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SMN2 splice modulators enhance U1–pre-mRNA association and rescue SMA mice

Nature Chemical Biology volume 11, pages 511–517 (2015)Cite this article

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A Corrigendum to this article was published on 18 March 2016

A Corrigendum to this article was published on 18 August 2015

This article has been updated

Abstract

Spinal muscular atrophy (SMA), which results from the loss of expression of the survival of motor neuron-1 (SMN1) gene, represents the most common genetic cause of pediatric mortality. A duplicate copy (SMN2) is inefficiently spliced, producing a truncated and unstable protein. We describe herein a potent, orally active, small-molecule enhancer of SMN2 splicing that elevates full-length SMN protein and extends survival in a severe SMA mouse model. We demonstrate that the molecular mechanism of action is via stabilization of the transient double-strand RNA structure formed by the SMN2 pre-mRNA and U1 small nuclear ribonucleic protein (snRNP) complex. The binding affinity of U1 snRNP to the 5′ splice site is increased in a sequence-selective manner, discrete from constitutive recognition. This new mechanism demonstrates the feasibility of small molecule–mediated, sequence-selective splice modulation and the potential for leveraging this strategy in other splicing diseases.

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Figure 1: Screening paradigm and molecules.
Figure 2: Small molecules modulate SMN levels in vivo.
Figure 3: Gene- and transcript-level changes in response to NVS-SM1.
Figure 4: NVS-SM2 sequence selectively alters exon splicing.
Figure 5: NVS-SM2 binds to and stabilizes the U1 snRNP:5′ss RNA complex.
Figure 6: Computational model and schematic of mechanism of action.

Change history

  • 15 July 2015

    The authors forgot to include a description of the measurement of SMN protein levels in tissue samples in the Online Methods. This description has been included in the HTML and PDF versions of the article.

  • 11 February 2016

    In the version of this article originally published online, the schematic for the construct in Figure 4a was incorrect. A corrected figure has been provided in the HTML and PDF versions of the article.

References

  1. Sugarman, E.A. et al. Pan-ethnic carrier screening and prenatal diagnosis for spinal muscular atrophy: clinical laboratory analysis of >72,400 specimens. Eur. J. Hum. Genet. 20, 27–32 (2012).

    Article  PubMed  Google Scholar 

  2. Lunn, M.R. & Wang, C.H. Spinal muscular atrophy. Lancet 371, 2120–2133 (2008).

    Article  PubMed  Google Scholar 

  3. Zhou, J., Zheng, X. & Shen, H. Targeting RNA-splicing for SMA treatment. Mol. Cells 33, 223–228 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Kolb, S.J. & Kissel, J.T. Spinal muscular atrophy: a timely review. Arch. Neurol. 68, 979–984 (2011).

    Article  PubMed  Google Scholar 

  5. Lefebvre, S. et al. Identification and characterization of a spinal muscular atrophy-determining gene. Cell 80, 155–165 (1995).

    Article  CAS  PubMed  Google Scholar 

  6. Monani, U.R. A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2. Hum. Mol. Genet. 8, 1177–1183 (1999).

    Article  CAS  PubMed  Google Scholar 

  7. Gavrilov, D.K., Shi, X., Das, K., Gilliam, T.C. & Wang, C.H. Differential SMN2 expression associated with SMA severity. Nat. Genet. 20, 230–231 (1998).

    Article  CAS  PubMed  Google Scholar 

  8. Lorson, C.L., Hahnen, E., Androphy, E.J. & Wirth, B. A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proc. Natl. Acad. Sci. USA 96, 6307–6311 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Le, T.T. et al. SMNΔ7, the major product of the centromeric survival motor neuron (SMN2) gene, extends survival in mice with spinal muscular atrophy and associates with full-length SMN. Hum. Mol. Genet. 14, 845–857 (2005).

    Article  CAS  PubMed  Google Scholar 

  10. Vitali, T. et al. Detection of the survival motor neuron (SMN) genes by FISH: further evidence for a role for SMN2 in the modulation of disease severity in SMA patients. Hum. Mol. Genet. 8, 2525–2532 (1999).

