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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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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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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The file contains Supplementary Text, Supplementary References, Supplementary Tables 1-3 and Supplementary Figures 1-16 with legends. (PDF 21889 kb)
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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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DOI: https://doi.org/10.1038/nature10171
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