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p53 is activated in response to disruption of the pre-mRNA splicing machinery

Oncogene volume 32, pages 1–14 (2013)Cite this article

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Abstract

In this study, we show that interfering with the splicing machinery results in activation of the tumour-suppressor p53. The spliceosome was targeted by small interfering RNA-mediated knockdown of proteins associated with different small nuclear ribonucleoprotein complexes and by using the small-molecule splicing modulator TG003. These interventions cause: the accumulation of p53, an increase in p53 transcriptional activity and can result in p53-dependent G1 cell cycle arrest. Mdm2 and MdmX are two key repressors of p53. We show that a decrease in MdmX protein level contributes to p53 activation in response to targeting the spliceosome. Interfering with the spliceosome also causes an increase in the rate of degradation of Mdm2. Alterations in splicing are linked with tumour development. There are frequently global changes in splicing in cancer. Our study suggests that p53 activation could participate in protection against potential tumour-promoting defects in the spliceosome. A number of known p53-activating agents affect the splicing machinery and this could contribute to their ability to upregulate p53. Preclinical studies indicate that tumours can be more sensitive than normal cells to small-molecule spliceosome inhibitors. Activation of p53 could influence the selective anti-tumour activity of this therapeutic approach.

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References

  1. Lane DP, Cheok CF, Lain S . p53-based cancer therapy. Cold Spring Harb Perspect Biol 2010; 2: a001222.

    PubMed  PubMed Central  Google Scholar 

  2. Cheok CF, Verma CS, Baselga J, Lane DP . Translating p53 into the clinic. Nat Rev Clin Oncol. 2011; 8: 25–37.

    Article  CAS  PubMed  Google Scholar 

  3. Brooks CL, Gu W . p53 regulation by ubiquitin. FEBS Lett 2011; 585: 2803–2809.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Hock A, Vousden KH . Regulation of the p53 pathway by ubiquitin and related proteins. Int J Biochem Cell Biol 2010; 42: 1618–1621.

    Article  CAS  PubMed  Google Scholar 

  5. Lenos K, Jochemsen AG . Functions of MDMX in the modulation of the p53-response. J Biomed Biotechnol 2011; 2011: 876173.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Wade M, Wang YV, Wahl GM . The p53 orchestra: Mdm2 and Mdmx set the tone. Trends Cell Biol 2010; 20: 299–309.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Marine JC, Dyer MA, Jochemsen AG . MDMX: from bench to bedside. J Cell Sci 2007; 120: 371–378.

    Article  CAS  PubMed  Google Scholar 

  8. Stommel JM, Wahl GM . Accelerated MDM2 auto-degradation induced by DNA-damage kinases is required for p53 activation. EMBO J 2004; 23: 1547–1556.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kawai H, Wiederschain D, Kitao H, Stuart J, Tsai KK, Yuan ZM . DNA damage-induced MDMX degradation is mediated by MDM2. J Biol Chem 2003; 278: 45946–45953.

    Article  CAS  PubMed  Google Scholar 

  10. Wang YV, Wade M, Wong E, Li YC, Rodewald LW, Wahl GM . Quantitative analyses reveal the importance of regulated Hdmx degradation for p53 activation. Proc Natl Acad Sci USA 2007; 104: 12365–12370.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Will CL, Luhrmann R . Spliceosome Structure and Function. Cold Spring Harb Perspect Biol 2011; 3: 1–23.

    Article  Google Scholar 

  12. Ritchie DB, Schellenberg MJ, MacMillan AM . Spliceosome structure: piece by piece. Biochim Biophys Acta 2009; 1789: 624–633.

    Article  CAS  PubMed  Google Scholar 

  13. Newman AJ, Nagai K . Structural studies of the spliceosome: blind men and an elephant. Curr Opin Struct Biol 2010; 20: 82–89.

    Article  CAS  PubMed  Google Scholar 

  14. Valadkhan S, Jaladat Y . The spliceosomal proteome: at the heart of the largest cellular ribonucleoprotein machine. Proteomics 2010; 10: 4128–4141.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Pan Q, Shai O, Lee LJ, Frey BJ, Blencowe BJ . Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet 2008; 40: 1413–1415.