    Article  CAS  PubMed  Google Scholar 

  11. Monani, U.R. Spinal muscular atrophy: a deficiency in a ubiquitous protein; a motor neuron-specific disease. Neuron 48, 885–896 (2005).

    Article  CAS  PubMed  Google Scholar 

  12. Sumner, C.J. Molecular mechanisms of spinal muscular atrophy. J. Child Neurol. 22, 979–989 (2007).

    Article  PubMed  Google Scholar 

  13. McAndrew, P.E. et al. Identification of proximal spinal muscular atrophy carriers and patients by analysis of SMNT and SMNC gene copy number. Am. J. Hum. Genet. 60, 1411–1422 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Matera, A.G. & Wang, Z. A day in the life of the spliceosome. Nat. Rev. Mol. Cell Biol. 15, 108–121 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Wang, E.T. et al. Alternative isoform regulation in human tissue transcriptomes. Nature 456, 470–476 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Wang, Z. & Burge, C.B. Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. RNA 14, 802–813 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Xiong, H.Y. et al. The human splicing code reveals new insights into the genetic determinants of disease. Science 347, 1254806 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Hua, Y., Vickers, T.A., Baker, B.F., Bennett, C.F. & Krainer, A.R. Enhancement of SMN2 exon 7 inclusion by antisense oligonucleotides targeting the exon. PLoS Biol. 5, e73 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Hua, Y., Vickers, T.A., Okunola, H.L., Bennett, C.F. & Krainer, A.R. Antisense masking of an hnRNP A1/A2 intronic splicing silencer corrects SMN2 splicing in transgenic mice. Am. J. Hum. Genet. 82, 834–848 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Naryshkin, N.A. et al. SMN2 splicing modifiers improve motor function and longevity in mice with spinal muscular atrophy. Science 345, 688–693 (2014).

    Article  CAS  PubMed  Google Scholar 

  21. Roca, X., Krainer, A.R. & Eperon, I.C. Pick one, but be quick: 5′ splice sites and the problems of too many choices. Genes Dev. 27, 129–144 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Lewandowska, M.A. The missing puzzle piece: splicing mutations. Int. J. Clin. Exp. Pathol. 6, 2675–2682 (2013).

    PubMed  PubMed Central  Google Scholar 

  23. Lara-Pezzi, E., Gómez-Salinero, J., Gatto, A. & García-Pavía, P. The alternative heart: impact of alternative splicing in heart disease. J. Cardiovasc. Transl. Res. 6, 945–955 (2013).

    Article  PubMed  Google Scholar 

  24. Wang, J., Zhang, J., Li, K., Zhao, W. & Cui, Q. SpliceDisease database: linking RNA splicing and disease. Nucleic Acids Res. 40, D1055–D1059 (2012).

    Article  CAS  PubMed  Google Scholar 

  25. Osborne, M. et al. Characterization of behavioral and neuromuscular junction phenotypes in a novel allelic series of SMA mouse models. Hum. Mol. Genet. 21, 4431–4447 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Burnett, B.G. et al. Regulation of SMN protein stability. Mol. Cell. Biol. 29, 1107–1115 (2009).

    Article  CAS  PubMed  Google Scholar 

  27. El-Khodor, B.F. et al. Identification of a battery of tests for drug candidate evaluation in the SMNΔ7 neonate model of spinal muscular atrophy. Exp. Neurol. 212, 29–43 (2008).

    Article  CAS  PubMed  Google Scholar 

  28. Le, T.T. et al. Temporal requirement for high SMN expression in SMA mice. Hum. Mol. Genet. 20, 3578–3591 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Kariya, S. et al. Requirement of enhanced Survival Motoneuron protein imposed during neuromuscular junction maturation. J. Clin. Invest. 124, 785–800 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Richard, H. et al. Prediction of alternative isoforms from exon expression levels in RNA-Seq experiments. Nucleic Acids Res. 38, e112 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Goina, E., Skoko, N. & Pagani, F. Binding of DAZAP1 and hnRNPA1/A2 to an exonic splicing silencer in a natural BRCA1 exon 18 mutant. Mol. Cell. Biol. 28, 3850–3860 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Singh, N.N., Singh, R.N. & Androphy, E.J. Modulating role of RNA structure in alternative splicing of a critical exon in the spinal muscular atrophy genes. Nucleic Acids Res. 35, 371–389 (2007).

