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Active nuclear import and cytoplasmic retention of activation-induced deaminase

Nature Structural & Molecular Biology volume 16, pages 517–527 (2009)Cite this article

Abstract

The enzyme activation-induced deaminase (AID) triggers antibody diversification in B cells by catalyzing deamination and consequently mutation of immunoglobulin genes. To minimize off-target deamination, AID is restrained by several regulatory mechanisms including nuclear exclusion, thought to be mediated exclusively by active nuclear export. Here we identify two other mechanisms involved in controlling AID subcellular localization. AID is unable to passively diffuse into the nucleus, despite its small size, and its nuclear entry requires active import mediated by a conformational nuclear localization signal. We also identify in its C terminus a determinant for AID cytoplasmic retention, which hampers diffusion to the nucleus, competes with nuclear import and is crucial for maintaining the predominantly cytoplasmic localization of AID in steady-state conditions. Blocking nuclear import alters the balance between these processes in favor of cytoplasmic retention, resulting in reduced isotype class switching.

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Figure 1: AID nuclear import is an active process.
Figure 2: Most of the AID protein is required to mediate efficient nuclear import.
Figure 3: Several noncontiguous, positively charged residues are crucial for AID nuclear import.
Figure 4: AID conformational NLS and interaction with importin-α.
Figure 5: AID cannot passively diffuse into the nucleus.
Figure 6: The C-terminal domain of AID contains a cytoplasmic retention determinant.
Figure 7: Altering the balance between AID subcellular localization mechanisms has functional consequences.

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References

  1. Muramatsu, M. et al. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102, 553–563 (2000).

    Article  CAS  Google Scholar 

  2. Revy, P. et al. Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2). Cell 102, 565–575 (2000).

    Article  CAS  Google Scholar 

  3. Peled, J.U. et al. The biochemistry of somatic hypermutation. Annu. Rev. Immunol. 26, 481–511 (2007).

    Article  Google Scholar 

  4. Di Noia, J.M. & Neuberger, M.S. Molecular mechanisms of antibody somatic hypermutation. Annu. Rev. Biochem. 76, 1–22 (2007).

    Article  CAS  Google Scholar 

  5. Chaudhuri, J. et al. Evolution of the immunoglobulin heavy chain class switch recombination mechanism. Adv. Immunol. 94, 157–214 (2007).

    Article  CAS  Google Scholar 

  6. Martin, A. & Scharff, M.D. Somatic hypermutation of the AID transgene in B and non-B cells. Proc. Natl. Acad. Sci. USA 99, 12304–12308 (2002).

    Article  CAS  Google Scholar 

  7. Okazaki, I.M. et al. Constitutive expression of AID leads to tumorigenesis. J. Exp. Med. 197, 1173–1181 (2003).

    Article  CAS  Google Scholar 

  8. Liu, M. et al. Two levels of protection for the B cell genome during somatic hypermutation. Nature 451, 841–845 (2008).

    Article  CAS  Google Scholar 

  9. Pasqualucci, L. et al. BCL-6 mutations in normal germinal center B cells: evidence of somatic hypermutation acting outside Ig loci. Proc. Natl. Acad. Sci. USA 95, 11816–11821 (1998).

    Article  CAS  Google Scholar 

  10. Shen, H.M., Peters, A., Baron, B., Zhu, X. & Storb, U. Mutation of BCL-6 gene in normal B cells by the process of somatic hypermutation of Ig genes. Science 280, 1750–1752 (1998).

    Article  CAS  Google Scholar 

  11. Dorsett, Y. et al. A role for AID in chromosome translocations between c-myc and the IgH variable region. J. Exp. Med. 204, 2225–2232 (2007).

    Article  CAS  Google Scholar 

  12. Ramiro, A.R. et al. AID is required for c-myc/IgH chromosome translocations in vivo. Cell 118, 431–438 (2004).

    Article  CAS  Google Scholar 

  13. Crouch, E.E. et al. Regulation of AID expression in the immune response. J. Exp. Med. 204, 1145–1156 (2007).

    Article  CAS  Google Scholar 

  14. de Yébenes, V.G. et al. miR-181b negatively regulates activation-induced cytidine deaminase in B cells. J. Exp. Med. 205, 2199–2206 (2008).

    Article  Google Scholar 

  15. Dorsett, Y. et al. MicroRNA-155 suppresses activation-induced cytidine deaminase-mediated Myc-Igh translocation. Immunity 28, 630–638 (2008).

