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:

A peptide motif in Raver1 mediates splicing repression by interaction with the PTB RRM2 domain

Nature Structural & Molecular Biology volume 13, pages 839–848 (2006)Cite this article

Abstract

Polypyrimidine tract–binding protein (PTB) is a regulatory splicing repressor. Raver1 acts as a PTB corepressor for splicing of α-tropomyosin (Tpm1) exon 3. Here we define a minimal region of Raver1 that acts as a repressor domain when recruited to RNA. A conserved [S/G][I/L]LGxxP motif is essential for splicing repressor activity and sufficient for interaction with PTB. An adjacent proline-rich region is also essential for repressor activity but not for PTB interaction. NMR analysis shows that LLGxxP peptides interact with a hydrophobic groove on the dorsal surface of the RRM2 domain of PTB, which constitutes part of the minimal repressor region of PTB. The requirement for the PTB-Raver1 interaction that we have characterized may serve to bring the additional repressor regions of both proteins into a configuration that allows them to synergistically effect exon skipping.

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: A proline-rich splicing repressor domain in Raver1.
Figure 2: Multiple PTB-interacting regions of Raver1.
Figure 3: A peptide motif essential for splicing repression and PTB binding.
Figure 4: [S/G][L/I]LgxxP is sufficient to bind PTB.
Figure 5: Mutation of the four PTB-binding motifs impairs Raver1 activity.
Figure 6: Interaction of Raver1491–511 and RNA with PTB RRM2.
Figure 7: Structural model of the complex of Raver1 peptide with PTB RRM2.

Similar content being viewed by others

References

  1. Lareau, L.F., Green, R.E., Bhatnagar, R.S. & Brenner, S.E. The evolving roles of alternative splicing. Curr. Opin. Struct. Biol. 14, 273–282 (2004).

    Article  CAS  Google Scholar 

  2. Cartegni, L., Chew, S.L. & Krainer, A.R. Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat. Rev. Genet. 3, 285–298 (2002).

    Article  CAS  Google Scholar 

  3. Matlin, A.J., Clark, F. & Smith, C.W. Understanding alternative splicing: towards a cellular code. Nat. Rev. Mol. Cell Biol. 6, 386–398 (2005).

    Article  CAS  Google Scholar 

  4. Wagner, E.J. & Garcia-Blanco, M.A. Polypyrimidine tract binding protein antagonizes exon definition. Mol. Cell. Biol. 21, 3281–3288 (2001).

    Article  CAS  Google Scholar 

  5. Conte, M.R. et al. Structure of tandem RNA recognition motifs from polypyrimidine tract binding protein reveals novel features of the RRM fold. EMBO J. 19, 3132–3141 (2000).

    Article  CAS  Google Scholar 

  6. Simpson, P.J. et al. Structure and RNA interactions of the N-terminal RRM domains of PTB. Structure 12, 1631–1643 (2004).

    Article  CAS  Google Scholar 

  7. Oberstrass, F.C. et al. Structure of PTB bound to RNA: specific binding and implications for splicing regulation. Science 309, 2054–2057 (2005).

    Article  CAS  Google Scholar 

  8. Perez, I., Lin, C.H., McAfee, J.G. & Patton, J.G. Mutation of PTB binding sites causes misregulation of alternative 3′ splice site selection in vivo. RNA 3, 764–778 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Amir-Ahmady, B., Boutz, P.L., Markovtsov, V., Phillips, M.L. & Black, D.L. Exon repression by polypyrimidine tract binding protein. RNA 11, 699–716 (2005).

    Article  CAS  Google Scholar 

  10. Shen, H., Kan, J.L., Ghigna, C., Biamonti, G. & Green, M.R. A single polypyrimidine tract binding protein (PTB) binding site mediates splicing inhibition at mouse IgM exons M1 and M2. RNA 10, 787–794 (2004).

    Article  CAS  Google Scholar 

  11. Izquierdo, J.M. et al. Regulation of Fas alternative splicing by antagonistic effects of TIA-1 and PTB on exon definition. Mol. Cell 19, 475–484 (2005).

    Article  CAS  Google Scholar 

  12. Wagner, E.J. & Garcia-Blanco, M.A. RNAi-mediated PTB depletion leads to enhanced exon definition. Mol. Cell 10, 943–949 (2002).

    Article  CAS  Google Scholar 

  13. Gooding, C., Roberts, G.C. & Smith, C.W. Role of an inhibitory pyrimidine element and polypyrimidine tract binding protein in repression of a regulated alpha-tropomyosin exon. RNA 4, 85–100 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Lin, C.H. & Patton, J.G. Regulation of alternative 3′ splice site selection by constitutive splicing factors. RNA 1, 234–245 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Singh, R., Valcarcel, J. & Green, M.R. Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins. Science 268, 1173–1176 (1995).

    Article  CAS  Google Scholar 

  16. Chou, M.Y., Underwood, J.G., Nikolic, J., Luu, M.H. & Black, D.L. Multisite RNA binding and release of polypyrimidine tract binding protein during the regulation of c-src neural-specific splicing. Mol. Cell 5, 949–957 (2000).

    Article  CAS  Google Scholar 

  17. Perez, I., McAfee, J.G. & Patton, J.G. Multiple RRMs contribute to RNA binding specificity and affinity for polypyrimidine tract binding protein. Biochemistry 36, 11881–11890 (1997).

    Article  CAS  Google Scholar 

  18. Oh, Y.L. et al. Determination of functional domains in polypyrimidine-tract-binding protein. Biochem. J. 331, 169–175 (1998).

    Article  CAS  Google Scholar 

  19. Monie, T.P. et al. The polypyrimidine tract binding protein is a monomer. RNA 11, 1803–1808 (2005).

    Article  CAS  Google Scholar 

  20. Zhang, W., Liu, H., Han, K. & Grabowski, P.J. Region-specific alternative splicing in the nervous system: implications for regulation by the RNA-binding protein NAPOR. RNA 8, 671–685 (2002).

    Article  CAS  Google Scholar 

  21. Charlet-B., N., Logan, P., Singh, G. & Cooper, T.A. Dynamic antagonism between ETR-3 and PTB regulates cell type-specific alternative splicing. Mol. Cell 9, 649–658 (2002).

    Article  CAS  Google Scholar 

  22. Gromak, N., Matlin, A.J., Cooper, T.A. & Smith, C.W. Antagonistic regulation of alpha-actinin alternative splicing by CELF proteins and polypyrimidine tract binding protein. RNA 9, 443–456 (2003).

    Article  CAS  Google Scholar 

  23. Polydorides, A.D., Okano, H.J., Yang, Y.Y., Stefani, G. & Darnell, R.B. A brain-enriched polypyrimidine tract-binding protein antagonizes the ability of Nova to regulate neuron-specific alternative splicing. Proc. Natl. Acad. Sci. USA 97, 6350–6355 (2000).

    Article  CAS  Google Scholar 

  24. Markovtsov, V. et al. Cooperative assembly of an hnRNP complex induced by a tissue-specific homolog of polypyrimidine tract binding protein. Mol. Cell. Biol. 20, 7463–7479 (2000).

    Article  CAS  Google Scholar 

  25. Yamamoto, H., Tsukahara, K., Kanaoka, Y., Jinno, S. & Okayama, H. Isolation of a mammalian homologue of a fission yeast differentiation regulator. Mol. Cell. Biol. 19, 3829–3841 (1999).

    Article  CAS  Google Scholar 

  26. Gooding, C., Kemp, P. & Smith, C.W. A novel polypyrimidine tract-binding protein paralog expressed in smooth muscle cells. J. Biol. Chem. 278, 15201–15207 (2003).

    Article  CAS  Google Scholar 

  27. Huttelmaier, S. et al. Raver1, a dual compartment protein, is a ligand for PTB/hnRNPI and microfilament attachment proteins. J. Cell Biol. 155, 775–786 (2001).

    Article  CAS  Google Scholar 

  28. Gromak, N. et al. The PTB interacting protein raver1 regulates alpha-tropomyosin alternative splicing. EMBO J. 22, 6356–6364 (2003).

    Article  CAS  Google Scholar 

  29. Robinson, F. & Smith, C.W.J. A splicing repressor domain in polypyrimidine tract binding protein. J. Biol. Chem. 281, 800–806 (2006).

    Article  CAS  Google Scholar 

  30. Xia, Z. & Liu, Y. Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophys. J. 81, 2395–2402 (2001).

    Article  CAS  Google Scholar 

  31. Hahm, B. et al. Polypyrimidine tract-binding protein interacts with HnRNP L. FEBS Lett. 425, 401–406 (1998).

    Article  CAS  Google Scholar 

  32. Dominguez, C., Boelens, R. & Bonvin, A.M. HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J. Am. Chem. Soc. 125, 1731–1737 (2003).

    Article  CAS  Google Scholar 

  33. Singh, R. & Valcarcel, J. Building specificity with nonspecific RNA-binding proteins. Nat. Struct. Mol. Biol. 12, 645–653 (2005).

    Article  CAS  Google Scholar 

  34. Selenko, P. et al. Structural basis for the molecular recognition between human splicing factors U2AF65 and SF1/mBBP. Mol. Cell 11, 965–976 (2003).

    Article  CAS  Google Scholar 

  35. Sharma, S., Falick, A.M. & Black, D.L. Polypyrimidine Tract Binding protein blocks the 5′ splice site dependent assembly of U2AF and the prespliceosomal E complex. Mol. Cell 19, 485–496 (2005).

    Article  CAS  Google Scholar 

  36. Chan, D.C., Bedford, M.T. & Leder, P. Formin binding proteins bear WWP/WW domains that bind proline-rich peptides and functionally resemble SH3 domains. EMBO J. 15, 1045–1054 (1996).

    Article  CAS  Google Scholar 

  37. Bedford, M.T., Chan, D.C. & Leder, P. FBP WW domains and the Abl SH3 domain bind to a specific class of proline-rich ligands. EMBO J. 16, 2376–2383 (1997).

    Article  CAS  Google Scholar 

  38. Bedford, M.T., Reed, R. & Leder, P. WW domain-mediated interactions reveal a spliceosome-associated protein that binds a third class of proline-rich motif: the proline glycine and methionine-rich motif. Proc. Natl. Acad. Sci. USA 95, 10602–10607 (1998).

    Article  CAS  Google Scholar 

  39. Wollerton, M.C., Gooding, C., Wagner, E.J., Garcia-Blanco, M.A. & Smith, C.W. Autoregulation of polypyrimidine tract binding protein by alternative splicing leading to nonsense-mediated decay. Mol. Cell 13, 91–100 (2004).

    Article  CAS  Google Scholar 

  40. Roberts, G.C., Gooding, C., Mak, H.Y., Proudfoot, N.J. & Smith, C.W. Co-transcriptional commitment to alternative splice site selection. Nucleic Acids Res. 26, 5568–5572 (1998).

    Article  CAS  Google Scholar 

  41. Tian, M. & Maniatis, T. A splicing enhancer exhibits both constitutive and regulated activities. Genes Dev. 8, 1703–1712 (1994).

    Article  CAS  Google Scholar 

  42. Kleinhenz, B. et al. Raver2, a new member of the hnRNP family. FEBS Lett. 579, 4254–4258 (2005).

    Article  CAS  Google Scholar 

  43. Gordon, G.W., Berry, G., Liang, X.H., Levine, B. & Herman, B. Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J. 74, 2702–2713 (1998).

    Article  CAS  Google Scholar 

  44. Johnson, B.A. & Blevins, R.A. NMRVIEW—a computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614 (1994).

    Article  CAS  Google Scholar 

  45. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Valcárcel for helpful comments on the manuscript, B. Jockusch for Raver1 monoclonal antibodies and I. Moss, of the Advanced Biotechnology Centre, Imperial College London, for technical advice and for preparation of the peptides. This work was funded by Wellcome Trust program grant 059879 to C.W.J.S. A.P.R. was supported by a Wellcome Trust Prize studentship. S.M. and S.C. are also indebted to the Wellcome Trust for grant support (078209). S.H. and M.L. were supported by US National Institutes of Health grant AR 41480 to R.H.S.

Author information

Authors and Affiliations

  1. Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, CB2 1GA, UK

    Alexis P Rideau, Clare Gooding & Christopher W J Smith

  2. Division of Molecular Biosciences, Imperial College, South Kensington Campus, Exhibition Road, London, SW7 2AZ, UK

    Peter J Simpson & Stephen Matthews

  3. Divisions of Molecular and Cell Biology, Imperial College, South Kensington Campus, Exhibition Road, London, SW7 2AZ, UK

    Tom P Monie & Stephen Curry

  4. Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, Dresden, 01307, Germany

    Mike Lorenz

  5. Medical Faculty, University of Halle-Wittenberg, Heinrich-Damerow-Str. 1, Halle (Saale), 06907, Germany

    Stefan Hüttelmaier

  6. Department of Anatomy & Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, 10461, New York, USA

    Robert H Singer

Authors
  1. Alexis P Rideau
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Clare Gooding
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Peter J Simpson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tom P Monie
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mike Lorenz
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Stefan Hüttelmaier
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Robert H Singer
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Stephen Matthews
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Stephen Curry
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Christopher W J Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.P.R. mapped the Raver1 repressor domain and identified and characterized the PTB-interacting motif. C.G. did pull-down experiments identifying multiple PTB-interacting segments of Raver1, and some transfections. C.W.J.S. directed these experiments, coordinated the collaborations and wrote the first draft of the manuscript. S.M. and S.C. coordinated design of structural experiments. T.P.M. expressed and purified all PTB constructs; P.J.S. and S.M. planned and executed NMR experiments and data analysis. R.H.S. coordinated the FRET experiments. S.H. tested FRET with full-length Raver1. M.L. carried out all other FRET experiments. All authors contributed to writing and redrafting the manuscript.

Corresponding author

Correspondence to Christopher W J Smith.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

R442–629-MS2 activity in the presence of mutations in PTBP-binding sites within the P3 pyrimidine tracts of TPM1. (PDF 122 kb)

Supplementary Fig. 2

NMR binding isotherms for the titration of different PTB constructs with Raver1 peptides and RNA. (PDF 169 kb)

Supplementary Fig. 3

Per-residue chemical shift change in RRM2 upon initial addition of Raver1 peptide and subsequent RNA titration. (PDF 239 kb)

Supplementary Fig. 4

2D 1H-15N HSQC titration of Raver1 peptide with PTBP1-2, illustrating progressive broadening of peptide backbone signals. (PDF 86 kb)

Supplementary Fig. 5

Titration of PTB1-2 with full-length peptide Raver1491–511 and a truncated version containing only the core motif SLLGEPPA, and paramagnetic relaxation enhancement for 15N-labeled PTBP-RRM2 in complex with spin-labeled Raver1 peptides. (PDF 894 kb)

Supplementary Fig. 6

The Raver1-binding site of PTBP1 RRM2, highlighting the position of Leu500 and Leu501 of Raver1. (PDF 416 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rideau, A., Gooding, C., Simpson, P. et al. A peptide motif in Raver1 mediates splicing repression by interaction with the PTB RRM2 domain. Nat Struct Mol Biol 13, 839–848 (2006). https://doi.org/10.1038/nsmb1137

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb1137

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