    Article  CAS  PubMed  Google Scholar 

  33. Crooks, G.E., Hon, G., Chandonia, J.-M. & Brenner, S.E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Bailey, T.L. et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 37, W202–W208 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Akerman, M., David-Eden, H., Pinter, R.Y. & Mandel-Gutfreund, Y. A computational approach for genome-wide mapping of splicing factor binding sites. Genome Biol. 10, R30 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Yeo, G. & Burge, C.B. Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J. Comput. Biol. 11, 377–394 (2004).

    Article  CAS  PubMed  Google Scholar 

  37. Salcius, M. et al. SEC-TID: a label-free method for small-molecule target identification. J. Biomol. Screen. 19, 917–927 (2014).

    Article  CAS  PubMed  Google Scholar 

  38. Kondo, Y., Oubridge, C., van Roon, A.-M.M. & Nagai, K. Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5′ splice site recognition. Elife 4, e04986 (2015).

    Article  PubMed Central  Google Scholar 

  39. Rigo, F. et al. Pharmacology of a central nervous system delivered 2′-O-methoxyethyl-modified survival of motor neuron splicing oligonucleotide in mice and nonhuman primates. J. Pharmacol. Exp. Ther. 350, 46–55 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Kotake, Y. et al. Splicing factor SF3b as a target of the antitumor natural product pladienolide. Nat. Chem. Biol. 3, 570–575 (2007).

    Article  CAS  PubMed  Google Scholar 

  41. Zhang, M.L., Lorson, C.L., Androphy, E.J. & Zhou, J. An in vivo reporter system for measuring increased inclusion of exon 7 in SMN2 mRNA: potential therapy of SMA. Gene Ther. 8, 1532–1538 (2001).

    Article  CAS  PubMed  Google Scholar 

  42. Cheung, A.K. et al. 1,4-disubstituted pyridazine analogs there of and methods for treating SMN-deficiency–related conditions. United States Patent 8,729,263 (2014).

  43. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    Article  CAS  PubMed  Google Scholar 

  44. Lacoste, A., Berenshteyn, F. & Brivanlou, A.H. An efficient and reversible transposable system for gene delivery and lineage-specific differentiation in human embryonic stem cells. Cell Stem Cell 5, 332–342 (2009).

    Article  CAS  PubMed  Google Scholar 

  45. Chambers, S.M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275–280 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Pruitt, K.D., Tatusova, T. & Maglott, D.R. NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res. 35, D61–D65 (2007).

    Article  CAS  PubMed  Google Scholar 

  47. Langmead, B. & Salzberg, S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. 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  PubMed  Google Scholar 

  49. Krainer, A.R. Pre-mRNA splicing by complementation with purified human U1, U2, U4/U6 and U5 snRNPs. Nucleic Acids Res. 16, 9415–9429 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Kastner, B. & Lührmann, R. Purification of U small nuclear ribonucleoprotein particles. Methods Mol. Biol. 118, 289–298 (1999).

    CAS  PubMed  Google Scholar 

  51. Frostell-Karlsson, A. et al. Biosensor analysis of the interaction between immobilized human serum albumin and drug compounds for prediction of human serum albumin binding levels. J. Med. Chem. 43, 1986–1992 (2000).

    Article  CAS  PubMed  Google Scholar 

  52. Parisien, M. & Major, F. The MC-Fold and MC-Sym pipeline infers RNA structure from sequence data. Nature 452, 51–55 (2008).

