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.

  • Article
  • Published:

Cotranscriptional exon skipping in the genotoxic stress response

Nature Structural & Molecular Biology volume 17, pages 1358–1366 (2010)Cite this article

Subjects

Abstract

Pre-mRNA splicing is functionally coupled to transcription, and genotoxic stresses can enhance alternative exon inclusion by affecting elongating RNA polymerase II. We report here that various genotoxic stress inducers, including camptothecin (CPT), inhibit the interaction between Ewing's sarcoma proto-oncoprotein (EWS), an RNA polymerase II–associated factor, and YB-1, a spliceosome-associated factor. This results in the cotranscriptional skipping of several exons of the MDM2 gene, which encodes the main p53 ubiquitin ligase. This reversible exon skipping participates in the regulation of MDM2 expression that may contribute to the accumulation of p53 during stress exposure and its rapid shut-off when stress is removed. Finally, a splicing-sensitive microarray identified numerous exons that are skipped in response to CPT and EWS–YB-1 depletion. These data demonstrate genotoxic stress-induced alteration of the communication between the transcriptional and splicing machineries, which results in widespread exon skipping and plays a central role in the genotoxic stress response.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: CPT treatment induces MDM2 exon skipping.
Figure 2: CPT effects on MDM2 splicing are reversible and p53 independent.
Figure 3: Cotranscriptional alteration of MDM2 splicing by CPT.
Figure 4: CPT-mediated effects on MDM2 splicing are not dependent on Pol II phosphorylation.
Figure 5: EWS and YB-1 depletion induces MDM2 exon skipping.
Figure 6: CPT impairs EWS and YB-1 interaction and YB-1 recruitment on the MDM2 gene.
Figure 7: Transcriptome-wide impact of CPT on exon skipping.
Figure 8: Model depicting the effects of CPT exposure and withdrawal on the MDM2-p53 loop.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

Gene Expression Omnibus

References

  1. Zorio, D.A. & Bentley, D.L. The link between mRNA processing and transcription: communication works both ways. Exp. Cell Res. 296, 91–97 (2004).

    Article  CAS  Google Scholar 

  2. Listerman, I., Sapra, A.K. & Neugebauer, K.M. Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells. Nat. Struct. Mol. Biol. 13, 815–822 (2006).

    Article  CAS  Google Scholar 

  3. Wetterberg, I., Bauren, G. & Wieslander, L. The intranuclear site of excision of each intron in Balbiani ring 3 pre-mRNA is influenced by the time remaining to transcription termination and different excision efficiencies for the various introns. RNA 2, 641–651 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Bittencourt, D. et al. Cotranscriptional splicing potentiates the mRNA production from a subset of estradiol-stimulated genes. Mol. Cell. Biol. 28, 5811–5824 (2008).

    Article  CAS  Google Scholar 

  5. Ghosh, S. & Garcia-Blanco, M.A. Coupled in vitro synthesis and splicing of RNA polymerase II transcripts. RNA 6, 1325–1334 (2000).

    Article  CAS  Google Scholar 

  6. Das, R. et al. Functional coupling of RNAP II transcription to spliceosome assembly. Genes Dev. 20, 1100–1109 (2006).

    Article  CAS  Google Scholar 

  7. Stamm, S. et al. Function of alternative splicing. Gene 344, 1–20 (2005).

    Article  CAS  Google Scholar 

  8. de la Mata, M. et al. A slow RNA polymerase II affects alternative splicing in vivo. Mol. Cell 12, 525–532 (2003).

    Article  CAS  Google Scholar 

  9. Muñoz, M.J. et al. DNA damage regulates alternative splicing through inhibition of RNA polymerase II elongation. Cell 137, 708–720 (2009).

    Article  Google Scholar 

  10. Auboeuf, D. et al. CoAA, a nuclear receptor coactivator protein at the interface of transcriptional coactivation and RNA splicing. Mol. Cell. Biol. 24, 442–453 (2004).

