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

Nature volume 475, pages 408–411 (2011)Cite this article

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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.

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Figure 1: Structure of the tandem RRM domains of U2AF65 free and when bound to a high-affinity Py tract.
Figure 2: Binding of Py tracts of different strength to U2AF65 RRM1–RRM2.
Figure 3: Spliceosome assembly as a function of Py tract strength.
Figure 4: Py tract recognition by U2AF.

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Protein Data Bank

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.

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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.).

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Authors and 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. Department Chemie, Munich Center for Integrated Protein Science and Chair Biomolecular NMR, 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

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  1. Cameron D. Mackereth
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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.

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Correspondence to Michael Sattler.

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The authors declare no competing financial interests.

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Mackereth, C., Madl, T., Bonnal, S. et al. Multi-domain conformational selection underlies pre-mRNA splicing regulation by U2AF. Nature 475, 408–411 (2011). https://doi.org/10.1038/nature10171

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