U2AF-homology motif interactions are required for alternative splicing regulation by SPF45

  • An Erratum to this article was published on 01 August 2007


The U2AF-homology motif (UHM) mediates protein-protein interactions between factors involved in constitutive RNA splicing. Here we report that the splicing factor SPF45 regulates alternative splicing of the apoptosis regulatory gene FAS (also called CD95). The SPF45 UHM is necessary for this activity and binds UHM-ligand motifs (ULMs) present in the 3′ splice site–recognizing factors U2AF65, SF1 and SF3b155. We describe a 2.1-Å crystal structure of SPF45-UHM in complex with a ULM peptide from SF3b155. Features distinct from those of previously described UHM-ULM structures allowed the design of mutations in the SPF45 UHM that selectively impair binding to individual ULMs. Splicing assays using the ULM-selective SPF45 variants demonstrate that individual UHM-ULM interactions are required for FAS splicing regulation by SPF45 in vivo. Our data suggest that networks of UHM-ULM interactions are involved in regulating alternative splicing.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: U2AF homology motifs and ligands in splicing factors.
Figure 2: SPF45 induces exon 6 skipping in a FAS minigene.
Figure 3: Analysis of SPF45-UHM–ULM interactions.
Figure 4: Crystal structure of SPF45-UHM bound to SF3b155-ULM5.
Figure 5: SPF45-UHM binds ULM peptides derived from U2AF65, SF1 and SF3b155.
Figure 6: GST pull-down binding experiments and FAS splicing assays using SPF45-UHM mutants.

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank


  1. 1

    Black, D.L. Mechanisms of alternative pre-messenger RNA splicing. Annu. Rev. Biochem. 72, 291–336 (2003).

    CAS  Article  Google Scholar 

  2. 2

    Bell, L.R., Horabin, J.I., Schedl, P. & Cline, T.W. Positive autoregulation of sex-lethal by alternative splicing maintains the female determined state in Drosophila. Cell 65, 229–239 (1991).

    CAS  Article  Google Scholar 

  3. 3

    Lallena, M.J., Chalmers, K.J., Llamazares, S., Lamond, A.I. & Valcarcel, J. Splicing regulation at the second catalytic step by Sex-lethal involves 3′ splice site recognition by SPF45. Cell 109, 285–296 (2002).

    CAS  Article  Google Scholar 

  4. 4

    Aravind, L. & Koonin, E.V. G-patch: a new conserved domain in eukaryotic RNA-processing proteins and type D retroviral polyproteins. Trends Biochem. Sci. 24, 342–344 (1999).

    CAS  Article  Google Scholar 

  5. 5

    Silverman, E.J. et al. Interaction between a G-patch protein and a spliceosomal DEXD/H-box ATPase that is critical for splicing. Mol. Cell. Biol. 24, 10101–10110 (2004).

    CAS  Article  Google Scholar 

  6. 6

    Svec, M., Bauerova, H., Pichova, I., Konvalinka, J. & Strisovsky, K. Proteinases of betaretroviruses bind single-stranded nucleic acids through a novel interaction module, the G-patch. FEBS Lett. 576, 271–276 (2004).

    CAS  Article  Google Scholar 

  7. 7

    Frenal, K. et al. Structural and functional characterization of the TgDRE multidomain protein, a DNA repair enzyme from Toxoplasma gondii. Biochemistry 45, 4867–4874 (2006).

    CAS  Article  Google Scholar 

  8. 8

    Chaouki, A.S. & Salz, H.K. Drosophila SPF45: a bifunctional protein with roles in both splicing and DNA repair. PLoS Genet. 2, e178 (2006).

    Article  Google Scholar 

  9. 9

    Kielkopf, C.L., Lucke, S. & Green, M.R. U2AF homology motifs: protein recognition in the RRM world. Genes Dev. 18, 1513–1526 (2004).

    CAS  Article  Google Scholar 

  10. 10

    Maris, C., Dominguez, C. & Allain, F.H. The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression. FEBS J. 272, 2118–2131 (2005).

    CAS  Article  Google Scholar 

  11. 11

    Kielkopf, C.L., Rodionova, N.A., Green, M.R. & Burley, S.K. A novel peptide recognition mode revealed by the X-ray structure of a core U2AF35/U2AF65 heterodimer. Cell 106, 595–605 (2001).

    CAS  Article  Google Scholar 

  12. 12

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

    CAS  Article  Google Scholar 

  13. 13

    Berglund, J.A., Abovich, N. & Rosbash, M. A cooperative interaction between U2AF65 and mBBP/SF1 facilitates branchpoint region recognition. Genes Dev. 12, 858–867 (1998).

    CAS  Article  Google Scholar 

  14. 14

    Rain, J.C., Rafi, Z., Rhani, Z., Legrain, P. & Krämer, A. Conservation of functional domains involved in RNA binding and protein- protein interactions in human and Saccharomyces cerevisiae pre-mRNA splicing factor SF1. RNA 4, 551–565 (1998).

    CAS  Article  Google Scholar 

  15. 15

    Rudner, D.Z., Kanaar, R., Breger, K.S. & Rio, D.C. Interaction between subunits of heterodimeric splicing factor U2AF is essential in vivo. Mol. Cell. Biol. 18, 1765–1773 (1998).

    CAS  Article  Google Scholar 

  16. 16

    Gozani, O., Potashkin, J. & Reed, R. A potential role for U2AF-SAP 155 interactions in recruiting U2 snRNP to the branch site. Mol. Cell. Biol. 18, 4752–4760 (1998).

    CAS  Article  Google Scholar 

  17. 17

    Das, R., Zhou, Z. & Reed, R. Functional association of U2 snRNP with the ATP-independent spliceosomal complex E. Mol. Cell 5, 779–787 (2000).

    CAS  Article  Google Scholar 

  18. 18

    Thickman, K.R., Swenson, M.C., Kabogo, J.M., Gryczynski, Z. & Kielkopf, C.L. Multiple U2AF65 binding sites within SF3b155: thermodynamic and spectroscopic characterization of protein-protein interactions among pre-mRNA splicing factors. J. Mol. Biol. 356, 664–683 (2006).

    CAS  Article  Google Scholar 

  19. 19

    Spadaccini, R. et al. Biochemical and NMR analyses of an SF3b155-p14–U2AF-RNA interaction network involved in branch point definition during pre-mRNA splicing. RNA 12, 410–425 (2006).

    CAS  Article  Google Scholar 

  20. 20

    Page-McCaw, P.S., Amonlirdviman, K. & Sharp, P.A. PUF60: a novel U2AF65-related splicing activity. RNA 5, 1548–1560 (1999).

    CAS  Article  Google Scholar 

  21. 21

    Van Buskirk, C. & Schupbach, T. Half pint regulates alternative splice site selection in Drosophila. Dev. Cell 2, 343–353 (2002).

    CAS  Article  Google Scholar 

  22. 22

    Jung, D.J., Na, S.Y., Na, D.S. & Lee, J.W. Molecular cloning and characterization of CAPER, a novel coactivator of activating protein-1 and estrogen receptors. J. Biol. Chem. 277, 1229–1234 (2002).

    CAS  Article  Google Scholar 

  23. 23

    Dowhan, D.H. et al. Steroid hormone receptor coactivation and alternative RNA splicing by U2AF65-related proteins CAPERalpha and CAPERbeta. Mol. Cell 17, 429–439 (2005).

    CAS  Article  Google Scholar 

  24. 24

    Cheng, J. et al. Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule. Science 263, 1759–1762 (1994).

    CAS  Article  Google Scholar 

  25. 25

    Krammer, P.H. CD95's deadly mission in the immune system. Nature 407, 789–795 (2000).

    CAS  Article  Google Scholar 

  26. 26

    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).

    CAS  Article  Google Scholar 

  27. 27

    Sampath, J. et al. Human SPF45, a splicing factor, has limited expression in normal tissues, is overexpressed in many tumors, and can confer a multidrug-resistant phenotype to cells. Am. J. Pathol. 163, 1781–1790 (2003).

    CAS  Article  Google Scholar 

  28. 28

    Allain, F.H. et al. Specificity of ribonucleoprotein interaction determined by RNA folding during complex formulation. Nature 380, 646–650 (1996).

    CAS  Article  Google Scholar 

  29. 29

    Wang, X. et al. Phosphorylation of splicing factor SF1 on Ser20 by cGMP-dependent protein kinase regulates spliceosome assembly. EMBO J. 18, 4549–4559 (1999).

    CAS  Article  Google Scholar 

  30. 30

    Boudrez, A., Beullens, M., Waelkens, E., Stalmans, W. & Bollen, M. Phosphorylation-dependent interaction between the splicing factors SAP155 and NIPP1. J. Biol. Chem. 277, 31834–31841 (2002).

    CAS  Article  Google Scholar 

  31. 31

    Wang, C. et al. Phosphorylation of spliceosomal protein SAP 155 coupled with splicing catalysis. Genes Dev. 12, 1409–1414 (1998).

    CAS  Article  Google Scholar 

  32. 32

    Shi, Y., Reddy, B. & Manley, J.L. PP1/PP2A phosphatases are required for the second step of Pre-mRNA splicing and target specific snRNP proteins. Mol. Cell 23, 819–829 (2006).

    CAS  Article  Google Scholar 

  33. 33

    Zhang, M., Zamore, P.D., Carmo-Fonseca, M., Lamond, A.I. & Green, M.R. Cloning and intracellular localization of the U2 small nuclear ribonucleoprotein auxiliary factor small subunit. Proc. Natl. Acad. Sci. USA 89, 8769–8773 (1992).

    CAS  Article  Google Scholar 

  34. 34

    Johnson, J.M. et al. Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays. Science 302, 2141–2144 (2003).

    CAS  Article  Google Scholar 

  35. 35

    McCoy, A.J., Grosse-Kunstleve, R.W, Storoni, L.C. & Rean, R.J. Likelihood-enhanced fast translation functions. Acta Crystallogr. D Biol. Crystallogr. 61, 458–464 (2005).

    Article  Google Scholar 

  36. 36

    Sali, A. & Blundell, T.L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993).

    CAS  Article  Google Scholar 

  37. 37

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  38. 38

    Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    CAS  Article  Google Scholar 

  39. 39

    Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283–291 (1993).

    CAS  Article  Google Scholar 

  40. 40

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

    CAS  Article  Google Scholar 

  41. 41

    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).

    CAS  Article  Google Scholar 

  42. 42

    Sattler, M., Schleucher, J. & Griesinger, C. Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog. Nucl. Magn. Reson. Spectrosc. 34, 93–158 (1999).

    CAS  Article  Google Scholar 

  43. 43

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

    CAS  Article  Google Scholar 

  44. 44

    Korzhnev, D.M., Skrynnikov, N.R., Millet, O., Torchia, D.A. & Kay, L.E. An NMR experiment for the accurate measurement of heteronuclear spin-lock relaxation rates. J. Am. Chem. Soc. 124, 10743–10753 (2002).

    CAS  Article  Google Scholar 

  45. 45

    Forch, P. et al. The apoptosis-promoting factor TIA-1 is a regulator of alternative pre-mRNA splicing. Mol. Cell 6, 1089–1098 (2000).

    CAS  Article  Google Scholar 

  46. 46

    Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Cryst. 26, 795–800 (1993).

    CAS  Article  Google Scholar 

Download references


We thank the staff at the European Synchrotron Radiation Facility and Swiss Light Source for assistance during data collection, V. Rybin (European Molecular Biology Laboratory, EMBL) for ITC measurements, I. Sinning and I. Tews (University of Heidelberg) for ITC measurement time, G. Stier (EMBL) for expression vectors, and B. Simon (EMBL) for help with NMR measurements. M.H. is a fellow of the Peter and Traudl Engelhorn Foundation. This work was supported by the Deutsche Forschungsgemeinschaft (Sa 823/5) and EU grant 3D Repertoire (LSHG-CT-2005-512028).

Author information




L.C. performed biochemistry, NMR experiments and data analysis; L.C. and J.B. performed crystallization and crystallographic data collection; L.C. and M.H. interpreted the crystallographic data; S.B. carried out molecular biology splicing activity assays; K.S. provided resources; M.S., J.V., L.C. and S.B. conceived the study and wrote the paper.

Corresponding author

Correspondence to Michael Sattler.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Expression control of SPF54 by western blot with anti-SPF45 (PDF 111 kb)

Supplementary Fig. 2

Isothermal calorimetry titrations of SPF45-UHM with U2AF65-ULM (PDF 117 kb)

Supplementary Fig. 3

In the cocrystals of SPF45-UHM and SF3b155-ULM5, the N terminus of the peptide is involved in lattice formation (PDF 152 kb)

Supplementary Fig. 4

15N T1p data, 1H-15N heteronuclear NOE data and 1H,15N correlation spectra of SPF45 (PDF 318 kb)

Supplementary Fig. 5

Supplementary GST pull-down data (PDF 346 kb)

Supplementary Methods (PDF 15 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Corsini, L., Bonnal, S., Basquin, J. et al. U2AF-homology motif interactions are required for alternative splicing regulation by SPF45. Nat Struct Mol Biol 14, 620–629 (2007). https://doi.org/10.1038/nsmb1260

Download citation

Further reading