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. 1999 Nov;5(11):1504–1508. doi: 10.1017/s1355838299991501

Resolution of the mammalian E complex and the ATP-dependent spliceosomal complexes on native agarose mini-gels.

R Das 1, R Reed 1
PMCID: PMC1369872  PMID: 10580479

Abstract

A great deal of progress in elucidating the mechanisms of spliceosome assembly has been achieved by analyzing the A, B, and C spliceosomal complexes on native polyacrylamide gels. In contrast, progress in understanding the earliest spliceosomal complex E has been hampered because this complex dissociates on native gels and is difficult to detect by other methods. Here we report conditions for resolving the spliceosomal complex E using a native horizontal agarose mini-gel system. This system also provides a simple alternative to polyacrylamide gels for resolving the ATP-dependent spliceosomal complexes.

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Selected References

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

  1. Bennett M., Piñol-Roma S., Staknis D., Dreyfuss G., Reed R. Differential binding of heterogeneous nuclear ribonucleoproteins to mRNA precursors prior to spliceosome assembly in vitro. Mol Cell Biol. 1992 Jul;12(7):3165–3175. doi: 10.1128/mcb.12.7.3165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brody E., Abelson J. The "spliceosome": yeast pre-messenger RNA associates with a 40S complex in a splicing-dependent reaction. Science. 1985 May 24;228(4702):963–967. doi: 10.1126/science.3890181. [DOI] [PubMed] [Google Scholar]
  3. Champion-Arnaud P., Gozani O., Palandjian L., Reed R. Accumulation of a novel spliceosomal complex on pre-mRNAs containing branch site mutations. Mol Cell Biol. 1995 Oct;15(10):5750–5756. doi: 10.1128/mcb.15.10.5750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Gozani O., Patton J. G., Reed R. A novel set of spliceosome-associated proteins and the essential splicing factor PSF bind stably to pre-mRNA prior to catalytic step II of the splicing reaction. EMBO J. 1994 Jul 15;13(14):3356–3367. doi: 10.1002/j.1460-2075.1994.tb06638.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Grabowski P. J., Seiler S. R., Sharp P. A. A multicomponent complex is involved in the splicing of messenger RNA precursors. Cell. 1985 Aug;42(1):345–353. doi: 10.1016/s0092-8674(85)80130-6. [DOI] [PubMed] [Google Scholar]
  7. Jamison S. F., Crow A., Garcia-Blanco M. A. The spliceosome assembly pathway in mammalian extracts. Mol Cell Biol. 1992 Oct;12(10):4279–4287. doi: 10.1128/mcb.12.10.4279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Konarska M. M., Grabowski P. J., Padgett R. A., Sharp P. A. Characterization of the branch site in lariat RNAs produced by splicing of mRNA precursors. Nature. 1985 Feb 14;313(6003):552–557. doi: 10.1038/313552a0. [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. Konarska M. M., Sharp P. A. Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes. Cell. 1987 Jun 19;49(6):763–774. doi: 10.1016/0092-8674(87)90614-3. [DOI] [PubMed] [Google Scholar]
  11. Krainer A. R., Maniatis T., Ruskin B., Green M. R. Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell. 1984 Apr;36(4):993–1005. doi: 10.1016/0092-8674(84)90049-7. [DOI] [PubMed] [Google Scholar]
  12. Lamond A. I., Konarska M. M., Sharp P. A. A mutational analysis of spliceosome assembly: evidence for splice site collaboration during spliceosome formation. Genes Dev. 1987 Aug;1(6):532–543. doi: 10.1101/gad.1.6.532. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. Reed R., Griffith J., Maniatis T. Purification and visualization of native spliceosomes. Cell. 1988 Jun 17;53(6):949–961. doi: 10.1016/s0092-8674(88)90489-8. [DOI] [PubMed] [Google Scholar]
  17. Reed R. Protein composition of mammalian spliceosomes assembled in vitro. Proc Natl Acad Sci U S A. 1990 Oct;87(20):8031–8035. doi: 10.1073/pnas.87.20.8031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]

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