snRNPs: Methods for Purification

Abstract

Small nuclear ribonucleoprotein particles (snRNPs) are essential cofactors for the nuclear splicing of pre‐mRNA introns. The method best suited to the purification of snRNPs is affinity chromatography combined with competitive elution.

Keywords: snRNP; pre‐mRNA splicing; chromatography; RNA–protein interaction

Figure 1.

Glycerol gradient centrifugation of snRNPs. snRNPs eluted from an anti‐m3G column were loaded on a 10–30% glycerol gradient and centrifuged in a Beckman SW 28 rotor for 16 h at 27 000 rpm. After fractionation the proteins were extracted and separated by electrophoresis on an SDS–polyacrylamide gel. The migration of the individual snRNAs is indicated at the bottom of the figure. The bands corresponding to the common proteins are marked on the left, and on the right the approximate molecular masses of several specific proteins are indicated. The Coomassie‐stained gel was kindly provided by Axel Badouin.

close

References

Bach M, Winkelmann G and Lührmann R (1989) 20S small nuclear ribonucleoprotein U5 shows a surprisingly complex protein composition. Proceedings of the National Academy of Sciences of the USA 86: 6038–6042.

Behrens S‐E and Lührmann R (1991) Immunoaffinity purification of a [U4/U6.U5] tri‐snRNP from human cells. Genes and Development 5: 1439–1452.

Behrens S‐E, Tyc K, Kastner B, Reichelt J and Lührmann R (1993) Small nuclear ribonucleoprotein (RNP) U2 contains numerous additional proteins and has a bipartite RNP structure under splicing conditions. Molecular and Cellular Biology 13: 307–319.

Dignam JD, Levovitz RM and Roeder RG (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Research 11: 1475–1589.

Fabrizio P, Esser S, Kastner B and Lührmann R (1994) Isolation of S. cerevisiae snRNPs: comparison of U1 and U4/U6.U5 to their human counterparts. Science 264: 261–265.

Gottschalk A, Tang J, Puig O et al. (1998) A comprehensive biochemical and genetic analysis of the yeast U1 snRNP reveals five novel proteins. RNA 4: 374–393.

Krämer A (1996) The structure and function of proteins involved in mammalian pre‐mRNA splicing. Annual Review of Biochemistry 65: 367–409.

Lamond AI and Sproat BS (1993) Isolation and characterisation of ribonucleoprotein complexes. In: Higgens SJ and Hames BD (eds) RNA Processing – A Practical Approach, vol. 1, pp 102–140. Oxford: IRL Press.

Moore MJ, Query CC and Sharp PA (1993) Splicing of precursor to mRNA by the spliceosome. In: Gesteland RF and Atkin JF (eds) The RNA World, pp. 303–357. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Will CL and Lührmann R (1997) Protein functions in pre‐mRNA splicing. Current Opinion in Cell Biology 9: 320–328.

Further Reading

Kastner B (1998) Purification and electron microscopy of spliceosomal snRNPs. In: Schenkel J (ed.) RNP. Particles, Splicing and Autoimmune Diseases, pp. 95–140. Berlin: Springer Verlag.

Krämer A (1990) Purification of small nuclear ribonucleoprotein particles active in RNA processing. Methods in Enzymology 181: 215–231.

Will CL, Kastner B and Lührmann R (1994) Analysis of ribonucleoprotein interactions. In: Higgens SJ and Hames BD (eds) RNA Processing. – A Practical Approach, vol. 1, pp. 141–177. Oxford: IRL Press.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Kastner, Berthold(Apr 2001) snRNPs: Methods for Purification. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0002605]