    Article  CAS  Google Scholar 

  11. Masuhiro, Y. et al. Splicing potentiation by growth factor signals via estrogen receptor phosphorylation. Proc. Natl. Acad. Sci. USA 102, 8126–8131 (2005).

    Article  CAS  Google Scholar 

  12. Ljungman, M. & Hanawalt, P.C. The anti-cancer drug camptothecin inhibits elongation but stimulates initiation of RNA polymerase II transcription. Carcinogenesis 17, 31–35 (1996).

    Article  CAS  Google Scholar 

  13. Collins, I., Weber, A. & Levens, D. Transcriptional consequences of topoisomerase inhibition. Mol. Cell. Biol. 21, 8437–8451 (2001).

    Article  CAS  Google Scholar 

  14. Jung, Y. & Lippard, S.J. RNA polymerase II blockage by cisplatin-damaged DNA. Stability and polyubiquitylation of stalled polymerase. J. Biol. Chem. 281, 1361–1370 (2006).

    Article  CAS  Google Scholar 

  15. Ljungman, M. & Paulsen, M.T. The cyclin-dependent kinase inhibitor roscovitine inhibits RNA synthesis and triggers nuclear accumulation of p53 that is unmodified at Ser15 and Lys382. Mol. Pharmacol. 60, 785–789 (2001).

    CAS  PubMed  Google Scholar 

  16. Ljungman, M. & Lane, D.P. Transcription - guarding the genome by sensing DNA damage. Nat. Rev. Cancer 4, 727–737 (2004).

    Article  CAS  Google Scholar 

  17. Meek, D.W. The p53 response to DNA damage. DNA Repair (Amst.) 3, 1049–1056 (2004).

    Article  CAS  Google Scholar 

  18. Zauberman, A., Flusberg, D., Haupt, Y., Barak, Y. & Oren, M. A functional p53-responsive intronic promoter is contained within the human mdm2 gene. Nucleic Acids Res. 23, 2584–2592 (1995).

    Article  CAS  Google Scholar 

  19. Wu, X., Bayle, J.H., Olson, D. & Levine, A.J. The p53-mdm-2 autoregulatory feedback loop. Genes Dev. 7, 1126–1132 (1993).

    Article  CAS  Google Scholar 

  20. Haupt, Y., Maya, R., Kazaz, A. & Oren, M. Mdm2 promotes the rapid degradation of p53. Nature 387, 296–299 (1997).

    Article  CAS  Google Scholar 

  21. Matsumoto, R. et al. Short alternative splice transcripts of the mdm2 oncogene correlate to malignancy in human astrocytic neoplasms. Cancer Res. 58, 609–613 (1998).

    CAS  PubMed  Google Scholar 

  22. Bartel, F., Taubert, H. & Harris, L.C. Alternative and aberrant splicing of MDM2 mRNA in human cancer. Cancer Cell 2, 9–15 (2002).

    Article  CAS  Google Scholar 

  23. Sigalas, I., Calvert, A.H., Anderson, J.J., Neal, D.E. & Lunec, J. Alternatively spliced mdm2 transcripts with loss of p53 binding domain sequences: transforming ability and frequent detection in human cancer. Nat. Med. 2, 912–917 (1996).

    Article  CAS  Google Scholar 

  24. Evans, S.C. et al. An alternatively spliced HDM2 product increases p53 activity by inhibiting HDM2. Oncogene 20, 4041–4049 (2001).

    Article  CAS  Google Scholar 

  25. Chandler, D.S., Singh, R.K., Caldwell, L.C., Bitler, J.L. & Lozano, G. Genotoxic stress induces coordinately regulated alternative splicing of the p53 modulators MDM2 and MDM4. Cancer Res. 66, 9502–9508 (2006).

    Article  CAS  Google Scholar 

  26. Dias, C.S., Liu, Y., Yau, A., Westrick, L. & Evans, S.C. Regulation of hdm2 by stress-induced hdm2alt1 in tumor and nontumorigenic cell lines correlating with p53 stability. Cancer Res. 66, 9467–9473 (2006).

