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.

  • Brief Communication
  • Published:

Structural modeling of protein–RNA complexes using crosslinking of segmentally isotope-labeled RNA and MS/MS

Nature Methods volume 14, pages 487–490 (2017)Cite this article

Subjects

Abstract

Ribonucleoproteins (RNPs) are key regulators of cellular function. We established an efficient approach, crosslinking of segmentally isotope-labeled RNA and tandem mass spectrometry (CLIR-MS/MS), to localize protein–RNA interactions simultaneously at amino acid and nucleotide resolution. The approach was tested on polypyrimidine tract binding protein 1 and U1 small nuclear RNP. Our method provides distance restraints to support integrative atomic-scale structural modeling and to gain mechanistic insights into RNP-regulated processes.

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: CLIR-MS/MS analysis of crosslinked PTBP1-peptides.
Figure 2: CLIR-MS/MS mapping of PTBP1 on EMCV-IRES domains D–F.
Figure 3: CLIR-MS/MS applied to U1snRNP.

Similar content being viewed by others

Accession codes

Primary accessions

Proteomics Identifications Database

Referenced accessions

Protein Data Bank

References

  1. Cooper, T.A., Wan, L. & Dreyfuss, G. Cell 136, 777–793 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Kim, E. et al. Cancer Cell 27, 617–630 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Leitner, A., Faini, M., Stengel, F. & Aebersold, R. Trends Biochem. Sci. 41, 20–32 (2016).

    Article  CAS  PubMed  Google Scholar 

  4. Kramer, K. et al. Nat. Methods 11, 1064–1070 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kwon, S.C. et al. Nat. Struct. Mol. Biol. 20, 1122–1130 (2013).

    Article  CAS  PubMed  Google Scholar 

  6. Beckmann, B.M. et al. Nat. Commun. 6, 10127 (2015).

    Article  CAS  PubMed  Google Scholar 

  7. Lelyveld, V.S., Björkbom, A., Ransey, E.M., Sliz, P. & Szostak, J.W. J. Am. Chem. Soc. 137, 15378–15381 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Spriggs, K.A., Bushell, M., Mitchell, S.A. & Willis, A.E. Cell Death Differ. 12, 585–591 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Romanelli, M.G., Diani, E. & Lievens, P.M. Int. J. Mol. Sci. 14, 22906–22932 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Oberstrass, F.C. et al. Science 309, 2054–2057 (2005).

    Article  CAS  PubMed  Google Scholar 

  11. Han, A. et al. PLoS Comput. Biol. 10, e1003442 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Kafasla, P. et al. Mol. Cell 34, 556–568 (2009).

    Article  CAS  PubMed  Google Scholar 

  13. Jang, S.K. & Wimmer, E. Genes Dev. 4, 1560–1572 (1990).

    Article  CAS  PubMed  Google Scholar 

  14. Rinner, O. et al. Nat. Methods 5, 315–318 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lamichhane, R. et al. Proc. Natl. Acad. Sci. USA 107, 4105–4110 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Gygi, S.P. et al. Nat. Biotechnol. 17, 994–999 (1999).

    Article  CAS  PubMed  Google Scholar 

  17. Ong, S.E. et al. Mol. Cell. Proteomics 1, 376–386 (2002).

    Article  CAS  PubMed  Google Scholar 

  18. Oda, Y., Huang, K., Cross, F.R., Cowburn, D. & Chait, B.T. Proc. Natl. Acad. Sci. USA 96, 6591–6596 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Müller, D.R. et al. Anal. Chem. 73, 1927–1934 (2001).

    Article  PubMed  CAS  Google Scholar 

  20. Duss, O., Diarra Dit Konté, N. & Allain, F.H. Methods Enzymol. 565, 537–562 (2015).

    Article  CAS  PubMed  Google Scholar 

  21. Liu, H., Sadygov, R.G. & Yates, J.R. III. Anal. Chem. 76, 4193–4201 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Daubner, G.M., Cléry, A., Jayne, S., Stevenin, J. & Allain, F.H. EMBO J. 31, 162–174 (2012).

    Article  CAS  PubMed  Google Scholar 

  23. Will, C.L. & Luhrmann, R. Cold Spring Harb. Perspect. Biol. 3, a003707 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kondo, Y., Oubridge, C., van Roon, A.M. & Nagai, K. eLife 4, e04986 (2015).

    Article  PubMed Central  Google Scholar 

  25. Pomeranz Krummel, D.A., Oubridge, C., Leung, A.K., Li, J. & Nagai, K. Nature 458, 475–480 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Dorn, G. et al. Mapping protein-RNA interactions with single residue resolution by CLIR-MS/MS. Protocol Exchange http://dx.doi.org/10.1038/protex.2017.031 (2017).

  27. Tugarinov, V., Kanelis, V. & Kay, L.E. Nat. Protoc. 1, 749–754 (2006).

    Article  CAS  PubMed  Google Scholar 

  28. Sambrook, J. & Russell, D.W. Molecular Cloning: A Laboratory Manual 3rd edn. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2001).

  29. Gasteiger, E. et al. in The Proteomics Protocols Handbook (ed. Walker, J.M.) 571–607 (Humana, Totowa, New Jersey, USA, 2005).

  30. Milligan, J.F., Groebe, D.R., Witherell, G.W. & Uhlenbeck, O.C. Nucleic Acids Res. 15, 8783–8798 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Kaminski, A. & Jackson, R.J. RNA 4, 626–638 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Duke, G.M., Hoffman, M.A. & Palmenberg, A.C. J. Virol. 66, 1602–1609 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Duss, O., Maris, C., von Schroetter, C. & Allain, F.H. Nucleic Acids Res. 38, e188 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Maniatis, T., Jeffrey, A. & van deSande, H. Biochemistry 14, 3787–3794 (1975).

    Article  CAS  PubMed  Google Scholar 

  35. Sharma, K. et al. Methods 89, 138–148 (2015).

    Article  CAS  PubMed  Google Scholar 

  36. Leitner, A. et al. Anal. Chem. 82, 2726–2733 (2010).

    Article  CAS  PubMed  Google Scholar 

  37. Chambers, M.C. et al. Nat. Biotechnol. 30, 918–920 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Walzthoeni, T. et al. Nat. Methods 12, 1185–1190 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Dominguez, C., Schubert, M., Duss, O., Ravindranathan, S. & Allain, F.H. Prog. Nucl. Magn. Reson. Spectrosc. 58, 1–61 (2011).

    Article  CAS  PubMed  Google Scholar 

  40. Lee, W., Revington, M.J., Arrowsmith, C. & Kay, L.E. FEBS Lett. 350, 87–90 (1994).

    Article  CAS  PubMed  Google Scholar 

  41. Peterson, R.D., Theimer, C.A., Wu, H. & Feigon, J. J. Biomol. NMR 28, 59–67 (2004).

    Article  CAS  PubMed  Google Scholar 

  42. Herrmann, T., Güntert, P. & Wüthrich, K. J. Biomol. NMR 24, 171–189 (2002).

    Article  CAS  PubMed  Google Scholar 

  43. Herrmann, T., Güntert, P. & Wüthrich, K. J. Mol. Biol. 319, 209–227 (2002).

    Article  CAS  PubMed  Google Scholar 

  44. Güntert, P. Methods Mol. Biol. 278, 353–378 (2004).

    PubMed  Google Scholar 

  45. Parisien, M. & Major, F. Nature 452, 51–55 (2008).

    Article  CAS  PubMed  Google Scholar 

  46. Case, D.A. et al. (University of California, San Francisco, 2012).

  47. Vizcaíno, J.A. et al. Nucleic Acids Res. 41, D1063–D1069 (2013).

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank V. Panse for the use of his UV device for crosslinking and N. Diarra dit Konte and O. Duss for advice concerning segmental isotope labeling of RNA. G.D. was supported by a PhD fellowship from the Boehringer Ingelheim Fond. This work was supported by the SNF project 31003A_149921 and the SNF-NCCR RNA and Disease to F.H.-T.A. and by ERC grants Proteomics v3.0 (AdvG grant #233226) and Proteomics4D (AdvG grant #670821) to R.A.

Author information

Author notes

  1. Georg Dorn and Alexander Leitner: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biology, Institute for Molecular Biology and Biophysics, ETH Zürich, Zürich, Switzerland

    Georg Dorn, Julien Boudet, Sébastien Campagne, Christine von Schroetter, Ahmed Moursy & Frédéric H-T Allain

  2. Department of Biology, Institute of Molecular Systems Biology, ETH Zürich, Zürich, Switzerland

    Alexander Leitner & Ruedi Aebersold

  3. Faculty of Science, University of Zurich, Zürich, Switzerland

    Ruedi Aebersold

Authors
  1. Georg Dorn
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alexander Leitner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Julien Boudet
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sébastien Campagne
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Christine von Schroetter
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ahmed Moursy
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ruedi Aebersold
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Frédéric H-T Allain
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed to the design of experiments, the discussion of the results and the writing of the manuscript. C.v.S. purified isotope-labeled nucleotides. G.D. and A.M. optimized crosslinking conditions. G.D. and S.C. prepared U1snRNP samples. G.D. prepared all other samples and performed and analyzed NMR experiments. A.L. and G.D. performed sample preparation for MS analysis. A.L. performed all LC-MS/MS experiments and analyzed the LC-MS/MS results with support from G.D. J.B. set up the modeling protocol and modeled the RRM2-EMCVF complex based on crosslinking data. G.D. implemented the modeling protocol and modeled the complexes of RRM1, RRM3 and RRM4.

Corresponding authors

Correspondence to Ruedi Aebersold or Frédéric H-T Allain.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Localization of crosslinks detected for the RRM1-EMCVE complex.

His62 was detected as the sole crosslinking site and is shown in red on the structure of RRM1 in complex with CU-RNA1. The detection of U- and UU-modifications suggests binding of RRM1 to nucleotides 324-326.

Supplementary Figure 2 Segmental labeling of EMCVDElinkF-RNA.

a, strategy for segmental labeling as employed in our study. Notably, only one aliquot of isotope labeled RNA is used independent of the size of the RNA and the number of isotope labeled segments. b, RNAse H digestion of EMCVDElinkF with chimSLD (lane 1), chimLinkF (lane 2), chimSLE (lane 3) and all three chimeras simultaneously (lane 5). The full-length RNA is shown in lane 4, an example for splinted RNA-ligation is shown in lane 6 (input) and 7 (ligation). Note that the isotope labeled RNA fragment is kept as limiting substrate in the reaction.

Supplementary Figure 3 NMR titration experiments for RRM1 (top) and RRM2 (bottom).

(a) EMCVE and (b) EMCVF. The chemical shift perturbations between 1H15N-signals of the free and the bound RRMs indicate binding. The signals of the UCUUU-pentaloop of EMCVF (right) shift upon addition of RRM1 (top) and RRM2 (bottom) due to binding.

Supplementary Figure 4 RRM2 binds preferentially to EMCVF.

a, Upon titration of RRM12 with EMCVF only the RRM2 moiety reaches saturation at a 1:1 ratio (EMCVF:RRM12) and signals overlap almost perfectly with signals of the RRM2-EMCVF complex (yellow). b, In the presence of SLE, RRM1 and RRM2 bind SLE and SLF, respectively, as indicated by the almost perfect superimposition of spectra collected on RRM1-EMCVE (green), RRM2-EMCVF (yellow) and RRM12-EMCVEmutF (blue; right) complexes.

Supplementary Figure 5 Comparison of fingerprint spectra of subcomplexes with their corresponding signals in the PTBP1-EMCVDElinkF complex.

a, the 1H-15N-TROSY signals and, b, the ILV-methyl-1H13C-signals of RRM1 (green), RRM2 (yellow) and RRM34 (magenta) in complex with EMCVE, EMCVF and EMCVDElink, respectively, overlap well with signals of the PTBP1-EMCVDElinkF complex (black) which is typical for identical binding of the RRMs in the subcomplexes.

