abstract

The U1 small nuclear ribonucleoprotein particle (snRNP)-specific 70K and A proteins are known to bind directly to stem-loops of the U1 snRNA, whereas the U1-C protein does not bind to naked U1 snRNA, but depends on other U1 snRNP protein components for its association. Focusing on the U1-70K and U1-C proteins, protein-protein interactions contributing to the association of these particle-specific proteins with the U1 snRNP were studied. Immunoprecipitation of complexes formed after incubation of naked U1 snRNA or purified U1 snRNPs lacking their specific proteins (core U1 snRNP) with in vitro translated U1-C protein, revealed that both common snRNP proteins and the U1-70K protein are required for the association of U1-C with the U1 snRNP. Binding studies with various in vitro translated U1-70K mutants demonstrated that the U1-70K N-terminal domain is necessary and sufficient for the interaction of U1-C with core U1 snRNPs. Surprisingly, several N-terminal fragments of the U1-70K protein, which lacked the U1-70K RNP-80 motif and did not bind naked U1 RNA, associated stably with core U1 snRNPs. This suggests that a new U1-70K binding site is generated upon association of common U1 snRNP proteins with U1 RNA. The interaction between the N-terminal domain of U1-70K and the core RNP domain was specific for the U1 snRNP; stable binding was not observed with core U2 or U5 snRNPs, suggesting essential structural differences among snRNP core domains. Evidence for direct protein-protein interactions between U1-specific proteins and common snRNP proteins was supported by chemical crosslinking experiments using purified U1 snRNPs. Individual crosslinks between the U1-70K and the common D2 or B'/B protein, as well as between U1-C and B'/B, were detected. A model for the assembly of U1 snRNP is presented in which the complex of common proteins on the RNA backbone functions as a platform for the association of the U1-specific proteins.

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Selected References

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

  1. Bach M., Bringmann P., Lührmann R. Purification of small nuclear ribonucleoprotein particles with antibodies against modified nucleosides of small nuclear RNAs. Methods Enzymol. 1990;181:232–257. doi: 10.1016/0076-6879(90)81125-e. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Behrens S. E., Lührmann R. Immunoaffinity purification of a [U4/U6.U5] tri-snRNP from human cells. Genes Dev. 1991 Aug;5(8):1439–1452. doi: 10.1101/gad.5.8.1439. [DOI] [PubMed] [Google Scholar]
  4. Behrens S. E., Tyc K., Kastner B., Reichelt J., Lührmann R. Small nuclear ribonucleoprotein (RNP) U2 contains numerous additional proteins and has a bipartite RNP structure under splicing conditions. Mol Cell Biol. 1993 Jan;13(1):307–319. doi: 10.1128/mcb.13.1.307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Branlant C., Krol A., Ebel J. P., Lazar E., Haendler B., Jacob M. U2 RNA shares a structural domain with U1, U4, and U5 RNAs. EMBO J. 1982;1(10):1259–1265. doi: 10.1002/j.1460-2075.1982.tb00022.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dreyfuss G., Swanson M. S., Piñol-Roma S. Heterogeneous nuclear ribonucleoprotein particles and the pathway of mRNA formation. Trends Biochem Sci. 1988 Mar;13(3):86–91. doi: 10.1016/0968-0004(88)90046-1. [DOI] [PubMed] [Google Scholar]
  7. Fischer U., Lührmann R. An essential signaling role for the m3G cap in the transport of U1 snRNP to the nucleus. Science. 1990 Aug 17;249(4970):786–790. doi: 10.1126/science.2143847. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Hamm J., Darzynkiewicz E., Tahara S. M., Mattaj I. W. The trimethylguanosine cap structure of U1 snRNA is a component of a bipartite nuclear targeting signal. Cell. 1990 Aug 10;62(3):569–577. doi: 10.1016/0092-8674(90)90021-6. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. Jarmolowski A., Mattaj I. W. The determinants for Sm protein binding to Xenopus U1 and U5 snRNAs are complex and non-identical. EMBO J. 1993 Jan;12(1):223–232. doi: 10.1002/j.1460-2075.1993.tb05648.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kastner B., Bach M., Lührmann R. Electron microscopy of small nuclear ribonucleoprotein (snRNP) particles U2 and U5: evidence for a common structure-determining principle in the major U snRNP family. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1710–1714. doi: 10.1073/pnas.87.5.1710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Kenan D. J., Query C. C., Keene J. D. RNA recognition: towards identifying determinants of specificity. Trends Biochem Sci. 1991 Jun;16(6):214–220. doi: 10.1016/0968-0004(91)90088-d. [DOI] [PubMed] [Google Scholar]
  19. Kohtz J. D., Jamison S. F., Will C. L., Zuo P., Lührmann R., Garcia-Blanco M. A., Manley J. L. Protein-protein interactions and 5'-splice-site recognition in mammalian mRNA precursors. Nature. 1994 Mar 10;368(6467):119–124. doi: 10.1038/368119a0. [DOI] [PubMed] [Google Scholar]
  20. 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]
  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. Lomant A. J., Fairbanks G. Chemical probes of extended biological structures: synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate). J Mol Biol. 1976 Jun 14;104(1):243–261. doi: 10.1016/0022-2836(76)90011-5. [DOI] [PubMed] [Google Scholar]
  23. Mattaj I. W., Boelens W., Izaurralde E., Jarmolowski A., Kambach C. Nucleocytoplasmic transport and snRNP assembly. Mol Biol Rep. 1993 Aug;18(2):79–83. doi: 10.1007/BF00986760. [DOI] [PubMed] [Google Scholar]
  24. Mattaj I. W. Cap trimethylation of U snRNA is cytoplasmic and dependent on U snRNP protein binding. Cell. 1986 Sep 12;46(6):905–911. doi: 10.1016/0092-8674(86)90072-3. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Query C. C., Bentley R. C., Keene J. D. A common RNA recognition motif identified within a defined U1 RNA binding domain of the 70K U1 snRNP protein. Cell. 1989 Apr 7;57(1):89–101. doi: 10.1016/0092-8674(89)90175-x. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. 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]
  31. Smith V., Barrell B. G. Cloning of a yeast U1 snRNP 70K protein homologue: functional conservation of an RNA-binding domain between humans and yeast. EMBO J. 1991 Sep;10(9):2627–2634. doi: 10.1002/j.1460-2075.1991.tb07805.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sumpter V., Kahrs A., Fischer U., Kornstädt U., Lührmann R. In vitro reconstitution of U1 and U2 snRNPs from isolated proteins and snRNA. Mol Biol Rep. 1992 Sep;16(4):229–240. doi: 10.1007/BF00419662. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. 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]
  36. Woppmann A., Will C. L., Kornstädt U., Zuo P., Manley J. L., Lührmann R. Identification of an snRNP-associated kinase activity that phosphorylates arginine/serine rich domains typical of splicing factors. Nucleic Acids Res. 1993 Jun 25;21(12):2815–2822. doi: 10.1093/nar/21.12.2815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wu J. Y., Maniatis T. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell. 1993 Dec 17;75(6):1061–1070. doi: 10.1016/0092-8674(93)90316-i. [DOI] [PubMed] [Google Scholar]

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