    Article  CAS  PubMed  Google Scholar 

  16. Wang ET, Sandberg R, Luo S, Khrebtukova I, Zhang L, Mayr C et al. Alternative isoform regulation in human tissue transcriptomes. Nature 2008; 456: 470–476.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Black DL . Protein diversity from alternative splicing: a challenge for bioinformatics and post-genome biology. Cell 2000; 103: 367–370.

    Article  CAS  PubMed  Google Scholar 

  18. Blencowe BJ . Alternative splicing: new insights from global analyses. Cell 2006; 126: 37–47.

    Article  CAS  PubMed  Google Scholar 

  19. Smith CW, Valcarcel J . Alternative pre-mRNA splicing: the logic of combinatorial control. Trends Biochem Sci 2000; 25: 381–388.

    Article  CAS  PubMed  Google Scholar 

  20. David CJ, Manley JL . Alternative pre-mRNA splicing regulation in cancer: pathways and programs unhinged. Genes Dev 2010; 24: 2343–2364.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ward AJ, Cooper TA . The pathobiology of splicing. J Pathol 2010; 220: 152–163.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Fackenthal JD, Godley LA . Aberrant RNA splicing and its functional consequences in cancer cells. Dis Model Mech 2008; 1: 37–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Ritchie W, Granjeaud S, Puthier D, Gautheret D . Entropy measures quantify global splicing disorders in cancer. PLoS Comput Biol 2008; 4: e1000011.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Reyal F, van Vliet MH, Armstrong NJ, Horlings HM, de Visser KE, Kok M et al. A comprehensive analysis of prognostic signatures reveals the high predictive capacity of the proliferation, immune response and RNA splicing modules in breast cancer. Breast Cancer Res 2008; 10: R93.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Grosso AR, Martins S, Carmo-Fonseca M . The emerging role of splicing factors in cancer. EMBO Rep 2008; 9: 1087–1093.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Andre F, Michiels S, Dessen P, Scott V, Suciu V, Uzan C et al. Exonic expression profiling of breast cancer and benign lesions: a retrospective analysis. Lancet Oncol 2009; 10: 381–390.

    Article  CAS  PubMed  Google Scholar 

  27. Kotake Y, Sagane K, Owa T, Mimori-Kiyosue Y, Shimizu H, Uesugi M et al. Splicing factor SF3b as a target of the antitumor natural product pladienolide. Nat Chem Biol 2007; 3: 570–575.

    Article  CAS  PubMed  Google Scholar 

  28. Kaida D, Motoyoshi H, Tashiro E, Nojima T, Hagiwara M, Ishigami K et al. Spliceostatin A targets SF3b and inhibits both splicing and nuclear retention of pre-mRNA. Nat Chem Biol 2007; 3: 576–583.

    Article  CAS  PubMed  Google Scholar 

  29. Grainger RJ, Beggs JD . Prp8 protein: at the heart of the spliceosome. RNA 2005; 11: 533–557.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Wilkinson CR, Dittmar GA, Ohi MD, Uetz P, Jones N, Finley D . Ubiquitin-like protein Hub1 is required for pre-mRNA splicing and localization of an essential splicing factor in fission yeast. Curr Biol 2004; 14: 2283–2288.

    Article  CAS  PubMed  Google Scholar 

  31. Alexander R, Beggs JD . Cross-talk in transcription, splicing and chromatin: who makes the first call? Biochem Soc Trans 2010; 38: 1251–1256.

    Article  CAS  PubMed  Google Scholar 

  32. Xiao SH, Manley JL . Phosphorylation of the ASF/SF2 RS domain affects both protein–protein and protein–RNA interactions and is necessary for splicing. Genes Dev 1997; 11: 334–344.

    Article  CAS  PubMed  Google Scholar 

  33. Cao W, Garcia-Blanco MA . A serine/arginine-rich domain in the human U1 70k protein is necessary and sufficient for ASF/SF2 binding. J Biol Chem 1998; 273: 20629–20635.

    Article  CAS  PubMed  Google Scholar 

  34. Hernandez H, Makarova OV, Makarov EM, Morgner N, Muto Y, Krummel DP et al. Isoforms of U1-70k control subunit dynamics in the human spliceosomal U1 snRNP. PLoS One 2009; 4: e7202.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Golas MM, Sander B, Will CL, Luhrmann R, Stark H . Molecular architecture of the multiprotein splicing factor SF3b. Science 2003; 300: 980–984.

