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:

Structure-function studies of nucleocytoplasmic transport of retroviral genomic RNA by mRNA export factor TAP

Abstract

mRNA export is mediated by the TAP–p15 heterodimer, which belongs to the family of NTF2-like export receptors. TAP–p15 heterodimers also bind to the constitutive transport element (CTE) present in simian type D retroviral RNAs, and they mediate the export of viral unspliced RNAs to the host cytoplasm. We have solved the crystal structure of the RNA recognition and leucine-rich repeat motifs of TAP bound to one symmetrical half of the CTE RNA. L-shaped conformations of protein and RNA are involved in a mutual molecular embrace on complex formation. We have monitored the impact of structure-guided mutations on binding affinities in vitro and transport assays in vivo. Our studies define the principles by which CTE RNA subverts the mRNA export receptor TAP, thereby facilitating the nuclear export of viral genomic RNAs, and, more generally, provide insights on cargo RNA recognition by mRNA export receptors.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structure of TAP-NTD bound to the hCTE RNA.
Figure 2: Fold of the internal loop and bulged bases in the hCTE–TAP complex.
Figure 3: Key interactions between hCTE RNA and the TAP-NTD in the complex.
Figure 4: In vitro ITC, and direct and competitive filter-binding data for TAP-NTD and hCTE RNA mutants.
Figure 5: In vivo RNA export assay.
Figure 6: Stoichiometry of TAP-NTD binding to CTE RNA.
Figure 7: Packing of two molecules of complex (TAP-NTD bound to hCTE) in the crystallographic asymmetric unit and a model of the 2:1 complex of TAP-NTD bound to full-length CTE.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Chook, Y.M. & Blobel, G. Karyopherins and nuclear import. Curr. Opin. Struct. Biol. 11, 703–715 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Conti, E. & Izaurralde, E. Nucleocytoplasmic transport enters the atomic age. Curr. Opin. Cell Biol. 13, 310–319 (2001).

    Article  CAS  PubMed  Google Scholar 

  3. Conti, E., Muller, C.W. & Stewart, M. Karyopherin flexibility in nucleocytoplasmic transport. Curr. Opin. Struct. Biol. 16, 237–244 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Cook, A., Bono, F., Jinek, M. & Conti, E. Structural biology of nucleocytoplasmic transport. Annu. Rev. Biochem. 76, 647–671 (2007).

    Article  CAS  PubMed  Google Scholar 

  5. Köhler, A. & Hurt, E. Exporting RNA from the nucleus to the cytoplasm. Nat. Rev. Mol. Cell Biol. 8, 761–773 (2007).

    Article  PubMed  Google Scholar 

  6. Stewart, M. Molecular mechanism of the nuclear protein import cycle. Nat. Rev. Mol. Cell Biol. 8, 195–208 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. Cook, A.G. & Conti, E. Nuclear export complexes in the frame. Curr. Opin. Struct. Biol. 20, 247–252 (2010).

    Article  CAS  PubMed  Google Scholar 

  8. Cook, A.G., Fukuhara, N., Jinek, M. & Conti, E. Structures of the tRNA export factor in the nuclear and cytosolic states. Nature 461, 60–65 (2009).

    Article  CAS  PubMed  Google Scholar 

  9. Okada, C. et al. A high-resolution structure of the pre-microRNA nuclear export machinery. Science 326, 1275–1279 (2009).

    Article  CAS  PubMed  Google Scholar 

  10. Braun, I.C., Rohrbach, E., Schmitt, C. & Izaurralde, E. TAP binds to the constitutive transport element (CTE) through a novel RNA-binding motif that is sufficient to promote CTE-dependent RNA export from the nucleus. EMBO J. 18, 1953–1965 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Grüter, P. et al. TAP, the human homolog of Mex67p, mediates CTE-dependent RNA export from the nucleus. Mol. Cell 1, 649–659 (1998).

    Article  PubMed  Google Scholar 

  12. Izaurralde, E. Friedrich Miescher Prize awardee lecture review. A conserved family of nuclear export receptors mediates the exit of messenger RNA to the cytoplasm. Cell. Mol. Life Sci. 58, 1105–1112 (2001).

    Article  CAS  PubMed  Google Scholar 

  13. Segref, A. et al. Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA and nuclear pores. EMBO J. 16, 3256–3271 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Fribourg, S., Braun, I.C., Izaurralde, E. & Conti, E. Structural basis for the recognition of a nucleoporin FG repeat by the NTF2-like domain of the TAP/p15 mRNA nuclear export factor. Mol. Cell 8, 645–656 (2001).