    Article  CAS  PubMed  Google Scholar 

  53. Rueda, M., Totrov, M. & Abagyan, R. ALiBERO: evolving a team of complementary pocket conformations rather than a single leader. J. Chem. Inf. Model. 52, 2705–2714 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Abagyan, R. & Totrov, M. Biased probability Monte Carlo conformational searches and electrostatic calculations for peptides and proteins. J. Mol. Biol. 235, 983–1002 (1994).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors wish to acknowledge members of the Novartis Institutes for BioMedical Research (NIBR) Leadership Spinal Muscular Atrophy Advisory Board (J. Hastewell, J. Bell, K. Briner, P. Bouchard, E. Beckman and G. Kwei), NIBR Project Management (D. Silva and C. Gauthier) and NIBR Translational Medicine (R. Roubenoff) for their advice and contributions to the drug discovery efforts; J.R. Kerrigan, D. Glass, P. Manos, F. Harbinski, C. Mickanin, R.E.J. Beckwith, R. Sun, W. Broom, S.J. Luchanksy, L. Murphy, M. Schirle, J. Duca, R. Chopra and K. Clark for their contributions to experimental efforts and insights on the manuscript; S.J. Burden (NYU School of Medicine) for his gift of SMNΔ7 mouse myoblasts; K. Mineev and A.S. Arseniev for their assistance with NMR peak assignments; A. Abrams for his artwork in the schematic diagram; the SMA Foundation (K. Chen, D. Kobayashi, S. Paushkin and L. Eng) for their advice and contributions to the drug discovery efforts; Psychogenics (S. Ramboz and K. Cirillo); and PharmOptima (D. Decker, R. Poorman and P. Zaworski) for their contributions to in vivo studies.

Author information

Author notes

  1. James Palacino, Susanne E Swalley and Cheng Song: These authors contributed equally to this work.

  2. These authors contributed equally to this work.

Authors and Affiliations

  1. Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA

    James Palacino, Susanne E Swalley, Cheng Song, Atwood K Cheung, Lei Shu, Xiaolu Zhang, Mailin Van Hoosear, Youngah Shin, Donovan N Chin, Nicole A Renaud, Thomas M Smith, Michael Salcius, Xiaoying Shi, Marc Hild, Rebecca Servais, Monish Jain, Lin Deng, Caroline Bullock, Michael McLellan, Leo Murphy, Cecile Blaustein, Frada Berenshteyn, Arnaud Lacoste, Jason R Thomas, Gregory A Michaud, Brian S Tseng, Jeffery A Porter, Vic E Myer, John A Tallarico, Lawrence G Hamann, Daniel Curtis, Mark C Fishman, William F Dietrich, Natalie A Dales & Rajeev Sivasankaran

  2. Novartis Institutes for Biomedical Research, Forum 1, Basel, Switzerland

    Caroline Gubser Keller, Martin Beibel, Sven Schuierer, Marcel J J Blommers & Guglielmo Roma

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  1. James Palacino
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Contributions

C.S., T.M.S. and R.S. performed the high-throughput screen. A.K.C., L.S., L.G.H. and N.A.D. performed the chemical synthesis. C.S., M.V.H., Y.S., C. Blaustein, F.B., A.L. and R.S. performed cell-based structure-activity experiments. M.V.H., Y.S., R.S., M.J., L.D., C. Bullock, M.M., W.F.D. and R.S. performed in vivo experiments. J.P., C.G.K., M.B., N.A.R., X.S., M.H., S.S., L.M., G.R. and R.S. performed the RNAseq experiments. J.P. and C.S. performed the chimera experiments. S.E.S., M.S. and J.R.T. performed the biochemistry and biophysics experiments. X.Z. and M.J.J.B. performed the NMR experiments. D.N.C. performed the computational modeling. L.G.H. and N.A.D. supervised the medicinal chemistry experiments. M.J., B.S.T., W.F.D. and R.S. provided intellectual input to the in vivo mouse biology experiments. B.S.T., J.A.P., D.C., M.C.F. and R.S. provided intellectual input to the overall drug discovery studies. G.A.M., J.A.P., V.E.M. and J.A.T. provided intellectual input to the mechanism of action studies. N.A.D. and R.S. directed the drug discovery experiments. J.P. and S.E.S. directed the mechanism-of-action experiments. J.P., S.E.S., J.A.T., N.A.D. and R.S. prepared the manuscript.

Corresponding authors

Correspondence to Susanne E Swalley or Rajeev Sivasankaran.

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Palacino, J., Swalley, S., Song, C. et al. SMN2 splice modulators enhance U1–pre-mRNA association and rescue SMA mice. Nat Chem Biol 11, 511–517 (2015). https://doi.org/10.1038/nchembio.1837

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