Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1990 Jan;9(1):241–249. doi: 10.1002/j.1460-2075.1990.tb08101.x

In vitro splicing of mutually exclusive exons from the chicken beta-tropomyosin gene: role of the branch point location and very long pyrimidine stretch.

M Goux-Pelletan 1, D Libri 1, Y d'Aubenton-Carafa 1, M Fiszman 1, E Brody 1, J Marie 1
PMCID: PMC551653  PMID: 2153077

Abstract

The chicken beta-tropomyosin gene contains 11 exons, two of which are spliced into mRNA only in skeletal muscle. One pair of alternative exons, 6A and 6B, is found in the middle of the gene; they are spliced in a mutually exclusive manner. The non-muscle splice 6A-7 is by far the predominant in vitro reaction in a HeLa cell nuclear extract. A minor product is the 6A-6B splice, which is excluded in all tissues. This minor product results from the use of a branch point located 105 nt upstream of the 3' end of the intron separating exons 6A and 6B. The region between the branch point sequence and the final AG contains a stretch of approximately 80 pyrimidines. We have examined the role of the distance of the branchpoint to the 3' splice site and of the sequences between these two elements. Our results suggest that at least two cis-acting elements contribute to the mutual exclusivity of exons 6A and 6B. The intron between exons 6A and 6B is intrinsically poorly 'spliceable' both because the branch point is too far upstream of the 3' end of the intron to give efficient splicing and because of the particular sequence lying between this branch point and the 3' splice site.

Full text

PDF
243

Images in this article

Selected References

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

  1. Aebi M., Hornig H., Padgett R. A., Reiser J., Weissmann C. Sequence requirements for splicing of higher eukaryotic nuclear pre-mRNA. Cell. 1986 Nov 21;47(4):555–565. doi: 10.1016/0092-8674(86)90620-3. [DOI] [PubMed] [Google Scholar]
  2. Beyer A. L., Osheim Y. N. Splice site selection, rate of splicing, and alternative splicing on nascent transcripts. Genes Dev. 1988 Jun;2(6):754–765. doi: 10.1101/gad.2.6.754. [DOI] [PubMed] [Google Scholar]
  3. Breitbart R. E., Andreadis A., Nadal-Ginard B. Alternative splicing: a ubiquitous mechanism for the generation of multiple protein isoforms from single genes. Annu Rev Biochem. 1987;56:467–495. doi: 10.1146/annurev.bi.56.070187.002343. [DOI] [PubMed] [Google Scholar]
  4. 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]
  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. Eperon L. P., Graham I. R., Griffiths A. D., Eperon I. C. Effects of RNA secondary structure on alternative splicing of pre-mRNA: is folding limited to a region behind the transcribing RNA polymerase? Cell. 1988 Jul 29;54(3):393–401. doi: 10.1016/0092-8674(88)90202-4. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Fu X. Y., Colgan J. D., Manley J. L. Multiple cis-acting sequence elements are required for efficient splicing of simian virus 40 small-t antigen pre-mRNA. Mol Cell Biol. 1988 Sep;8(9):3582–3590. doi: 10.1128/mcb.8.9.3582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fu X. Y., Manley J. L. Factors influencing alternative splice site utilization in vivo. Mol Cell Biol. 1987 Feb;7(2):738–748. doi: 10.1128/mcb.7.2.738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Helfman D. M., Cheley S., Kuismanen E., Finn L. A., Yamawaki-Kataoka Y. Nonmuscle and muscle tropomyosin isoforms are expressed from a single gene by alternative RNA splicing and polyadenylation. Mol Cell Biol. 1986 Nov;6(11):3582–3595. doi: 10.1128/mcb.6.11.3582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Helfman D. M., Ricci W. M., Finn L. A. Alternative splicing of tropomyosin pre-mRNAs in vitro and in vivo. Genes Dev. 1988 Dec;2(12A):1627–1638. doi: 10.1101/gad.2.12a.1627. [DOI] [PubMed] [Google Scholar]
  14. Jacob M., Gallinaro H. The 5' splice site: phylogenetic evolution and variable geometry of association with U1RNA. Nucleic Acids Res. 1989 Mar 25;17(6):2159–2180. doi: 10.1093/nar/17.6.2159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Krämer A., Frick M., Keller W. Separation of multiple components of HeLa cell nuclear extracts required for pre-messenger RNA splicing. J Biol Chem. 1987 Dec 25;262(36):17630–17640. [PubMed] [Google Scholar]
  17. Leff S. E., Rosenfeld M. G., Evans R. M. Complex transcriptional units: diversity in gene expression by alternative RNA processing. Annu Rev Biochem. 1986;55:1091–1117. doi: 10.1146/annurev.bi.55.070186.005303. [DOI] [PubMed] [Google Scholar]
  18. Libri D., Lemonnier M., Meinnel T., Fiszman M. Y. A single gene codes for the beta subunits of smooth and skeletal muscle tropomyosin in the chicken. J Biol Chem. 1989 Feb 15;264(5):2935–2944. [PubMed] [Google Scholar]
  19. Libri D., Marie J., Brody E., Fiszman M. Y. A subfragment of the beta tropomyosin gene is alternatively spliced when transfected into differentiating muscle cells. Nucleic Acids Res. 1989 Aug 25;17(16):6449–6462. doi: 10.1093/nar/17.16.6449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. MacLeod A. R., Houlker C., Reinach F. C., Smillie L. B., Talbot K., Modi G., Walsh F. S. A muscle-type tropomyosin in human fibroblasts: evidence for expression by an alternative RNA splicing mechanism. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7835–7839. doi: 10.1073/pnas.82.23.7835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mount S. M. A catalogue of splice junction sequences. Nucleic Acids Res. 1982 Jan 22;10(2):459–472. doi: 10.1093/nar/10.2.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Munroe S. H. Antisense RNA inhibits splicing of pre-mRNA in vitro. EMBO J. 1988 Aug;7(8):2523–2532. doi: 10.1002/j.1460-2075.1988.tb03100.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ohshima Y., Gotoh Y. Signals for the selection of a splice site in pre-mRNA. Computer analysis of splice junction sequences and like sequences. J Mol Biol. 1987 May 20;195(2):247–259. doi: 10.1016/0022-2836(87)90647-4. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Padgett R. A., Konarska M. M., Aebi M., Hornig H., Weissmann C., Sharp P. A. Nonconsensus branch-site sequences in the in vitro splicing of transcripts of mutant rabbit beta-globin genes. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8349–8353. doi: 10.1073/pnas.82.24.8349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Reed R., Maniatis T. Intron sequences involved in lariat formation during pre-mRNA splicing. Cell. 1985 May;41(1):95–105. doi: 10.1016/0092-8674(85)90064-9. [DOI] [PubMed] [Google Scholar]
  27. Ruskin B., Greene J. M., Green M. R. Cryptic branch point activation allows accurate in vitro splicing of human beta-globin intron mutants. Cell. 1985 Jul;41(3):833–844. doi: 10.1016/s0092-8674(85)80064-7. [DOI] [PubMed] [Google Scholar]
  28. Schmitt P., Gattoni R., Keohavong P., Stévenin J. Alternative splicing of E1A transcripts of adenovirus requires appropriate ionic conditions in vitro. Cell. 1987 Jul 3;50(1):31–39. doi: 10.1016/0092-8674(87)90659-3. [DOI] [PubMed] [Google Scholar]
  29. Smith C. W., Nadal-Ginard B. Mutually exclusive splicing of alpha-tropomyosin exons enforced by an unusual lariat branch point location: implications for constitutive splicing. Cell. 1989 Mar 10;56(5):749–758. doi: 10.1016/0092-8674(89)90678-8. [DOI] [PubMed] [Google Scholar]
  30. Solnick D. Alternative splicing caused by RNA secondary structure. Cell. 1985 Dec;43(3 Pt 2):667–676. doi: 10.1016/0092-8674(85)90239-9. [DOI] [PubMed] [Google Scholar]
  31. Solnick D., Lee S. I. Amount of RNA secondary structure required to induce an alternative splice. Mol Cell Biol. 1987 Sep;7(9):3194–3198. doi: 10.1128/mcb.7.9.3194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wieringa B., Hofer E., Weissmann C. A minimal intron length but no specific internal sequence is required for splicing the large rabbit beta-globin intron. Cell. 1984 Jul;37(3):915–925. doi: 10.1016/0092-8674(84)90426-4. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES