Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

An LKB1 AT-AC intron mutation causes Peutz-Jeghers syndrome via splicing at noncanonical cryptic splice sites

Abstract

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disorder associated with gastrointestinal polyposis and an increased cancer risk. PJS is caused by germline mutations in the tumor suppressor gene LKB1. One such mutation, IVS2+1A>G, alters the second intron 5′ splice site, which has sequence features of a U12-type AT-AC intron. We report that in patients, LKB1 RNA splicing occurs from the mutated 5′ splice site to several cryptic, noncanonical 3′ splice sites immediately adjacent to the normal 3′ splice site. In vitro splicing analysis demonstrates that this aberrant splicing is mediated by the U12-dependent spliceosome. The results indicate that the minor spliceosome can use a variety of 3′ splice site sequences to pair to a given 5′ splice site, albeit with tight constraints for maintaining the 3′ splice site position. The unusual splicing defect associated with this PJS-causing mutation uncovers differences in splice-site recognition between the major and minor pre-mRNA splicing pathways.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Splicing of LKB1 IVS2+1A>G RNA from PJS patients.
Figure 2: Aberrant spliced products arise from LKB1 IVS2+1A>G and are subject to NMD.
Figure 3: In vitro splicing analysis of LKB1 IVS2+1A>G pre-mRNA.

Similar content being viewed by others

References

  1. Yoo, L.I., Chung, D.C. & Yuan, J. LKB1—a master tumour suppressor of the small intestine and beyond. Nat. Rev. Cancer 2, 529–535 (2002).

    Article  CAS  Google Scholar 

  2. Lim W. et al. Further observations on LKB1/STK11 status and cancer risk in Peutz-Jeghers syndrome. Br. J. Cancer 89, 308–313 (2003).

    Article  CAS  Google Scholar 

  3. Hemminki, A. et al. A serine/threonine kinase gene defective in Peutz-Jeghers syndrome. Nature 391, 184–187 (1998).

    Article  CAS  Google Scholar 

  4. Jenne, D.E. et al. Peutz-Jeghers syndrome is caused by mutations in a novel serine threonine kinase. Nat. Genet. 18, 38–43 (1998).

    Article  CAS  Google Scholar 

  5. Bardeesy, N. et al. Loss of the Lkb1 tumour suppressor provokes intestinal polyposis but resistance to transformation. Nature 419, 162–167 (2002).

    Article  CAS  Google Scholar 

  6. Shaw, R.J. et al. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc. Natl. Acad. Sci. USA 101, 3329–3335 (2004).

    Article  CAS  Google Scholar 

  7. Lizcano, J.M. et al. LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J. 23, 833–843 (2004).

    Article  CAS  Google Scholar 

  8. Corradetti, M.N., Inoki, K., Bardeesy, N., DePinho, R.A. & Guan, K.-L. Regulation of the TSC pathway by LKB1: evidence of a molecular link between tuberous sclerosis complex and Peutz-Jeghers syndrome. Genes Dev. 18, 1533–1538 (2004).

    Article  CAS  Google Scholar 

  9. Resta, N. et al. Two novel mutations and a new STK11/LKB1 gene isoform in Peutz-Jeghers patients. Hum. Mutat. 20, 78–79 (2002).

    Article  CAS  Google Scholar 

  10. Hastings, M.L. & Krainer, A.R. Splicing in the new millennium. Curr. Opin. Cell Biol. 13, 302–309 (2001).

    Article  CAS  Google Scholar 

  11. Patel, A.A. & Steitz, J.A. Splicing double: insights from the second spliceosome. Nat. Rev. Mol. Cell Biol. 4, 960–970 (2003).

    Article  CAS  Google Scholar 

  12. Levine, A. & Durbin, R. A computational scan for U12-dependent introns in the human genome sequence. Nucleic Acids Res. 29, 4006–4013 (2001).

    Article  CAS  Google Scholar 

  13. Burset, M., Seledtsov, I.A. & Solovyev, V.V. Analysis of canonical and non-canonical splice sites in mammalian genomes. Nucleic Acids Res. 28, 4364–4375 (2000).

    Article  CAS  Google Scholar 

  14. Maquat, L.E. Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics. Nat. Rev. Mol. Cell Biol. 5, 89–99 (2004).

    Article  CAS  Google Scholar 

  15. Nakai, K. & Sakamoto H. Construction of a novel database containing aberrant splicing mutations of mammalian genes. Gene 141, 171–177 (1994).

    Article  CAS  Google Scholar 

  16. Roca, X., Sachidanandam, R. & Krainer, A.R. Intrinsic differences between authentic and cryptic 5′ splice sites. Nucleic Acids Res. 31, 6321–6333 (2003).

