Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Dec;86(23):9243–9247. doi: 10.1073/pnas.86.23.9243

Identification, purification, and biochemical characterization of U2 small nuclear ribonucleoprotein auxiliary factor.

P D Zamore 1, M R Green 1
PMCID: PMC298470  PMID: 2531895

Abstract

Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA branch site is an early step in spliceosome assembly and appears to commit a pre-mRNA to the splicing pathway. We have shown previously that this ATP-dependent binding requires a non-rnRNP factor, U2 snRNP auxiliary factor (U2AF), in addition to U2 snRNP. In this report we have identified U2AF, purified it to homogeneity, and characterized its biochemical properties. Purified U2AF comprises roughly equimolar quantities of two polypeptides, approximately 65 kDa and approximately 35 kDa, which appear to be associated. Measured by ultraviolet crosslinking, the 65-kDa polypeptide binds specifically to the polypyrimidine tract/3' splice site region. U2AF binds rapidly at 4 degrees C in the absence of ATP and remains associated with the pre-mRNA following U2 snRNP binding. Thus, the simple binding of U2AF initiates mammalian spliceosome assembly by facilitating the ATP-dependent binding of U2 snRNP.

Full text

PDF
9243

Images in this article

9244Image
on p.9244
9244Image
on p.9244
9244Image
on p.9244
9245Image
on p.9245
9246Image
on p.9246
9246Image
on p.9246
9246Image
on p.9246
9247Image
on p.9247
9247Image
on p.9247

Selected References

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

  1. Bindereif A., Green M. R. An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly. EMBO J. 1987 Aug;6(8):2415–2424. doi: 10.1002/j.1460-2075.1987.tb02520.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bindereif A., Green M. R. Ribonucleoprotein complex formation during pre-mRNA splicing in vitro. Mol Cell Biol. 1986 Jul;6(7):2582–2592. doi: 10.1128/mcb.6.7.2582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Black D. L., Chabot B., Steitz J. A. U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing. Cell. 1985 Oct;42(3):737–750. doi: 10.1016/0092-8674(85)90270-3. [DOI] [PubMed] [Google Scholar]
  4. Cheng S. C., Abelson J. Spliceosome assembly in yeast. Genes Dev. 1987 Nov;1(9):1014–1027. doi: 10.1101/gad.1.9.1014. [DOI] [PubMed] [Google Scholar]
  5. 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]
  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. Grabowski P. J., Sharp P. A. Affinity chromatography of splicing complexes: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles in the spliceosome. Science. 1986 Sep 19;233(4770):1294–1299. doi: 10.1126/science.3638792. [DOI] [PubMed] [Google Scholar]
  8. Green M. R. Pre-mRNA splicing. Annu Rev Genet. 1986;20:671–708. doi: 10.1146/annurev.ge.20.120186.003323. [DOI] [PubMed] [Google Scholar]
  9. Konarska M. M., Sharp P. A. Electrophoretic separation of complexes involved in the splicing of precursors to mRNAs. Cell. 1986 Sep 12;46(6):845–855. doi: 10.1016/0092-8674(86)90066-8. [DOI] [PubMed] [Google Scholar]
  10. Krämer A. Analysis of RNase-A-resistant regions of adenovirus 2 major late precursor-mRNA in splicing extracts reveals an ordered interaction of nuclear components with the substrate RNA. J Mol Biol. 1987 Aug 5;196(3):559–573. doi: 10.1016/0022-2836(87)90032-5. [DOI] [PubMed] [Google Scholar]
  11. Lamond A. I., Konarska M. M., Grabowski P. J., Sharp P. A. Spliceosome assembly involves the binding and release of U4 small nuclear ribonucleoprotein. Proc Natl Acad Sci U S A. 1988 Jan;85(2):411–415. doi: 10.1073/pnas.85.2.411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Legrain P., Seraphin B., Rosbash M. Early commitment of yeast pre-mRNA to the spliceosome pathway. Mol Cell Biol. 1988 Sep;8(9):3755–3760. doi: 10.1128/mcb.8.9.3755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Maniatis T., Reed R. The role of small nuclear ribonucleoprotein particles in pre-mRNA splicing. Nature. 1987 Feb 19;325(6106):673–678. doi: 10.1038/325673a0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Pikielny C. W., Rymond B. C., Rosbash M. Electrophoresis of ribonucleoproteins reveals an ordered assembly pathway of yeast splicing complexes. 1986 Nov 27-Dec 3Nature. 324(6095):341–345. doi: 10.1038/324341a0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Ruskin B., Green M. R. Specific and stable intron-factor interactions are established early during in vitro pre-mRNA splicing. Cell. 1985 Nov;43(1):131–142. doi: 10.1016/0092-8674(85)90018-2. [DOI] [PubMed] [Google Scholar]
  19. Ruskin B., Zamore P. D., Green M. R. A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly. Cell. 1988 Jan 29;52(2):207–219. doi: 10.1016/0092-8674(88)90509-0. [DOI] [PubMed] [Google Scholar]
  20. Sharp P. A. Splicing of messenger RNA precursors. Science. 1987 Feb 13;235(4790):766–771. doi: 10.1126/science.3544217. [DOI] [PubMed] [Google Scholar]
  21. Zapp M. L., Berget S. M. Evidence for nuclear factors involved in recognition of 5' splice sites. Nucleic Acids Res. 1989 Apr 11;17(7):2655–2674. doi: 10.1093/nar/17.7.2655. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES