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.

  • Article
  • Published:

Structural insights into the U-box, a domain associated with multi-ubiquitination

Nature Structural & Molecular Biology volume 10, pages 250–255 (2003)Cite this article

Abstract

The structure of the U-box in the essential Saccharomyces cerevisiae pre-mRNA splicing factor Prp19p has been determined by NMR. The conserved zinc-binding sites supporting the cross-brace arrangement in RING-finger domains are replaced by hydrogen-bonding networks in the U-box. These hydrogen-bonding networks are necessary for the structural stabilization and activity of the U-box. A conservative Val→Ile point mutation in the Prp19p U-box domain leads to pre-mRNA splicing defects in vivo. NMR analysis of this mutant shows that the substitution disrupts structural integrity of the U-box domain. Furthermore, comparison of the Prp19p U-box domain with known RING–E2 complex structures demonstrates that both U-box and RING-fingers contain a conserved interaction surface. Mutagenesis of residues at this interface, while not perturbing the structure of the U-box, abrogates Prp19p function in vivo. These comparative structural and functional analyses imply that the U-box and its associated ubiquitin ligase activity are critical for Prp19p function in vivo.

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: The U-box and RING finger domains share a conserved fold.
Figure 2: U-box stability is mediated by hydrogen-bonding networks.
Figure 3: Mutational analysis of putative E2-interacting residues in the U-box.
Figure 4: The U-box domain is required for Prp19p function.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Pickart, C.M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70, 503–533 (2001).

    Article  CAS  Google Scholar 

  2. Cyr, D.M., Hohfeld, J. & Patterson, C. Protein quality control: U-box-containing E3 ubiquitin ligases join the fold. Trends Biochem. Sci. 27, 368–375 (2002).

    Article  CAS  Google Scholar 

  3. Patterson, C. A new gun in town: the U box is a ubiquitin ligase domain. Sci. STKE [online] <http://stke.sciencemag.org/cgi/content/full/ OC_sigtraus;2002/116/pe4> (2002).

  4. Koegl, M. et al. A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly. Cell 96, 635–644 (1999).

    Article  CAS  Google Scholar 

  5. Aravind, L. & Koonin, E.V. The U box is a modified RING finger — a common domain in ubiquitination. Curr. Biol. 10, R132–R134 (2000).

    Article  CAS  Google Scholar 

  6. Tarn, W.Y., Lee, K.R. & Cheng, S.C. Yeast precursor mRNA processing protein PRP19 associates with the spliceosome concomitant with or just after dissociation of U4 small nuclear RNA. Proc. Natl. Acad. Sci. USA 90, 10821–10825 (1993).

    Article  CAS  Google Scholar 

  7. McDonald, W.H., Ohi, R., Smelkova, N., Frendewey, D. & Gould, K.L. Myb-related fission yeast Cdc5p is a component of a 40S snRNP-containing complex and is essential for pre-mRNA splicing. Mol. Cell. Biol. 19, 5352–5362 (1999).

    Article  CAS  Google Scholar 

  8. Chen, H.R. et al. Snt309p, a component of the Prp19p-associated complex that interacts with Prp19p and associates with the spliceosome simultaneously with or immediately after dissociation of U4 in the same manner as Prp19p. Mol. Cell. Biol. 18, 2196–2204 (1998).

    Article  CAS  Google Scholar 

  9. Ohi, M.D. & Gould, K.L. Characterization of interactions among the Cef1p-Prp19p-associated splicing complex. RNA 8, 798–815 (2002).

    Article  CAS  Google Scholar 

  10. Cavanagh, J., Fairbrother, W.J., Palmer, A.G. & Skelton, N.J. Protein NMR Spectroscopy: Principles and Practice (Academic Press, San Diego; 1996).

    Google Scholar 

  11. Gervais, V. et al. Solution structure of the N-terminal domain of the human TFIIH MAT1 subunit: new insights into the RING finger family. J. Biol. Chem. 276, 7457–7464 (2001).

    Article  CAS  Google Scholar 

  12. Hanzawa, H. et al. The structure of the C4C4 ring finger of human NOT4 reveals features distinct from those of C3HC4 RING fingers. J. Biol. Chem. 276, 10185–10190 (2001).

    Article  CAS  Google Scholar 

  13. Bellon, S.F., Rodgers, K.K., Schatz, D.G., Coleman, J.E. & Steitz, T.A. Crystal structure of the RAG1 dimerization domain reveals multiple zinc-binding motifs including a novel zinc binuclear cluster. Nat. Struct. Biol. 4, 586–591 (1997).

    Article  CAS  Google Scholar 

  14. Zheng, N., Wang, P., Jeffrey, P.D. & Pavletich, N.P. Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases. Cell 102, 533–539 (2000).

    Article  CAS  Google Scholar 

  15. Zheng, N. et al. Structure of the Cul1–Rbx1–Skp1–F boxSkp2 SCF ubiquitin ligase complex. Nature 416, 703–709 (2002).

    Article  CAS  Google Scholar 

  16. Brzovic, P.S., Rajagopal, P., Hoyt, D.W., King, M.C. & Klevit, R.E. Structure of a BRCA1–BARD1 heterodimeric RING–RING complex. Nat. Struct. Biol. 8, 833–837 (2001).

    Article  CAS  Google Scholar 

  17. Hatakeyama, S., Yada, M., Matsumoto, M., Ishida, N. & Nakayama, K.I. U box proteins as a new family of ubiquitin-protein ligases. J. Biol. Chem. 276, 33111–33120 (2001).

    Article  CAS  Google Scholar 

  18. Huang, L. et al. Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade. Science 286, 1321–1326 (1999).

    Article  CAS  Google Scholar 

  19. Pringa, E., Martinez-Noel, G., Muller, U. & Harbers, K. Interaction of the ring finger-related U-box motif of a nuclear dot protein with ubiquitin-conjugating enzymes. J. Biol. Chem. 276, 19617–19623 (2001).

    Article  CAS  Google Scholar 

  20. Chen, H.R. et al. Snt309p, a component of the Prp19p-associated complex that interacts with Prp19p and associates with the spliceosome simultaneously with or immediately after dissociation of U4 in the same manner as Prp19p. Mol. Cell. Biol. 18, 2196–2204 (1998).

    Article  CAS  Google Scholar 

  21. Vijayraghavan, U., Company, M. & Abelson, J. Isolation and characterization of pre-mRNA splicing mutants of Saccharomyces cerevisiae. Genes Dev. 3, 1206–1216 (1989).

    Article  CAS  Google Scholar 

  22. Jiang, J. et al. CHIP is a U-box-dependent E3 ubiquitin ligase: identification of Hsc70 as a target for ubiquitylation. J. Biol. Chem. 276, 42938–42944 (2001).

    Article  CAS  Google Scholar 

  23. Murata, S., Minami, Y., Minami, M., Chiba, T. & Tanaka, K. CHIP is a chaperone-dependent E3 ligase that ubiquitylates unfolded protein. EMBO Rep. 2, 1133–1138 (2001).

    Article  CAS  Google Scholar 

  24. Meacham, G.C., Patterson, C., Zhang, W., Younger, J.M. & Cyr, D.M. The Hsc70 co-chaperone CHIP targets immature CFTR for proteasomal degradation. Nat. Cell. Biol. 3, 100–105 (2001).

    Article  CAS  Google Scholar 

  25. Imai, Y. et al. CHIP is associated with Parkin, a gene responsible for familial Parkinson's disease, and enhances its ubiquitin ligase activity. Mol. Cell 10, 55–67 (2002).

    Article  CAS  Google Scholar 

  26. Tarn, W.Y. et al. Functional association of essential splicing factor(s) with PRP19 in a protein complex. EMBO J. 13, 2421–2431 (1994).

    Article  CAS  Google Scholar 

  27. Bartels, C., Xia, T., Billeter, M., Guntert, P. & Wuthrich, K. XEASY. J. Biomol. NMR 6, 1–10 (1995).

    Article  CAS  Google Scholar 

  28. 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).

    Article  CAS  Google Scholar 

  29. Guntert, P., Mumenthaler, C. & Wuthrich, K. Torsion angle dynamics for NMR structure calculation with the new program DYANA. J. Mol. Biol. 273, 283–298 (1997).

    Article  CAS  Google Scholar 

  30. Pearlman, D.A. et al. AMBER 4.1 (San Francisco, CA.: University of California, San Francisco; 1995).

    Google Scholar 

  31. Maler, L., Potts, B.C. & Chazin, W.J. High resolution solution structure of apo calcyclin and structural variations in the S100 family of calcium-binding proteins. J. Biomol. NMR 13, 233–247 (1999).

    Article  CAS  Google Scholar 

  32. Maler, L., Blankenship, J., Rance, M. & Chazin, W.J. Site-site communication in the EF-hand Ca2+-binding protein calbindin D9k. Nat. Struct. Biol. 7, 245–250 (2000).

    Article  CAS  Google Scholar 

  33. Sastry, M. et al. The three-dimensional structure of Ca2+-bound calcyclin: implications for Ca2+-signal transduction by S100 proteins. Structure 6, 223–231 (1998).

    Article  CAS  Google Scholar 

  34. Lackner, P., Koppensteiner, W.A., Sippl, M.J. & Domingues, F.S. ProSup: a refined tool for protein structure alignment. Protein Eng. 13, 745–752 (2000).

    Article  CAS  Google Scholar 

  35. Koradi, R., Billeter, M. & Wuthrich, K. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51–55 (1996).

    Article  CAS  Google Scholar 

  36. Laskowski, R.A., Rullman, J.A., MacArthur, M.W., Kaptein, R. & Thornton, J.M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–486 (1996).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Hatakeyama and K.I. Nakayama from the Department of Molecular and Cellular Biology at Kyushu University for the kind gift of GST-Ubc3. We also gratefully acknowledge J.A. Smith for valuable technical assistance with structure calculations and molecular graphics. Work in our laboratories was supported by the National Institutes of Health in the form of operating grants to K.L.G. and W.J.C., training grant positions to M.D.O., C.W.V.K. and J.A.R., and core facility grants to the Vanderbilt Ingram Cancer Center and the Vanderbilt Center in Molecular Toxicology. K.L.G. is an Associate Investigator of the Howard Hughes Medical Institute.

Author information

Author notes

  1. Melanie D. Ohi, Craig W. Vander Kooi and Walter J. Chazin: These authors contributed equally to this work.

Authors and Affiliations

  1. Howard Hughes Medical Institute School of Medicine, Vanderbilt University, Nashville, 37232, Tennessee, USA

    Melanie D. Ohi, Joshua A. Rosenberg & Kathleen L. Gould

  2. Department of Cell Biology, School of Medicine, Vanderbilt University, Nashville, 37232, Tennessee, USA

    Melanie D. Ohi, Joshua A. Rosenberg & Kathleen L. Gould

  3. Department of Biochemistry, Schools of Medicine and Arts & Science, Vanderbilt University, Nashville, 37232, Tennessee, USA

    Craig W. Vander Kooi & Walter J. Chazin

  4. Center for Structural Biology, Schools of Medicine and Arts & Science, Vanderbilt University, Nashville, 37232, Tennessee, USA

    Craig W. Vander Kooi & Walter J. Chazin

  5. Department of Physics, Schools of Medicine and Arts & Science, Vanderbilt University, Nashville, 37232, Tennessee, USA

    Walter J. Chazin

Authors
  1. Melanie D. Ohi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Craig W. Vander Kooi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joshua A. Rosenberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Walter J. Chazin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kathleen L. Gould
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Walter J. Chazin or Kathleen L. Gould.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ohi, M., Vander Kooi, C., Rosenberg, J. et al. Structural insights into the U-box, a domain associated with multi-ubiquitination. Nat Struct Mol Biol 10, 250–255 (2003). https://doi.org/10.1038/nsb906

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsb906

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