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. 1996 Dec 1;24(23):4614–4623. doi: 10.1093/nar/24.23.4614

In vitro reconstitution of mammalian U1 snRNPs active in splicing: the U1-C protein enhances the formation of early (E) spliceosomal complexes.

C L Will 1, S Rümpler 1, J Klein Gunnewiek 1, W J van Venrooij 1, R Lührmann 1
PMCID: PMC146292  PMID: 8972845

Abstract

We have established an in vitro reconstitution/splicing complementation system which has allowed the investigation of the role of mammalian U1 snRNP components both in splicing and at the early stages of spliceosome formation. U1 snRNPs reconstituted from purified, native snRNP proteins and either authentic or in vitro transcribed U1 snRNA restored both early (E) splicing complex formation and splicing-activity to U1-depleted extracts. In vitro reconstituted U1 snRNPs possessing an m3G or ApppG cap were equally active in splicing, demonstrating that a physiological cap structure is not absolutely required for U1 function. However, the presence of an m7GpppG or GpppG cap was deleterious to splicing, most likely due to competition for the m7G cap binding proteins. No significant reduction in splicing or E complex formation was detected with U1 snRNPs reconstituted from U1 snRNA lacking the RNA binding sites of the U1-70K or U1-A protein (i.e., stem-loop I and II, respectively). Complementation studies with purified HeLa U1 snRNPs lacking subsets of the U1-specific proteins demonstrated a role for the U1-C, but not U1-A, protein in the formation and/or stabilization of early splicing complexes. Studies with recombinant U1-C protein mutants indicated that the N-terminal domain of U1-C is necessary and sufficient for the stimulation of E complex formation.

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Selected References

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  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. 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]
  3. Colot H. V., Stutz F., Rosbash M. The yeast splicing factor Mud13p is a commitment complex component and corresponds to CBP20, the small subunit of the nuclear cap-binding complex. Genes Dev. 1996 Jul 1;10(13):1699–1708. doi: 10.1101/gad.10.13.1699. [DOI] [PubMed] [Google Scholar]
  4. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Frendewey D., Keller W. Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences. Cell. 1985 Aug;42(1):355–367. doi: 10.1016/s0092-8674(85)80131-8. [DOI] [PubMed] [Google Scholar]
  7. Fu X. D. The superfamily of arginine/serine-rich splicing factors. RNA. 1995 Sep;1(7):663–680. [PMC free article] [PubMed] [Google Scholar]
  8. Gunnewiek J. M., van Aarssen Y., Wassenaar R., Legrain P., van Venrooij W. J., Nelissen R. L. Homodimerization of the human U1 snRNP-specific protein C. Nucleic Acids Res. 1995 Dec 11;23(23):4864–4871. doi: 10.1093/nar/23.23.4864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Hamm J., Mattaj I. W. Monomethylated cap structures facilitate RNA export from the nucleus. Cell. 1990 Oct 5;63(1):109–118. doi: 10.1016/0092-8674(90)90292-m. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Hilleren P. J., Kao H. Y., Siliciano P. G. The amino-terminal domain of yeast U1-70K is necessary and sufficient for function. Mol Cell Biol. 1995 Nov;15(11):6341–6350. doi: 10.1128/mcb.15.11.6341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Izaurralde E., Lewis J., McGuigan C., Jankowska M., Darzynkiewicz E., Mattaj I. W. A nuclear cap binding protein complex involved in pre-mRNA splicing. Cell. 1994 Aug 26;78(4):657–668. doi: 10.1016/0092-8674(94)90530-4. [DOI] [PubMed] [Google Scholar]
  15. Jamison S. F., Pasman Z., Wang J., Will C., Lührmann R., Manley J. L., Garcia-Blanco M. A. U1 snRNP-ASF/SF2 interaction and 5' splice site recognition: characterization of required elements. Nucleic Acids Res. 1995 Aug 25;23(16):3260–3267. doi: 10.1093/nar/23.16.3260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Lewis J. D., Izaurralde E., Jarmolowski A., McGuigan C., Mattaj I. W. A nuclear cap-binding complex facilitates association of U1 snRNP with the cap-proximal 5' splice site. Genes Dev. 1996 Jul 1;10(13):1683–1698. doi: 10.1101/gad.10.13.1683. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Michaud S., Reed R. A functional association between the 5' and 3' splice site is established in the earliest prespliceosome complex (E) in mammals. Genes Dev. 1993 Jun;7(6):1008–1020. doi: 10.1101/gad.7.6.1008. [DOI] [PubMed] [Google Scholar]
  21. Michaud S., Reed R. An ATP-independent complex commits pre-mRNA to the mammalian spliceosome assembly pathway. Genes Dev. 1991 Dec;5(12B):2534–2546. doi: 10.1101/gad.5.12b.2534. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. 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]
  25. Patton J. R., Patterson R. J., Pederson T. Reconstitution of the U1 small nuclear ribonucleoprotein particle. Mol Cell Biol. 1987 Nov;7(11):4030–4037. doi: 10.1128/mcb.7.11.4030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Patton J. R., Pederson T. The Mr 70,000 protein of the U1 small nuclear ribonucleoprotein particle binds to the 5' stem-loop of U1 RNA and interacts with Sm domain proteins. Proc Natl Acad Sci U S A. 1988 Feb;85(3):747–751. doi: 10.1073/pnas.85.3.747. [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. Rossi F., Forné T., Antoine E., Tazi J., Brunel C., Cathala G. Involvement of U1 small nuclear ribonucleoproteins (snRNP) in 5' splice site-U1 snRNP interaction. J Biol Chem. 1996 Sep 27;271(39):23985–23991. doi: 10.1074/jbc.271.39.23985. [DOI] [PubMed] [Google Scholar]
  29. Ruby S. W., Abelson J. An early hierarchic role of U1 small nuclear ribonucleoprotein in spliceosome assembly. Science. 1988 Nov 18;242(4881):1028–1035. doi: 10.1126/science.2973660. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Seraphin B., Rosbash M. Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing. Cell. 1989 Oct 20;59(2):349–358. doi: 10.1016/0092-8674(89)90296-1. [DOI] [PubMed] [Google Scholar]
  32. Siliciano P. G., Guthrie C. 5' splice site selection in yeast: genetic alterations in base-pairing with U1 reveal additional requirements. Genes Dev. 1988 Oct;2(10):1258–1267. doi: 10.1101/gad.2.10.1258. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. 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]
  36. Ségault V., Will C. L., Sproat B. S., Lührmann R. In vitro reconstitution of mammalian U2 and U5 snRNPs active in splicing: Sm proteins are functionally interchangeable and are essential for the formation of functional U2 and U5 snRNPs. EMBO J. 1995 Aug 15;14(16):4010–4021. doi: 10.1002/j.1460-2075.1995.tb00072.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wersig C., Bindereif A. Reconstitution of functional mammalian U4 small nuclear ribonucleoprotein: Sm protein binding is not essential for splicing in vitro. Mol Cell Biol. 1992 Apr;12(4):1460–1468. doi: 10.1128/mcb.12.4.1460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Wolff T., Bindereif A. Reconstituted mammalian U4/U6 snRNP complements splicing: a mutational analysis. EMBO J. 1992 Jan;11(1):345–359. doi: 10.1002/j.1460-2075.1992.tb05057.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zhuang Y., Weiner A. M. A compensatory base change in U1 snRNA suppresses a 5' splice site mutation. Cell. 1986 Sep 12;46(6):827–835. doi: 10.1016/0092-8674(86)90064-4. [DOI] [PubMed] [Google Scholar]
  41. 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]

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