    Article  CAS  Google Scholar 

  16. Teng, G. et al. MicroRNA-155 is a negative regulator of activation-induced cytidine deaminase. Immunity 28, 621–629 (2008).

    Article  CAS  Google Scholar 

  17. Ito, S. et al. Activation-induced cytidine deaminase shuttles between nucleus and cytoplasm like apolipoprotein B mRNA editing catalytic polypeptide 1. Proc. Natl. Acad. Sci. USA 101, 1975–1980 (2004).

    Article  CAS  Google Scholar 

  18. McBride, K.M., Barreto, V.M., Ramiro, A.R., Stavropoulos, P. & Nussenzweig, M.C. Somatic hypermutation is limited by CRM1-dependent nuclear export of activation-induced deaminase. J. Exp. Med. 199, 1235–1244 (2004).

    Article  CAS  Google Scholar 

  19. Aoufouchi, S. et al. Proteasomal degradation restricts the nuclear lifespan of AID. J. Exp. Med. 205, 1357–1368 (2008).

    Article  CAS  Google Scholar 

  20. Basu, U. et al. The AID antibody diversification enzyme is regulated by protein kinase A phosphorylation. Nature 438, 508–511 (2005).

    Article  CAS  Google Scholar 

  21. McBride, K.M. et al. Regulation of hypermutation by activation-induced cytidine deaminase phosphorylation. Proc. Natl. Acad. Sci. USA 103, 8798–8803 (2006).

    Article  CAS  Google Scholar 

  22. Pasqualucci, L., Kitaura, Y., Gu, H. & Dalla-Favera, R. PKA-mediated phosphorylation regulates the function of activation-induced deaminase (AID) in B cells. Proc. Natl. Acad. Sci. USA 103, 395–400 (2006).

    Article  CAS  Google Scholar 

  23. Rada, C., Jarvis, J.M. & Milstein, C. AID-GFP chimeric protein increases hypermutation of Ig genes with no evidence of nuclear localization. Proc. Natl. Acad. Sci. USA 99, 7003–7008 (2002).

    Article  CAS  Google Scholar 

  24. Brar, S.S., Watson, M. & Diaz, M. Activation-induced cytosine deaminase (AID) is actively exported out of the nucleus but retained by the induction of DNA breaks. J. Biol. Chem. 279, 26395–26401 (2004).

    Article  CAS  Google Scholar 

  25. Richardson, W.D., Mills, A.D., Dilworth, S.M., Laskey, R.A. & Dingwall, C. Nuclear protein migration involves two steps: rapid binding at the nuclear envelope followed by slower translocation through nuclear pores. Cell 52, 655–664 (1988).

    Article  CAS  Google Scholar 

  26. Newmeyer, D.D. & Forbes, D.J. Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation. Cell 52, 641–653 (1988).

    Article  CAS  Google Scholar 

  27. Guiochon-Mantel, A. et al. Nucleocytoplasmic shuttling of the progesterone receptor. EMBO J. 10, 3851–3859 (1991).

    Article  CAS  Google Scholar 

  28. Larijani, M. et al. AID associates with single-stranded DNA with high affinity and a long complex half-life in a sequence-independent manner. Mol. Cell. Biol. 27, 20–30 (2007).

    Article  CAS  Google Scholar 

  29. Macara, I.G. Transport into and out of the nucleus. Microbiol. Mol. Biol. Rev. 65, 570–594 (2001).

    Article  CAS  Google Scholar 

  30. Görlich, D. & Kutay, U. Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell Dev. Biol. 15, 607–660 (1999).

    Article  Google Scholar 

  31. Chen, K.M. et al. Structure of the DNA deaminase domain of the HIV-1 restriction factor APOBEC3G. Nature 452, 116–119 (2008).

    Article  CAS  Google Scholar 

  32. Holden, L.G. et al. Crystal structure of the anti-viral APOBEC3G catalytic domain and functional implications. Nature 456, 121–124 (2008).

    Article  CAS  Google Scholar 

  33. Prochnow, C. et al. The APOBEC-2 crystal structure and functional implications for the deaminase AID. Nature 445, 447–451 (2007).

    Article  CAS  Google Scholar 

  34. Nilsen, H. et al. Analysis of uracil-DNA glycosylases from the murine Ung gene reveals differential expression in tissues and in embryonic development and a subcellular sorting pattern that differs from the human homologues. Nucleic Acids Res. 28, 2277–2285 (2000).

    Article  CAS  Google Scholar 

  35. Conticello, S.G. et al. Interaction between antibody-diversification enzyme AID and spliceosome-associated factor CTNNBL1. Mol. Cell 31, 474–484 (2008).

    Article  CAS  Google Scholar 

  36. Lange, A. et al. Classical nuclear localization signals: definition, function, and interaction with importin α. J. Biol. Chem. 282, 5101–5105 (2007).

    Article  CAS  Google Scholar 

  37. Miyamoto, Y. et al. Cellular stresses induce the nuclear accumulation of importin α and cause a conventional nuclear import block. J. Cell Biol. 165, 617–623 (2004).

    Article  CAS  Google Scholar 

  38. Kodiha, M., Chu, A., Matusiewicz, N. & Stochaj, U. Multiple mechanisms promote the inhibition of classical nuclear import upon exposure to severe oxidative stress. Cell Death Differ. 11, 862–874 (2004).

    Article  CAS  Google Scholar 

  39. Shivarov, V., Shinkura, R. & Honjo, T. Dissociation of in vitro DNA deamination activity and physiological functions of AID mutants. Proc. Natl. Acad. Sci. USA 105, 15866–15871 (2008).

    Article  CAS  Google Scholar 

  40. Doi, T. et al. The C-terminal region of activation-induced cytidine deaminase is responsible for a recombination function other than DNA cleavage in class switch recombination. Proc. Natl. Acad. Sci. USA 106, 2758–2763 (2009).

    Article  CAS  Google Scholar 

  41. Barreto, V., Reina-San-Martin, B., Ramiro, A.R., McBride, K.M. & Nussenzweig, M.C. C-terminal deletion of AID uncouples class switch recombination from somatic hypermutation and gene conversion. Mol. Cell 12, 501–508 (2003).

    Article  CAS  Google Scholar 

  42. Ta, V. et al. AID mutant analyses indicate requirement for class-switch-specific cofactors. Nat. Immunol. 4, 843–848 (2003).

    Article  CAS  Google Scholar 

  43. Shinkura, R. et al. Separate domains of AID are required for somatic hypermutation and class-switch recombination. Nat. Immunol. 5, 707–712 (2004).

    Article  CAS  Google Scholar 

  44. LaCasse, E.C. & Lefebvre, Y.A. Nuclear localization signals overlap DNA- or RNA-binding domains in nucleic acid-binding proteins. Nucleic Acids Res. 23, 1647–1656 (1995).

    Article  CAS  Google Scholar 

  45. Chester, A. et al. The apolipoprotein B mRNA editing complex performs a multifunctional cycle and suppresses nonsense-mediated decay. EMBO J. 22, 3971–3982 (2003).

    Article  CAS  Google Scholar 

  46. Yang, Y. & Smith, H.C. Multiple protein domains determine the cell type-specific nuclear distribution of the catalytic subunit required for apolipoprotein B mRNA editing. Proc. Natl. Acad. Sci. USA 94, 13075–13080 (1997).

    Article  CAS  Google Scholar 

  47. Dickerson, S.K., Market, E., Besmer, E. & Papavasiliou, F.N. AID mediates hypermutation by deaminating single stranded DNA. J. Exp. Med. 197, 1291–1296 (2003).

    Article  CAS  Google Scholar 

  48. Yang, G. et al. Activation-induced deaminase cloning, localization, and protein extraction from young VH-mutant rabbit appendix. Proc. Natl. Acad. Sci. USA 102, 17083–17088 (2005).

    Article  CAS  Google Scholar 

  49. Bennett, R.P., Presnyak, V., Wedekind, J.E. & Smith, H.C. Nuclear exclusion of the HIV-1 host defense factor APOBEC3G requires a novel cytoplasmic retention signal and is not dependent on RNA binding. J. Biol. Chem. 283, 7320–7327 (2008).

    Article  CAS  Google Scholar 

  50. Lau, P.P., Zhu, H.J., Baldini, A., Charnsangavej, C. & Chan, L. Dimeric structure of a human apolipoprotein B mRNA editing protein and cloning and chromosomal localization of its gene. Proc. Natl. Acad. Sci. USA 91, 8522–8526 (1994).

    Article  CAS  Google Scholar 

  51. Stenglein, M.D., Matsuo, H. & Harris, R.S. Two regions within the amino-terminal half of APOBEC3G cooperate to determine cytoplasmic localization. J. Virol. 82, 9591–9599 (2008).

    Article  CAS  Google Scholar 

  52. Wu, X., Geraldes, P., Platt, J.L. & Cascalho, M. The double-edged sword of activation-induced cytidine deaminase. J. Immunol. 174, 934–941 (2005).

    Article  CAS  Google Scholar 

  53. Cattoretti, G. et al. Nuclear and cytoplasmic AID in extrafollicular and germinal center B cells. Blood 107, 3967–3975 (2006).

    Article  CAS  Google Scholar 

  54. Greiner, A. et al. Differential expression of activation-induced cytidine deaminase (AID) in nodular lymphocyte-predominant and classical Hodgkin lymphoma. J. Pathol. 205, 541–547 (2005).

    Article  CAS  Google Scholar 

  55. Pasqualucci, L. et al. Expression of the AID protein in normal and neoplastic B cells. Blood 104, 3318–3325 (2004).

    Article  CAS  Google Scholar 

  56. Petersen-Mahrt, S.K., Harris, R.S. & Neuberger, M.S. AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification. Nature 418, 99–104 (2002).

    Article  CAS  Google Scholar 

  57. Di Noia, J.M. et al. Dependence of antibody gene diversification on uracil excision. J. Exp. Med. 204, 3209–3219 (2007).

    Article  CAS  Google Scholar 

  58. Kitamura, T. et al. Retrovirus-mediated gene transfer and expression cloning: powerful tools in functional genomics. Exp. Hematol. 31, 1007–1014 (2003).

    Article  CAS  Google Scholar 

  59. Zhang, W. et al. Clonal instability of V region hypermutation in the Ramos Burkitt's lymphoma cell line. Int. Immunol. 13, 1175–1184 (2001).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank S. Conticello and M. Neuberger (Medical Research Council Laboratory of Molecular Biology), I. Macara (University of Virginia School of Medicine), M. Malim (King's College London School of Medicine), N. Navaratnam (Imperial College London), A. Martin (Univeristy of Toronto), S. Swaminathan (University of Florida), J. Archambault, E. Cohen, T. Möröy and Y. Guindon (Institut de Recherches Cliniques de Montréal (IRCM)) for providing reagents and T. Honjo (Kyoto University School of Medicine) for the AID-deficient mice through A. Lamarre (INRS-Institute Armand-Frappier). We thank E.-L. Thivierge and C. Toulouse for expert animal care and D. Filion for help with confocal microscopy. We are grateful to H. Krokan and R. Harris for discussions and to S. Conticello, C. Buscaglia, D. Muñoz and J.F. Côté for critically reading the manuscript. This work was supported by the Canadian Institutes of Health Research (MOP 84543) and a Canada Research Chair (to J.M.D.). A.O. was supported by a fellowship from the Canadian Institutes of Health Research Cancer Training Program at the IRCM. V.A.C. was supported in part by a Michel Saucier fellowship from the Louis-Pasteur Canadian Fund through the University of Montreal.

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Authors and Affiliations

  1. Institut de Recherches Cliniques de Montréal, Montréal, Quebec, Canada

    Anne-Marie Patenaude, Alexandre Orthwein, Yi Hu, Vanina A Campo & Javier M Di Noia

  2. Department of Microbiology and Immunology, University of Montreal, Montréal, Quebec, Canada

    Alexandre Orthwein & Javier M Di Noia

  3. Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway

    Yi Hu & Bodil Kavli

  4. Institut Pasteur de Montevideo, Unidad de Cristalografia de Proteinas, Montevideo, Uruguay

    Alejandro Buschiazzo

  5. Département de Biologie Structurale & Chimie, Institut Pasteur, Paris, France

    Alejandro Buschiazzo

  6. Department of Medicine, University of Montreal, Montréal, Quebec, Canada

    Javier M Di Noia

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A.-M.P., A.O., Y.H., V.A.C., A.B. and J.M.D. performed research; all authors analyzed data and discussed results; J.M.D. designed research and wrote the paper.

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Correspondence to Javier M Di Noia.

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Patenaude, AM., Orthwein, A., Hu, Y. et al. Active nuclear import and cytoplasmic retention of activation-induced deaminase. Nat Struct Mol Biol 16, 517–527 (2009). https://doi.org/10.1038/nsmb.1598

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