    Article  CAS  Google Scholar 

  27. Pochampally, R. et al. A 60 kd MDM2 isoform is produced by caspase cleavage in non-apoptotic tumor cells. Oncogene 17, 2629–2636 (1998).

    Article  CAS  Google Scholar 

  28. Rigo, F. & Martinson, H.G. Functional coupling of last-intron splicing and 3′-end processing to transcription in vitro: the poly(A) signal couples to splicing before committing to cleavage. Mol. Cell. Biol. 28, 849–862 (2008).

    Article  CAS  Google Scholar 

  29. Kornblihtt, A.R. Promoter usage and alternative splicing. Curr. Opin. Cell Biol. 17, 262–268 (2005).

    Article  CAS  Google Scholar 

  30. Rossi, F. et al. Specific phosphorylation of SR proteins by mammalian DNA topoisomerase I. Nature 381, 80–82 (1996).

    Article  CAS  Google Scholar 

  31. Bertolotti, A. et al. EWS, but not EWS-FLI-1, is associated with both TFIID and RNA polymerase II: interactions between two members of the TET family, EWS and hTAFII68, and subunits of TFIID and RNA polymerase II complexes. Mol. Cell. Biol. 18, 1489–1497 (1998).

    Article  CAS  Google Scholar 

  32. Yang, L., Chansky, H.A. & Hickstein, D.D. EWS.Fli-1 fusion protein interacts with hyperphosphorylated RNA polymerase II and interferes with serine-arginine protein-mediated RNA splicing. J. Biol. Chem. 275, 37612–37618 (2000).

    Article  CAS  Google Scholar 

  33. Chansky, H.A., Hu, M., Hickstein, D.D. & Yang, L. Oncogenic TLS/ERG and EWS/Fli-1 fusion proteins inhibit RNA splicing mediated by YB-1 protein. Cancer Res. 61, 3586–3590 (2001).

    CAS  PubMed  Google Scholar 

  34. Petermann, R. et al. Oncogenic EWS-Fli1 interacts with hsRPB7, a subunit of human RNA polymerase II. Oncogene 17, 603–610 (1998).

    Article  CAS  Google Scholar 

  35. Liang, H., Atkins, H., Abdel-Fattah, R., Jones, S.N. & Lunec, J. Genomic organisation of the human MDM2 oncogene and relationship to its alternatively spliced mRNAs. Gene 338, 217–223 (2004).

    Article  CAS  Google Scholar 

  36. Brand, M. et al. UV-damaged DNA-binding protein in the TFTC complex links DNA damage recognition to nucleosome acetylation. EMBO J. 20, 3187–3196 (2001).

    Article  CAS  Google Scholar 

  37. Martinez, E. et al. Human staga complex is a chromatin-acetylating transcription coactivator that interacts with pre-mrna splicing and dna damage-binding factors in vivo. Mol. Cell. Biol. 21, 6782–6795 (2001).

    Article  CAS  Google Scholar 

  38. Cavellán, E., Asp, P., Percipalle, P. & Farrants, A.K. The WSTF-SNF2h chromatin remodeling complex interacts with several nuclear proteins in transcription. J. Biol. Chem. 281, 16264–16271 (2006).

    Article  Google Scholar 

  39. Clark, T.A. et al. Discovery of tissue-specific exons using comprehensive human exon microarrays. Genome Biol. 8, R64 (2007).

    Article  Google Scholar 

  40. Dutertre, M. et al. Exon-based clustering of murine breast tumor transcriptomes reveals alternative exons whose expression is associated with metastasis. Cancer Res. 70, 896–905 (2010).

    Article  CAS  Google Scholar 

  41. Solier, S. et al. Topoisomerase I and II inhibitors control caspase-2 pre-messenger RNA splicing in human cells. Mol. Cancer Res. 2, 53–61 (2004).

    CAS  PubMed  Google Scholar 

  42. Barbier, J. et al. Regulation of H-ras splice variant expression by cross talk between the p53 and nonsense-mediated mRNA decay pathways. Mol. Cell. Biol. 27, 7315–7333 (2007).