    Article  CAS  Google Scholar 

  17. Hastings, M.L. & Krainer, A.R. Functions of SR proteins in the U12-dependent AT-AC pre-mRNA splicing pathway. RNA 7, 471–482 (2001).

    Article  CAS  Google Scholar 

  18. Senapathy, P., Shapiro, M.B. & Harris, N.L. Splice junctions, branch point sites, and exons: sequence statistics, identification, and applications to genome project. Methods Enzymol. 183, 252–278 (1990).

  19. Krainer, A.R., Maniatis, T., Ruskin, B. & Green, M.R. Normal and mutant human β-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell 36, 993–1005 (1984).

    Article  CAS  Google Scholar 

  20. Frilander, M.J. & Steitz, J.A. Initial recognition of U12-dependent introns requires both U11/5′ splice site and U12/branchpoint interactions. Genes Dev. 13, 851–863 (1999).

    Article  CAS  Google Scholar 

  21. Will, C.L. et al. The human 18S U11/U12 snRNP contains a set of novel proteins not found in the U2-dependent spliceosome. RNA 10, 929–941 (2004).

    Article  CAS  Google Scholar 

  22. McConnell, T.S., Cho, S.-J., Frilander, M.J. & Steitz, J.A. Branchpoint selection in the splicing of U12-dependent introns in vitro. RNA 8, 579–586 (2002).

    Article  CAS  Google Scholar 

  23. Dietrich, R.C., Incorvaia, R. & Padgett, R.A. Terminal intron dinucleotide sequences do not distinguish between U2- and U12- dependent introns. Mol. Cell 1, 151–160 (1997).

    Article  CAS  Google Scholar 

  24. Dietrich, R.C., Peris, M.J., Seyboldt, A.S. & Padgett, R.A. Role of the 3′ splice site in U12-dependent intron splicing. Mol. Cell. Biol. 21, 1942–1952 (2001).

    Article  CAS  Google Scholar 

  25. Collins, C.A. & Guthrie, C. Allele-specific genetic interactions between Prp8 and RNA active site residues suggest a function for Prp8 at the catalytic core of the spliceosome. Genes Dev. 13, 1970–1982 (1999).

    Article  CAS  Google Scholar 

  26. Siatecka, M., Reyes, J.L., & Konarska, M.M. Functional interactions of Prp8 with both splice sites at the spliceosomal catalytic center. Genes Dev. 13, 1983–1993 (1999).

    Article  CAS  Google Scholar 

  27. Luo, H.R., Moreau, G.A., Levin, N. & Moore, M.J. The human Prp8 protein is a component of both U2- and U12-dependent spliceosomes. RNA 5, 8893–8908 (1999).

    Article  Google Scholar 

  28. Wu, Q. & Krainer, A.R. AT-AC pre-mRNA splicing mechanisms and conservation of minor introns in voltage-gated ion channel genes. Mol. Cell. Biol. 19, 3225–3236 (1999).

    Article  CAS  Google Scholar 

  29. Burge, C.B., Padgett, R.A. & Sharp P.A. Evolutionary fates and origins of U12-type introns. Mol. Cell 2, 773–785 (1998).

    Article  CAS  Google Scholar 

  30. Clark, F. & Thanaraj, T.A. Categorization and characterization of transcript-confirmed constitutively and alternatively spliced introns and exons from human. Hum. Mol. Gen. 11, 451–464 (2002).

    Article  CAS  Google Scholar 

  31. Otake, L.R., Scamborova, P., Hashimoto, C. & Steitz, J.A. The divergent U12-type spliceosome is required for pre-mRNA splicing and is essential for development in Drosophila. Mol. Cell 9, 439–446 (2002).

    Article  CAS  Google Scholar 

  32. Kohrman, D.C., Harris, J.B. & Meisler, M.H. Mutation detection in the med and medJ alleles of the sodium channel Scn8a. J. Biol. Chem. 271, 17576–17581 (1996).

    Article  CAS  Google Scholar 

  33. Shaw, M.A. et al. Identification of three novel SEDL mutations, including mutation in the rare, non-canonical splice site of exon 4. Clin. Genet. 64, 235–242 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by US National Institutes of Health grant GM42699 (A.R.K.) and MIUR-FIRB RBAU01SZHB-001 (G.G.). M.L.H. was supported by an American Cancer Society postdoctoral fellowship. We thank L. Manche and A. Quagliarella for technical assistance.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Adrian R Krainer.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hastings, M., Resta, N., Traum, D. et al. An LKB1 AT-AC intron mutation causes Peutz-Jeghers syndrome via splicing at noncanonical cryptic splice sites. Nat Struct Mol Biol 12, 54–59 (2005). https://doi.org/10.1038/nsmb873

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb873

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing