Purification and visualization of native spliceosomes
- PMID: 2968159
- DOI: 10.1016/s0092-8674(88)90489-8
Purification and visualization of native spliceosomes
Abstract
Mammalian spliceosomes were purified in preparative amounts by gel filtration chromatography and shown to be functional by in vitro complementation experiments. The column fractions containing spliceosomes are enriched in the snRNAs U1, U2, U4, U5, and U6 and a subset of proteins present in the nuclear extract. Splicing intermediates, the entire set of snRNAs, and the enriched proteins can be immunoprecipitated with three different monoclonal antibodies that recognize snRNP determinants. At least one U1 snRNP is present in each spliceosome since the particles are quantitatively immunoprecipitated by an anti-U1 snRNP monoclonal antibody. Examination of the spliceosome fractions by EM revealed a relatively homogeneous population of 40-60 nm particles with a striking morphology. Evidence that these particles are spliceosomes is their sensitivity to micrococcal nuclease, their ATP-dependent assembly, and their immunoprecipitation with a trimethyl cap monoclonal antibody. In addition, pre-mRNA was visualized in the particles by EM.
Similar articles
-
Purification of the spliceosome A-complex and its visualization by electron microscopy.J Biol Chem. 1995 Jun 30;270(26):15515-22. doi: 10.1074/jbc.270.26.15515. J Biol Chem. 1995. PMID: 7797545
-
Small nuclear ribonucleoproteins and heterogeneous nuclear ribonucleoproteins in the amphibian germinal vesicle: loops, spheres, and snurposomes.J Cell Biol. 1991 May;113(3):465-83. doi: 10.1083/jcb.113.3.465. J Cell Biol. 1991. PMID: 1826687 Free PMC article.
-
Factor required for mammalian spliceosome assembly is localized to discrete regions in the nucleus.Nature. 1990 Feb 1;343(6257):437-41. doi: 10.1038/343437a0. Nature. 1990. PMID: 2137203
-
The spliceosome.Bioessays. 1993 Sep;15(9):595-603. doi: 10.1002/bies.950150905. Bioessays. 1993. PMID: 8240312 Review.
-
Cryo-electron microscopy of spliceosomal components.Annu Rev Biophys Biomol Struct. 2006;35:435-57. doi: 10.1146/annurev.biophys.35.040405.101953. Annu Rev Biophys Biomol Struct. 2006. PMID: 16689644 Review.
Cited by
-
A block in mammalian splicing occurring after formation of large complexes containing U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins.Mol Cell Biol. 1989 Jan;9(1):259-67. doi: 10.1128/mcb.9.1.259-267.1989. Mol Cell Biol. 1989. PMID: 2522588 Free PMC article.
-
Alterations of RNase H sensitivity of the 3' splice site region during the in vitro splicing reaction.Nucleic Acids Res. 1991 Jul 25;19(14):3953-8. doi: 10.1093/nar/19.14.3953. Nucleic Acids Res. 1991. PMID: 1650457 Free PMC article.
-
Possible involvement of (2'5')oligoadenylate synthetase activity in pre-mRNA splicing.Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10377-81. doi: 10.1073/pnas.88.23.10377. Proc Natl Acad Sci U S A. 1991. PMID: 1720538 Free PMC article.
-
Nuclear-mitotic apparatus protein: a structural protein interface between the nucleoskeleton and RNA splicing.Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1505-9. doi: 10.1073/pnas.91.4.1505. Proc Natl Acad Sci U S A. 1994. PMID: 8108437 Free PMC article.
-
Electron microscopic identification of the yeast spliceosome.EMBO J. 1988 Dec 1;7(12):3829-36. doi: 10.1002/j.1460-2075.1988.tb03268.x. EMBO J. 1988. PMID: 3061806 Free PMC article.
Publication types
MeSH terms
Substances
Grants and funding
LinkOut - more resources
Full Text Sources
Miscellaneous