    Article  CAS  PubMed  Google Scholar 

  15. Herold, A. et al. TAP (NXF1) belongs to a multigene family of putative RNA export factors with a conserved modular architecture. Mol. Cell. Biol. 20, 8996–9008 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Liker, E., Fernandez, E., Izaurralde, E. & Conti, E. The structure of the mRNA export factor TAP reveals a cis arrangement of a non-canonical RNP domain and an LRR domain. EMBO J. 19, 5587–5598 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ho, D.N., Coburn, G.A., Kang, Y., Cullen, B.R. & Georgiadis, M.M. The crystal structure and mutational analysis of a novel RNA-binding domain found in the human Tap nuclear mRNA export factor. Proc. Natl. Acad. Sci. USA 99, 1888–1893 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Grant, R.P., Neuhaus, D. & Stewart, M. Structural basis for the interaction between the Tap/NXF1 UBA domain and FG nucleoporins at 1 resolution. J. Mol. Biol. 326, 849–858 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. Bachi, A. et al. The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates. RNA 6, 136–158 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ernst, R.K., Bray, M., Rekosh, D. & Hammarskjold, M.L. A structured retroviral RNA element that mediates nucleocytoplasmic export of intron-containing RNA. Mol. Cell. Biol. 17, 135–144 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Tabernero, C., Zolotukhin, A.S., Valentin, A., Pavlakis, G.N. & Felber, B.K. The posttranscriptional control element of the simian retrovirus type 1 forms an extensive RNA secondary structure necessary for its function. J. Virol. 70, 5998–6011 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Cullen, B.R. Viral RNAs: lessons from the enemy. Cell 136, 592–597 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Li, Y. et al. An intron with a constitutive transport element is retained in a Tap messenger RNA. Nature 443, 234–237 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. Kang, Y. & Cullen, B.R. The human Tap protein is a nuclear mRNA export factor that contains novel RNA-binding and nucleocytoplasmic transport sequences. Genes Dev. 13, 1126–1139 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Braun, I.C., Herold, A., Rode, M., Conti, E. & Izaurralde, E. Overexpression of TAP/p15 heterodimers bypasses nuclear retention and stimulates nuclear mRNA export. J. Biol. Chem. 276, 20536–20543 (2001).

    Article  CAS  PubMed  Google Scholar 

  26. Jin, L., Guzik, B.W., Bor, Y.C., Rekosh, D. & Hammarskjold, M.L. Tap and NXT promote translation of unspliced mRNA. Genes Dev. 17, 3075–3086 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Serganov, A., Polonskaia, A., Ehresmann, B., Ehresmann, C. & Patel, D.J. Ribosomal protein S15 represses its own translation via adaptation of an rRNA-like fold within its mRNA. EMBO J. 22, 1898–1908 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Batey, R.T., Rambo, R.P. & Doudna, J.A. Tertiary motifs in RNA structure and folding. Angew. Chem. Int. Edn Engl. 38, 2326–2343 (1999).

    Article  CAS  Google Scholar 

  29. Hermann, T. & Patel, D.J. Adaptive recognition by nucleic acid aptamers. Science 287, 820–825 (2000).

    Article  CAS  PubMed  Google Scholar 

  30. Tereshko, V., Skripkin, E. & Patel, D.J. Encapsulating streptomycin within a small 40-mer RNA. Chem. Biol. 10, 175–187 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Dibrov, S.M., Johnston-Cox, H., Weng, Y.H. & Hermann, T. Functional architecture of HCV IRES domain II stabilized by divalent metal ions in the crystal and in solution. Angew. Chem. Int. Edn Engl. 46, 226–229 (2007).

    Article  CAS  Google Scholar 

  32. Leontis, N.B. & Westhof, E. Geometric nomenclature and classification of RNA base pairs. RNA 7, 499–512 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Hermann, T. & Patel, D.J. RNA bulges as architectural and recognition motifs. Structure 8, R47–R54 (2000).

    Article  CAS  PubMed  Google Scholar 

  34. Jiang, F. et al. Anchoring an extended HTLV-1 Rex peptide within an RNA major groove containing junctional base triples. Structure 7, 1461–1472 (1999).

    Article  CAS  PubMed  Google Scholar 

  35. Cléry, A., Blatter, M. & Allain, F.H. RNA recognition motifs: boring? Not quite. Curr. Opin. Struct. Biol. 18, 290–298 (2008).