    Article  CAS  Google Scholar 

  43. Marengo, M.S. & Wassarman, D.A.A. DNA damage signal activates and derepresses exon inclusion in Drosophila TAF1 alternative splicing. RNA 14, 1681–1695 (2008).

    Article  CAS  Google Scholar 

  44. Sanchez, G. et al. Alteration of cyclin D1 transcript elongation by a mutated transcription factor up-regulates the oncogenic D1b splice isoform in cancer. Proc. Natl. Acad. Sci. USA 105, 6004–6009 (2008).

    Article  CAS  Google Scholar 

  45. Law, W.J., Cann, K.L. & Hicks, G.G. TLS, EWS and TAF15: a model for transcriptional integration of gene expression. Brief. Funct. Genomic Proteomic 5, 8–14 (2006).

    Article  CAS  Google Scholar 

  46. Stickeler, E. et al. The RNA binding protein YB-1 binds A/C-rich exon enhancers and stimulates splicing of the CD44 alternative exon v4. EMBO J. 20, 3821–3830 (2001).

    Article  CAS  Google Scholar 

  47. Hartmuth, K. et al. Protein composition of human prespliceosomes isolated by a tobramycin affinity-selection method. Proc. Natl. Acad. Sci. USA 99, 16719–16724 (2002).

    Article  CAS  Google Scholar 

  48. Deckert, J. et al. Protein composition and electron microscopy structure of affinity-purified human spliceosomal B complexes isolated under physiological conditions. Mol. Cell. Biol. 26, 5528–5543 (2006).

    Article  CAS  Google Scholar 

  49. Allemand, E., Hastings, M.L., Murray, M.V., Myers, M.P. & Krainer, A.R. Alternative splicing regulation by interaction of phosphatase PP2Cgamma with nucleic acid-binding protein YB-1. Nat. Struct. Mol. Biol. 14, 630–638 (2007).

    Article  CAS  Google Scholar 

  50. Solier, S. et al. Genome-wide analysis of novel splice variants induced by topoisomerase I poisoning shows preferential occurrence in genes encoding splicing factors. Cancer Res. published online 3 September 2010, doi:10.1158/0008-5472.CAN-10-2491.

    Article  CAS  Google Scholar 

  51. Pagani, F., Stuani, C., Zuccato, E., Kornblihtt, A.R. & Baralle, F.E. Promoter architecture modulates CFTR exon 9 skipping. J. Biol. Chem. 278, 1511–1517 (2003).

    Article  CAS  Google Scholar 

  52. Pleiss, J.A., Whitworth, G.B., Bergkessel, M. & Guthrie, C. Rapid, transcript-specific changes in splicing in response to environmental stress. Mol. Cell 27, 928–937 (2007).

    Article  CAS  Google Scholar 

  53. Zhang, N. et al. The Pso4 mRNA splicing and DNA repair complex interacts with WRN for processing of DNA interstrand cross-links. J. Biol. Chem. 280, 40559–40567 (2005).

    Article  CAS  Google Scholar 

  54. Singh, R.K., Tapia-Santos, A., Bebee, T.W. & Chandler, D.S. Conserved sequences in the final intron of MDM2 are essential for the regulation of alternative splicing of MDM2 in response to stress. Exp. Cell Res. 315, 3419–3432 (2009).

    Article  CAS  Google Scholar 

  55. Tanackovic, G. & Kramer, A. Human splicing factor SF3a, but not SF1, is essential for pre-mRNA splicing in vivo. Mol. Biol. Cell 16, 1366–1377 (2005).

    Article  CAS  Google Scholar 

  56. Hastings, M.L., Allemand, E., Duelli, D.M., Myers, M.P. & Krainer, A.R. Control of pre-mRNA splicing by the general splicing factors PUF60 and U2AF65. PLoS ONE 2, e538 (2007).