    Article  CAS  PubMed  Google Scholar 

  36. Cass DM, Berglund JA . The SF3b155 N-terminal domain is a scaffold important for splicing. Biochemistry 2006; 45: 10092–10101.

    Article  CAS  PubMed  Google Scholar 

  37. Makarova OV, Makarov EM, Luhrmann R . The 65 and 110 kDa SR-related proteins of the U4/U6.U5 tri-snRNP are essential for the assembly of mature spliceosomes. EMBO J 2001; 20: 2553–2563.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Schaffert N, Hossbach M, Heintzmann R, Achsel T, Luhrmann R . RNAi knockdown of hPrp31 leads to an accumulation of U4/U6 di-snRNPs in Cajal bodies. EMBO J 2004; 23: 3000–3009.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Corrionero A, Minana B, Valcarcel J . Reduced fidelity of branch point recognition and alternative splicing induced by the anti-tumor drug spliceostatin A. Genes Dev 2011; 25: 445–459.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Muraki M, Ohkawara B, Hosoya T, Onogi H, Koizumi J, Koizumi T et al. Manipulation of alternative splicing by a newly developed inhibitor of Clks. J Biol Chem 2004; 279: 24246–24254.

    Article  CAS  PubMed  Google Scholar 

  41. Ghosh G, Adams JA . Phosphorylation mechanism and structure of serine-arginine protein kinases. FEBS J 2011; 278: 587–597.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Bartel F, Harris LC, Wurl P, Taubert H . MDM2 and its splice variant messenger RNAs: expression in tumors and down-regulation using antisense oligonucleotides. Mol Cancer Res 2004; 2: 29–35.

    CAS  PubMed  Google Scholar 

  43. Bonner WM, Redon CE, Dickey JS, Nakamura AJ, Sedelnikova OA, Solier S et al. GammaH2AX and cancer. Nat Rev Cancer 2008; 8: 957–967.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Ljungman M . The transcription stress response. Cell Cycle 2007; 6: 2252–2257.

    Article  CAS  PubMed  Google Scholar 

  45. Svejstrup JQ . Contending with transcriptional arrest during RNAPII transcript elongation. Trends Biochem Sci 2007; 32: 165–171.

    Article  CAS  PubMed  Google Scholar 

  46. Brody Y, Neufeld N, Bieberstein N, Causse SZ, Bohnlein EM, Neugebauer KM et al. The in vivo kinetics of RNA polymerase II elongation during co-transcriptional splicing. PLoS Biol 2011; 9: e1000573.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Singh J, Padgett RA . Rates of in situ transcription and splicing in large human genes. Nat Struct Mol Biol 2009; 16: 1128–1133.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Kim MY, Hur J, Jeong S . Emerging roles of RNA and RNA-binding protein network in cancer cells. BMB Rep 2009; 42: 125–130.

    Article  CAS  PubMed  Google Scholar 

  49. Venables JP, Klinck R, Koh C, Gervais-Bird J, Bramard A, Inkel L et al. Cancer-associated regulation of alternative splicing. Nat Struct Mol Biol 2009; 16: 670–676.

    Article  CAS  PubMed  Google Scholar 

  50. Kim E, Goren A, Ast G . Insights into the connection between cancer and alternative splicing. Trends Genet 2008; 24: 7–10.

    Article  CAS  PubMed  Google Scholar 

  51. Utans U, Behrens SE, Luhrmann R, Kole R, Kramer A . A splicing factor that is inactivated during in vivo heat shock is functionally equivalent to the [U4/U6.U5] triple snRNP-specific proteins. Genes Dev 1992; 6: 631–641.

    Article  CAS  PubMed  Google Scholar 

  52. Disher K, Skandalis A . Evidence of the modulation of mRNA splicing fidelity in humans by oxidative stress and p53. Genome 2007; 50: 946–953.

    Article  CAS  PubMed  Google Scholar 

  53. Zhao X, Yu YT . Incorporation of 5-fluorouracil into U2 snRNA blocks pseudouridylation and pre-mRNA splicing in vivo. Nucleic Acids Res 2007; 35: 550–558.