    Article  PubMed  Google Scholar 

  36. Lunde, B.M., Moore, C. & Varani, G. RNA-binding proteins: modular design for efficient function. Nat. Rev. Mol. Cell Biol. 8, 479–490 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Serganov, A. & Patel, D.J. Towards deciphering the principles underlying an mRNA recognition code. Curr. Opin. Struct. Biol. 18, 120–129 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Price, S.R., Evans, P.R. & Nagai, K. Crystal structure of the spliceosomal U2B-U2A′ protein complex bound to a fragment of U2 small nuclear RNA. Nature 394, 645–650 (1998).

    Article  CAS  PubMed  Google Scholar 

  39. Teplova, M. et al. Structural basis for recognition and sequestration of UUU(OH) 3′ temini of nascent RNA polymerase III transcripts by La, a rheumatic disease autoantigen. Mol. Cell 21, 75–85 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Liu, L. et al. Structural basis of toll-like receptor 3 signaling with double-stranded RNA. Science 320, 379–381 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Hautbergue, G.M., Hung, M.L., Golovanov, A.P., Lian, L.Y. & Wilson, S.A. Mutually exclusive interactions drive handover of mRNA from export adaptors to TAP. Proc. Natl. Acad. Sci. USA 105, 5154–5159 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Stutz, F. & Izaurralde, E. The interplay of nuclear mRNP assembly, mRNA surveillance and export. Trends Cell Biol. 13, 319–327 (2003).

    Article  CAS  PubMed  Google Scholar 

  43. Walsh, M.J., Hautbergue, G.M. & Wilson, S.A. Structure and function of mRNA export adaptors. Biochem. Soc. Trans. 38, 232–236 (2010).

    Article  CAS  PubMed  Google Scholar 

  44. Huang, Y., Gattoni, R., Stevenin, J. & Steitz, J.A. SR splicing factors serve as adapter proteins for TAP-dependent mRNA export. Mol. Cell 11, 837–843 (2003).

    Article  CAS  PubMed  Google Scholar 

  45. CCP4. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

  46. Pikovskaya, O., Serganov, A.A., Polonskaia, A., Serganov, A. & Patel, D.J. Preparation and crystallization of riboswitch-ligand complexes. Methods Mol. Biol. 540, 115–128 (2009).

    Article  CAS  PubMed  Google Scholar 

  47. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  PubMed  Google Scholar 

  48. Navaza, J. AMoRe: an automated package for molecular replacement. Acta Crystallogr. A 50, 157–163 (1994).

    Article  Google Scholar 

  49. 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).

    Article  CAS  PubMed  Google Scholar 

  50. Winn, M.D., Isupov, M.N. & Murshudov, G.N. Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallogr. D Biol. Crystallogr. 57, 122–133 (2001).

    Article  CAS  PubMed  Google Scholar 

  51. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  Google Scholar 

  53. Robertson, M.P. & Scott, W.G. A general method for phasing novel complex RNA crystal structures without heavy-atom derivatives. Acta Crystallogr. D Biol. Crystallogr. D64, 738–744 (2008).

    Article  PubMed  Google Scholar 

  54. Terwilliger, T.C. et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr. D Biol. Crystallogr. 64, 61–69 (2008).

    Article  CAS  PubMed  Google Scholar 

  55. Huang, X.J. et al. Minimal Rev-response element for type 1 human immunodeficiency virus. J. Virol. 65, 2131–2134 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This research was supported by funds from the US National Institutes of Health (CA049982) to D.J.P. and the Max-Planck Society to E.I. X-ray data were collected at the X-29 beamline at the National Synchrotron Light Source at the Brookhaven National Laboratory and we are grateful to the staff for their assistance.

Author information

Authors and Affiliations

Authors

Contributions

Constructs design, protein and RNA preparation and purification, crystallization of complex and its structure determination and in vitro binding assays were undertaken by M.T. with the assistance of N.W.K. under the supervision of D.J.P. The in vivo transport assays were performed by L.W. under the supervision of E.I. The paper was written by M.T., D.J.P. and E.I. with input from the other authors.

Corresponding author

Correspondence to Dinshaw J Patel.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Methods (PDF 716 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Teplova, M., Wohlbold, L., Khin, N. et al. Structure-function studies of nucleocytoplasmic transport of retroviral genomic RNA by mRNA export factor TAP. Nat Struct Mol Biol 18, 990–998 (2011). https://doi.org/10.1038/nsmb.2094

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2094

This article is cited by

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