    Article  Google Scholar 

  57. Pacheco, T.R., Coelho, M.B., Desterro, J.M., Mollet, I. & Carmo-Fonseca, M. In vivo requirement of the small subunit of U2AF for recognition of a weak 3′ splice site. Mol. Cell. Biol. 26, 8183–8190 (2006).

    Article  CAS  Google Scholar 

  58. Stommel, J.M. & Wahl, G.M. A new twist in the feedback loop: stress-activated MDM2 destabilization is required for p53 activation. Cell Cycle 4, 411–417 (2005).

    Article  CAS  Google Scholar 

  59. Bargou, R.C. et al. Nuclear localization and increased levels of transcription factor YB-1 in primary human breast cancers are associated with intrinsic MDR1 gene expression. Nat. Med. 3, 447–450 (1997).

    Article  CAS  Google Scholar 

  60. Shibahara, K. et al. Targeted disruption of one allele of the Y-box binding protein-1 (YB-1) gene in mouse embryonic stem cells and increased sensitivity to cisplatin and mitomycin C. Cancer Sci. 95, 348–353 (2004).

    Article  CAS  Google Scholar 

  61. de la Grange, P., Dutertre, M., Correa, M. & Auboeuf, D. A new advance in alternative splicing databases: from catalogue to detailed analysis of regulation of expression and function of human alternative splicing variants. BMC Bioinformatics 8, 180 (2007).

    Article  Google Scholar 

Download references

Acknowledgements

We are grateful to Angela Krämer (University of Geneva) and Robin Fahraeus (Institut National de la Santé et de la Recherche Médicale U940) for providing antibodies. This work was supported by the EC (NoE EURASNET), Association pour la recherche sur le cancer, Ligue nationale contre le cancer, Institut national du cancer and Agence Nationale de la Recherche. G.S. was supported by La Chancellerie des Universités de Paris; M.D. by l'Institut National de la Santé et de la Recherche Médicale; J.B. by the French Ministry of Education. M.-C.D.C. was supported by a fellowship from the Brittany Region; G.D. by Vaincre la Mucoviscidose; L.C. by Brest University, the IFR 148-ScInBioS.

Author information

Author notes

  1. Marie-Cécile De Cian

    Present address: Present address: Centre national de la recherche scientifique UMR 6134, Faculté des Sciences, Campus Grossetti, Corte, France.,

  2. Martin Dutertre and Gabriel Sanchez: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut National de la Santé et de la Recherche Médicale U590, Centre Léon Bérard, Lyon, France

    Martin Dutertre, Gabriel Sanchez, Jérôme Barbier, Etienne Dardenne, Lise Gratadou & Didier Auboeuf

  2. Institut National de la Santé et de la Recherche Médicale U613, Faculté de Médecine, Brest, France

    Marie-Cécile De Cian, Gwendal Dujardin, Catherine Le Jossic-Corcos & Laurent Corcos

Authors
  1. Martin Dutertre
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gabriel Sanchez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marie-Cécile De Cian
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jérôme Barbier
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Etienne Dardenne
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lise Gratadou
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Gwendal Dujardin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Catherine Le Jossic-Corcos
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Laurent Corcos
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Didier Auboeuf
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.D., G.S., M.-C.D.C., L.C. and D.A. designed the experiments; M.D., L.C. and D.A. supervised the project; M.D., G.S., M.-C.D.C., J.B., E.D., L.G., G.D. and C.L.-C. conducted the experiments; M.D., G.S., M.-C.D.C., C.L.-C., M.-C.D.C., L.C. and D.A. performed the data analysis; M.D., G.S., M.-C.D.C., L.C. and D.A. wrote the manuscript.

Corresponding authors

Correspondence to Martin Dutertre or Didier Auboeuf.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 and Supplementary Table 1 (PDF 914 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dutertre, M., Sanchez, G., De Cian, MC. et al. Cotranscriptional exon skipping in the genotoxic stress response. Nat Struct Mol Biol 17, 1358–1366 (2010). https://doi.org/10.1038/nsmb.1912

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1912

This article is cited by

Search

Advanced 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