    Article  CAS  PubMed  Google Scholar 

  54. Lain S, Midgley C, Sparks A, Lane EB, Lane DP . An inhibitor of nuclear export activates the p53 response and induces the localization of HDM2 and p53 to U1A-positive nuclear bodies associated with the PODs. Exp Cell Res 1999; 248: 457–472.

    Article  CAS  PubMed  Google Scholar 

  55. Sleeman J . A regulatory role for CRM1 in the multi-directional trafficking of splicing snRNPs in the mammalian nucleus. J Cell Sci 2007; 120: 1540–1550.

    Article  CAS  PubMed  Google Scholar 

  56. Busa R, Sette C . An emerging role for nuclear RNA-mediated responses to genotoxic stress. RNA Biol 2010; 7: 390–396.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Matsuoka S, Ballif BA, Smogorzewska A, McDonald 3rd ER, Hurov KE, Luo J et al. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science 2007; 316: 1160–1166.

    CAS  PubMed  Google Scholar 

  58. Katzenberger RJ, Marengo MS, Wassarman DA . ATM and ATR pathways signal alternative splicing of Drosophila TAF1 pre-mRNA in response to DNA damage. Mol Cell Biol 2006; 26: 9256–9267.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Munoz MJ, Perez Santangelo MS, Paronetto MP, de la Mata M, Pelisch F, Boireau S et al. DNA damage regulates alternative splicing through inhibition of RNA polymerase II elongation. Cell 2009; 137: 708–720.

    Article  CAS  PubMed  Google Scholar 

  60. Bourdon JC, Fernandes K, Murray-Zmijewski F, Liu G, Diot A, Xirodimas DP et al. p53 isoforms can regulate p53 transcriptional activity. Genes Dev 2005; 19: 2122–2137.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Marcel V, Perrier S, Aoubala M, Ageorges S, Groves MJ, Diot A et al. Delta160p53 is a novel N-terminal p53 isoform encoded by Delta133p53 transcript. FEBS Lett 2010; 584: 4463–4468.

    Article  CAS  PubMed  Google Scholar 

  62. Chandler DS, Singh RK, Caldwell LC, Bitler JL, Lozano G . Genotoxic stress induces coordinately regulated alternative splicing of the p53 modulators MDM2 and MDM4. Cancer Res 2006; 66: 9502–9508.

    Article  CAS  PubMed  Google Scholar 

  63. Egecioglu DE, Chanfreau G . Proofreading and spellchecking: a two-tier strategy for pre-mRNA splicing quality control. RNA 2011; 17: 383–389.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Dayal S, Sparks A, Jacob J, Allende-Vega N, Lane DP, Saville MK . Suppression of the deubiquitinating enzyme USP5 causes the accumulation of unanchored polyubiquitin and the activation of p53. J Biol Chem 2009; 284: 5030–5041.

    Article  CAS  PubMed  Google Scholar 

  65. Allende-Vega N, Sparks A, Lane DP, Saville MK . MdmX is a substrate for the deubiquitinating enzyme USP2a. Oncogene 2010; 29: 432–441.

    Article  CAS  PubMed  Google Scholar 

  66. Stevenson LF, Sparks A, Allende-Vega N, Xirodimas DP, Lane DP, Saville MK . The deubiquitinating enzyme USP2a regulates the p53 pathway by targeting Mdm2. EMBO J 2007; 26: 976–986.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Giglio S, Mancini F, Gentiletti F, Sparaco G, Felicioni L, Barassi F et al. Identification of an Aberrantly Spliced Form of HDMX in Human Tumors: A New Mechanism for HDM2 Stabilization. Cancer Res 2005; 65: 9687–9694.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was funded by Cancer Research UK.

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Authors and Affiliations

  1. Division of Cancer Research, Medical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Angus, UK

    N Allende-Vega, S Dayal, U Agarwala, A Sparks, J-C Bourdon & M K Saville

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  1. N Allende-Vega
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  2. S Dayal
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Correspondence to M K Saville.

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Allende-Vega, N., Dayal, S., Agarwala, U. et al. p53 is activated in response to disruption of the pre-mRNA splicing machinery. Oncogene 32, 1–14 (2013). https://doi.org/10.1038/onc.2012.38

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