Supplementary Figure 6 NMR titration of RRM34-EMCVDElink with RRM1.

1H-15N resonances of RRM34-EMCVDElink are not affected by RRM1 addition (color code as indicated). The RRM1-moiety shows the signature of the RRM1-EMCVE complex.

Supplementary Figure 7 Cyana-based modeling strategy using CLIR-MS/MS-derived restraints.

a, peptides and modification sites are identified by CLIR-MS/MS, the mass of the modification infers on its composition as exemplarily shown for RRM3 crosslinks detected in “D”. b, CLIR-MS/MS data are converted to ambiguous upper proton-proton distance limits (illustrated for simplicity as red, dashed lines between carbon atoms) and, c, fed to Cyana 3.02 noeassign. Relevant structural information from published RNA-protein complexes and structure predictions serve to generate complementary ambiguous and unambiguous distance restraints. Calculated atomic resolution models were further refined under amber 12 force-field3. d, representation of the final CLIR-MS/MS restraints on the model of RRM3 in complex with nucleotides 299-306 of SLD.

Supplementary Figure 8 Ensembles of the RRM-RNA complexes modeled using CLIR-MS/MS-derived distance restraints.

Shown are the bundles of the 20 final structures of the, a, RRM1-EMCVE, b, RRM2-EMCVF, c, RRM3-EMCVD and, d, RRM4-EMCVlink complex. Only nucleotides 354-364, 300-305 and 340-344 are displayed in A, C and D, respectively, as these represent the well-defined binding site. RMSD values (Deviation of coordinates to their mean) are calculated for amino acids 47-153 (RRM1), 181-244 and 255-273 (RRM2) and 336-531 (RRM34 tandem domain) and nucleotides 321-336 (EMCVE), 354-364 (EMCVF), 302-305 (EMCVD) and 341-344 (EMCVlink).

Supplementary Figure 9 Recognition of the EMCV-IRES domains D–F by PTBP1-RRMs according to CLIR-MS/MS-based models.

Hydrophobic and specific hydrogen bond interactions are depicted for each binding pocket of the RRMs as derived from the CLIR-MS/MS-based models and compared with the published interactions detected in an RRM1-stemloop complex (pdb: 3N2O), the NMR derived model of RRM2-EMCVF and in the complex of RRM34 with ssRNA1. Recognition of nucleotides in the reference structures is shown in gray if different. Please note that U360 is looped out and not recognized by RRM2.

Supplementary Figure 10 Comparison of the RRM2-EMCVF models derived from NMR and CLIR-MS/MS.

RRM2 recognizes EMCVF by contacting C358 and U359 in pocket 1 and 2 by base-specific hydrogen bonds in both, a, NMR and, b, CLIR-MS/MS derived models. While U360 does not contact RRM2, U361 stacks on Tyr 267.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Tables 1–3 and Supplementary Notes 1–6 (PDF 2178 kb)

Supplementary Table 4

Modeling restraints (XLSX 519 kb)

Supplementary Data 1

CLIR-MS/MS derived model of RRM1 bound to stem loop E (ZIP 798 kb)

Supplementary Data 2

CLIR-MS/MS derived model of RRM2 bound to stem loop F (ZIP 931 kb)

Supplementary Data 3

CLIR-MS/MS derived model of RRM3 bound to stem loop D (ZIP 1175 kb)

Supplementary Data 4

CLIR-MS/MS derived model of RRM4 bound to linker sequence E-F (ZIP 1206 kb)

Supplementary Data 5

NMR derived model of RRM2 bound to stem loop F (ZIP 1008 kb)

Supplementary Protocol

Mapping protein-RNA interactions with single residue resolution by CLIR-MS/MS (PDF 389 kb)

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Dorn, G., Leitner, A., Boudet, J. et al. Structural modeling of protein–RNA complexes using crosslinking of segmentally isotope-labeled RNA and MS/MS. Nat Methods 14, 487–490 (2017). https://doi.org/10.1038/nmeth.4235

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.4235

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