Skip to main content
SpringerLink
Log in

Purification of U Small Nuclear Ribonucleoprotein Particles

  • Protocol
RNA-Protein Interaction Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 118))

Abstract

The spliceosome is the catalytic entity that removes the introns from the primary transcripts in eukaryotes. The spliceosome consists of four small nuclear ribonucleoproteins (snRNPs), named U1,U2, U4/U6, and U5 snRNP, and numerous non-snRNP proteins. Each snRNP consists of one (U1, U2, and U5 snRNP) or two (U4/U6 snRNP) RNA molecules and a large number of different proteins. Within the U4/U6 snRNP, U4 and U6 snRNA are basepaired together. Under conditions of in vitro splicing reactions in HeLa cell nuclear extracts (i.e., at about 150 mM salt concentration), the spliceosomal snRNPs are organized in three RNP forms: 12S U1, 17S U2, and 25S [U4/U6.U5] trisnRNP. These snRNP complexes may be considered as functional subunits of the spliceosome.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Moore, M. J., Query, C. C., and Sharp, P. A. (1993) Splicing of precursors to messenger RNAs by the spliceosome, in RNA World (Gesteland, R. F. and Atkins, J. F., eds.), Cold Spring Harbor Press, Cold Spring Harbor, NY, pp. 303–358.

    Google Scholar 

  2. Krämer, A. (1996) The structure and function of proteins involved in mammalian pre-mRNA splicing. Annu. Rev. Biochem. 65, 367–409.

    Article  PubMed  Google Scholar 

  3. Lührmann, R., Kastner, B., and Bach, M. (1990) Structure of spliceosomal snRNPs and their role in pre-mRNA splicing. Biochim. Biophys. Acta Gene Struct. Expression 1087, 265–292.

    Article  Google Scholar 

  4. Will, C. L. and Lührmann, R. (1997) snRNP structure and function, in Eukaryotic mRNA Processing (Krainer, A. R., ed.), IRC Press, Oxford, pp. 130–173.

    Google Scholar 

  5. Will, C. L., Kastner, B., and Lührmann, B. (1993) Analysis of ribonucleoprotein interactions, in RNA Processing — A Practical Approach, vol. 2 (Higgens, S. J. and Hames, B. D., eds.), IRL Press, Oxford, pp. 141–177.

    Google Scholar 

  6. Bochnig, P., Reuter, R., Bringmann, P., and Lührmann, R. (1987) A monoclonal antibody against 2,2,7-trimethylguanosine that reacts with intact U snRNPs as well as with 7-methylguanosine capped RNAs. Eur. J. Biochem. 168, 461–467.

    Article  PubMed  CAS  Google Scholar 

  7. Bringmann, P., Rinke, J., Appel, B., Reuter, R., and Lührmann R. (1983) Purification of snRNPs U1, U2, U4, U5 and U6 with 2,2,7-trimethylguanosine-specific antibody and definition of their constituent proteins reacting with anti-Sm and anti-(U1)RNP antisera. EMBO J. 2, 1129–1135.

    PubMed  CAS  Google Scholar 

  8. Krainer, A. R., Maniatis, T., Ruskin, B., and Green, M. (1984) Normal and mutant human β-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell 36, 993–1005.

    Article  PubMed  CAS  Google Scholar 

  9. Bearden, J. C. (1978) Quantitation of submicrogram quantities of proteins by an improved protein-dye binding assay. Biochim. Biophys. Acta 553, 525–529.

    Google Scholar 

  10. Bach, M., Winkelmann, G., and Lührmann, R. (1989) 20 S small nuclear ribonucleoprotein U5 shows a surprisingly complex protein composition. Proc. Natl. Acad. Sci. USA 86, 6038–6042.

    Article  PubMed  CAS  Google Scholar 

  11. Reuter, R. and Lührmann, R. (1986) Immunisation of mice with purified U1 small nuclear ribonucleoprotein (RNP) induces a pattern of antibody specificities characteristic of the anti-Sm and anti-RNP autoimmune response of patients with lupus erythematosus, as measured by monoclonal antibodies. Proc. Natl. Acad. Sci. USA 83, 8689–8693.

    Article  PubMed  CAS  Google Scholar 

  12. Behrens, S. E. and Lührmann, R. (1991) Immunoaffinity purification of a [U4/U6. U5] tri-snRNP complex from human cells. Genes Dev. 5, 1429–1452.

    Article  Google Scholar 

  13. Behrens, S. E., Tyc, K., Kastner, B., Reichelt, J., and Lührmann, R. (1992) Small nuclear ribonucleoprotein (RNP) U2 contains numerous additional proteins and has a bipartite RNP structure under splicing conditions. Mol. Cell. Biol. 13, 307–309.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institut für Molekularbiologie und Tumorforschung, Philipps Universität Marburg, Marburg, Germany

    Berthold Kastner & Reinhard Lührmann

Authors
  1. Berthold Kastner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Reinhard Lührmann
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Uniformed Services University of the Health Sciences, Bethesda, Maryland

    Susan R. Haynes

Rights and permissions

Reprints and permissions

Copyright information

© 1999 Humana Press Inc.

About this protocol

Cite this protocol

Kastner, B., Lührmann, R. (1999). Purification of U Small Nuclear Ribonucleoprotein Particles. In: Haynes, S.R. (eds) RNA-Protein Interaction Protocols. Methods in Molecular Biology™, vol 118. Humana Press. https://doi.org/10.1385/1-59259-676-2:289

Download citation

  • DOI: https://doi.org/10.1385/1-59259-676-2:289

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-568-3

  • Online ISBN: 978-1-59259-676-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation