Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1990 Aug;87(16):6253–6257. doi: 10.1073/pnas.87.16.6253

Mechanism for cryptic splice site activation during pre-mRNA splicing.

K K Nelson 1, M R Green 1
PMCID: PMC54511  PMID: 2143583

Abstract

The 5' splice site of a pre-mRNA is recognized by U1 small nuclear ribonucleoprotein particles (snRNP) through base pairing with the 5' end of U1 small nuclear RNA (snRNA). Single-base substitutions within a 9-nucleotide 5'-splice-site sequence can abolish or attenuate use of that site and, in higher eukaryotes, can also activate nearby "cryptic" 5' splice sites. Here we show that the effects of single-base substitutions within a 5' splice site can be completely or partially suppressed by cis mutations that improve the overall complementarity of the site to U1 snRNA. We further show that in the presence of the normal 5' splice site, a cryptic 5' splice site can be activated by increasing its complementarity to U1 snRNA. U1 snRNP binding experiments confirm that cryptic 5' splice sites are activated when their affinity for U1 snRNP approaches that of the authentic 5' splice site. Based upon these results, we propose a spliceosome competition model for 5'-splice-site selection and cryptic 5'-splice-site activation. We discuss our results with regard to the factors involved in 5'-splice-site recognition.

Full text

PDF
6253

Images in this article

6254Image
on p.6254
6255Image
on p.6255
6255Image
on p.6255
6255Image
on p.6255

Selected References

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

  1. Aebi M., Hornig H., Weissmann C. 5' cleavage site in eukaryotic pre-mRNA splicing is determined by the overall 5' splice region, not by the conserved 5' GU. Cell. 1987 Jul 17;50(2):237–246. doi: 10.1016/0092-8674(87)90219-4. [DOI] [PubMed] [Google Scholar]
  2. Bindereif A., Green M. R. An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly. EMBO J. 1987 Aug;6(8):2415–2424. doi: 10.1002/j.1460-2075.1987.tb02520.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Black D. L., Chabot B., Steitz J. A. U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing. Cell. 1985 Oct;42(3):737–750. doi: 10.1016/0092-8674(85)90270-3. [DOI] [PubMed] [Google Scholar]
  4. Chabot B., Black D. L., LeMaster D. M., Steitz J. A. The 3' splice site of pre-messenger RNA is recognized by a small nuclear ribonucleoprotein. Science. 1985 Dec 20;230(4732):1344–1349. doi: 10.1126/science.2933810. [DOI] [PubMed] [Google Scholar]
  5. Chabot B., Steitz J. A. Recognition of mutant and cryptic 5' splice sites by the U1 small nuclear ribonucleoprotein in vitro. Mol Cell Biol. 1987 Feb;7(2):698–707. doi: 10.1128/mcb.7.2.698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Favaloro J., Treisman R., Kamen R. Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol. 1980;65(1):718–749. doi: 10.1016/s0076-6879(80)65070-8. [DOI] [PubMed] [Google Scholar]
  7. Goldsmith M. E., Humphries R. K., Ley T., Cline A., Kantor J. A., Nienhuis A. W. "Silent" nucleotide substitution in a beta+-thalassemia globin gene activates splice site in coding sequence RNA. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2318–2322. doi: 10.1073/pnas.80.8.2318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  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. 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]
  11. Krämer A. Analysis of RNase-A-resistant regions of adenovirus 2 major late precursor-mRNA in splicing extracts reveals an ordered interaction of nuclear components with the substrate RNA. J Mol Biol. 1987 Aug 5;196(3):559–573. doi: 10.1016/0022-2836(87)90032-5. [DOI] [PubMed] [Google Scholar]
  12. Lawn R. M., Efstratiadis A., O'Connell C., Maniatis T. The nucleotide sequence of the human beta-globin gene. Cell. 1980 Oct;21(3):647–651. doi: 10.1016/0092-8674(80)90428-6. [DOI] [PubMed] [Google Scholar]
  13. Lillie J. W., Green M., Green M. R. An adenovirus E1a protein region required for transformation and transcriptional repression. Cell. 1986 Sep 26;46(7):1043–1051. doi: 10.1016/0092-8674(86)90704-x. [DOI] [PubMed] [Google Scholar]
  14. Maniatis T., Reed R. The role of small nuclear ribonucleoprotein particles in pre-mRNA splicing. Nature. 1987 Feb 19;325(6106):673–678. doi: 10.1038/325673a0. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Mount S. M., Pettersson I., Hinterberger M., Karmas A., Steitz J. A. The U1 small nuclear RNA-protein complex selectively binds a 5' splice site in vitro. Cell. 1983 Jun;33(2):509–518. doi: 10.1016/0092-8674(83)90432-4. [DOI] [PubMed] [Google Scholar]
  17. Nelson K. K., Green M. R. Splice site selection and ribonucleoprotein complex assembly during in vitro pre-mRNA splicing. Genes Dev. 1988 Mar;2(3):319–329. doi: 10.1101/gad.2.3.319. [DOI] [PubMed] [Google Scholar]
  18. Nelson K. K., Green M. R. Splice site selection and ribonucleoprotein complex assembly during in vitro pre-mRNA splicing. Genes Dev. 1988 Mar;2(3):319–329. doi: 10.1101/gad.2.3.319. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Orkin S. H., Kazazian H. H., Jr, Antonarakis S. E., Ostrer H., Goff S. C., Sexton J. P. Abnormal RNA processing due to the exon mutation of beta E-globin gene. Nature. 1982 Dec 23;300(5894):768–769. doi: 10.1038/300768a0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Ruby S. W., Abelson J. An early hierarchic role of U1 small nuclear ribonucleoprotein in spliceosome assembly. Science. 1988 Nov 18;242(4881):1028–1035. doi: 10.1126/science.2973660. [DOI] [PubMed] [Google Scholar]
  23. Ruskin B., Krainer A. R., Maniatis T., Green M. R. Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro. Cell. 1984 Aug;38(1):317–331. doi: 10.1016/0092-8674(84)90553-1. [DOI] [PubMed] [Google Scholar]
  24. Shapiro M. B., Senapathy P. RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res. 1987 Sep 11;15(17):7155–7174. doi: 10.1093/nar/15.17.7155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sharp P. A. Splicing of messenger RNA precursors. Science. 1987 Feb 13;235(4790):766–771. doi: 10.1126/science.3544217. [DOI] [PubMed] [Google Scholar]
  26. Séraphin B., Kretzner L., Rosbash M. A U1 snRNA:pre-mRNA base pairing interaction is required early in yeast spliceosome assembly but does not uniquely define the 5' cleavage site. EMBO J. 1988 Aug;7(8):2533–2538. doi: 10.1002/j.1460-2075.1988.tb03101.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Treisman R., Orkin S. H., Maniatis T. Specific transcription and RNA splicing defects in five cloned beta-thalassaemia genes. Nature. 1983 Apr 14;302(5909):591–596. doi: 10.1038/302591a0. [DOI] [PubMed] [Google Scholar]
  28. Zhuang Y., Weiner A. M. A compensatory base change in U1 snRNA suppresses a 5' splice site mutation. Cell. 1986 Sep 12;46(6):827–835. doi: 10.1016/0092-8674(86)90064-4. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES