Letter | Published:

Multi-domain conformational selection underlies pre-mRNA splicing regulation by U2AF

Nature volume 475, pages 408411 (21 July 2011) | Download Citation

Abstract

Many cellular functions involve multi-domain proteins, which are composed of structurally independent modules connected by flexible linkers. Although it is often well understood how a given domain recognizes a cognate oligonucleotide or peptide motif, the dynamic interaction of multiple domains in the recognition of these ligands remains to be characterized. Here we have studied the molecular mechanisms of the recognition of the 3′-splice-site-associated polypyrimidine tract RNA by the large subunit of the human U2 snRNP auxiliary factor (U2AF65)1,2,3 as a key early step in pre-mRNA splicing4. We show that the tandem RNA recognition motif domains of U2AF65 adopt two remarkably distinct domain arrangements in the absence or presence of a strong (that is, high affinity) polypyrimidine tract. Recognition of sequence variations in the polypyrimidine tract RNA involves a population shift between these closed and open conformations. The equilibrium between the two conformations functions as a molecular rheostat that quantitatively correlates the natural variations in polypyrimidine tract nucleotide composition, length and functional strength to the efficiency to recruit U2 snRNP to the intron during spliceosome assembly1,5,6,7,8. Mutations that shift the conformational equilibrium without directly affecting RNA binding modulate splicing activity accordingly. Similar mechanisms of cooperative multi-domain conformational selection may operate more generally in the recognition of degenerate nucleotide or amino acid motifs by multi-domain proteins9,10.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Data deposits

The coordinates of the open RNA-bound conformation of RRM1–RRM2 and the closed conformation in the absence of RNA are deposited in the Protein Data Bank with accession codes 2YH1 and 2YH0, respectively. All structural ensembles with explicit spin labels are available from the authors upon request.

References

  1. 1.

    , & Cloning and domain structure of the mammalian splicing factor U2AF. Nature 355, 609–614 (1992)

  2. 2.

    , , & Sex lethal and U2 small nuclear ribonucleoprotein auxiliary factor (U2AF65) recognize polypyrimidine tracts using multiple modes of binding. RNA 9, 88–99 (2003)

  3. 3.

    et al. The conserved RNA recognition motif 3 of U2 snRNA auxiliary factor (U2AF65) is essential in vivo but dispensible for activity in vitro. RNA 10, 240–253 (2004)

  4. 4.

    , & The spliceosome: design principles of a dynamic RNP machine. Cell 136, 701–718 (2009)

  5. 5.

    The organization of 3′ splice-site sequences in mammalian introns. Genes Dev. 3, 2113–2123 (1989)

  6. 6.

    , & A mutational analysis of the polypyrimidine tract of introns. Effects of sequence differences in pyrimidine tracts on splicing. J. Biol. Chem. 268, 11222–11229 (1993)

  7. 7.

    , & Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins. Science 268, 1173–1176 (1995)

  8. 8.

    , & Functional analysis of the polypyrimidine tract in pre-mRNA splicing. Nucleic Acids Res. 25, 888–896 (1997)

  9. 9.

    , , , & How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers. Nature Struct. Mol. Biol. 14, 1025–1040 (2007)

  10. 10.

    , , & Reading protein modifications with interaction domains. Nature Rev. Mol. Cell Biol. 7, 473–483 (2006)

  11. 11.

    , , & Solution structures of the first and second RNA-binding domains of human U2 small nuclear ribonucleoprotein particle auxiliary factor (U2AF(65)). EMBO J. 18, 4523–4534 (1999)

  12. 12.

    et al. Structural basis for polypyrimidine tract recognition by the essential pre-mRNA splicing factor U2AF65. Mol. Cell 23, 49–59 (2006)

  13. 13.

    , , , & An efficient protocol for NMR-spectroscopy-based structure determination of protein complexes in solution. Angew. Chem. Int. Edn Engl. 49, 1967–1970 (2010)

  14. 14.

    , & The role of dynamic conformational ensembles in biomolecular recognition. Nature Chem. Biol. 5, 789–796 (2009)

  15. 15.

    , , & Visualizing spatially correlated dynamics that directs RNA conformational transitions. Nature 450, 1263–1267 (2007)

  16. 16.

    et al. Recognition dynamics up to microseconds revealed from an RDC-derived ubiquitin ensemble in solution. Science 320, 1471–1475 (2008)

  17. 17.

    , , & Internal dynamics control activation and activity of the autoinhibited Vav DH domain. Nature Struct. Mol. Biol. 15, 613–618 (2008)

  18. 18.

    , , , & A transient and low-populated protein-folding intermediate at atomic resolution. Science 329, 1312–1316 (2010)

  19. 19.

    et al. A hierarchy of timescales in protein dynamics is linked to enzyme catalysis. Nature 450, 913–916 (2007)

  20. 20.

    , & Speeding molecular recognition by using the folding funnel: the fly-casting mechanism. Proc. Natl Acad. Sci. USA 97, 8868–8873 (2000)

  21. 21.

    , & Conformational selection or induced fit: a flux description of reaction mechanism. Proc. Natl Acad. Sci. USA 106, 13737–13741 (2009)

  22. 22.

    et al. Jmjd6 catalyses lysyl-hydroxylation of U2AF65, a protein associated with RNA splicing. Science 325, 90–93 (2009)

  23. 23.

    , , , & Intron removal requires proofreading of U2AF/3′ splice site recognition by DEK. Science 312, 1961–1965 (2006)

  24. 24.

    & Alignment of biological macromolecules in novel nonionic liquid crystalline media for NMR experiments. J. Am. Chem. Soc. 122, 7793–7797 (2000)

  25. 25.

    , , , & Dual function for U2AF(35) in AG-dependent pre-mRNA splicing. Mol. Cell. Biol. 21, 7673–7681 (2001)

  26. 26.

    , & Functional analysis of splicing factors and regulators. In mRNA Formation and Function 31–53 (Elsevier, 1997)

  27. 27.

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

  28. 28.

    & NMRView: a computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614 (1994)

  29. 29.

    et al. Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. Biochemistry 33, 5984–6003 (1994)

  30. 30.

    , , , & TROSY-based HNCO pulse sequences for the measurement of 1HN-15N, 15N-13CO, 1HN-13CO, 13CO-13Cα and 1HN-13Cα dipolar couplings in 15N, 13C, 2H-labelled proteins. J. Biomol. NMR 14, 333–343 (1999)

  31. 31.

    , & Ensemble approach for NMR structure refinement against 1H paramagnetic relaxation enhancement data arising from a flexible paramagnetic group attached to a macromolecule. J. Am. Chem. Soc. 126, 5879–5896 (2004)

  32. 32.

    & A new method to detect long-range protein-RNA contacts: NMR detection of electron-proton relaxation induced by nitroxide spin-labeled RNA. J. Am. Chem. Soc. 120, 10992–10993 (1998)

  33. 33.

    Calculation of protein structures with ambiguous distance restraints. Automated assignment of ambiguous NOE crosspeaks and disulphide connectivities. J. Mol. Biol. 245, 645–660 (1995)

  34. 34.

    et al. Crystallography 1 NMR system : A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

  35. 35.

    , & Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR 13, 289–302 (1999)

  36. 36.

    et al. The resonant mirror: a novel optical sensor for direct sensing of biomolecular interactions part II: applications. Biosens. Bioelectron. 8, 355–363 (1993)

Download references

Acknowledgements

We thank F. Gabel, M. Nilges, C. Griesinger, J. Müller and K. Scheffzek for discussions, and H. Tilgner for analysis of natural Py tract sequences. C.D.M. acknowledges support by EMBO Long Term Fellowship, ICSN and Aquitaine regional government. T.M. thanks the Austrian Science Fund (FWF) and EMBO for postdoctoral fellowships. We thank the EU NMR LSF in Frankfurt and the Bavarian NMR Centre (BNMRZ) in Munich for NMR measurement time. This work was supported by the European Commission, grants 3D Repertoire, FSG-V-RNA and NIM3 No. 226507 (M.S.), EURASNET, AICR and Fundación Marcelino Botín (J.V.).

Author information

Affiliations

  1. Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstrasse 1, 85764 Neuherberg, Germany

    • Cameron D. Mackereth
    • , Tobias Madl
    •  & Michael Sattler
  2. Institut Européen de Chimie et Biologie and Université de Bordeaux, 2 rue Robert Escarpit, 33607 Pessac, France

    • Cameron D. Mackereth
  3. Structural and Computational Biology, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany

    • Cameron D. Mackereth
    • , Bernd Simon
    • , Katia Zanier
    • , Alexander Gasch
    • , Vladimir Rybin
    •  & Michael Sattler
  4. Munich Center for Integrated Protein Science and Chair Biomolecular NMR, Department Chemie, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany

    • Tobias Madl
    •  & Michael Sattler
  5. Centre de Regulació Genòmica, Universitat Pompeu Fabra, Dr. Aiguader 88, 08003 Barcelona, Spain

    • Sophie Bonnal
    •  & Juan Valcárcel
  6. Institució Catalana de Recerca i Estudis Avançats, Dr. Aiguader 88, 08003 Barcelona, Spain

    • Juan Valcárcel

Authors

  1. Search for Cameron D. Mackereth in:

  2. Search for Tobias Madl in:

  3. Search for Sophie Bonnal in:

  4. Search for Bernd Simon in:

  5. Search for Katia Zanier in:

  6. Search for Alexander Gasch in:

  7. Search for Vladimir Rybin in:

  8. Search for Juan Valcárcel in:

  9. Search for Michael Sattler in:

Contributions

C.D.M., S.B., K.Z. and A.G. cloned and purified native and nitroxyl-labelled proteins. C.D.M., K.Z., B.S. and T.M. collected, processed and analysed NMR spectroscopy data. C.D.M., B.S. and T.M. calculated and analysed structural ensembles. S.B. performed in vitro splicing assays. V.R. performed ITC. J.V. and M.S. contributed to study design. C.D.M. and M.S. wrote the paper. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Michael Sattler.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    The file contains Supplementary Text, Supplementary References, Supplementary Tables 1-3 and Supplementary Figures 1-16 with legends.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature10171

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.