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Structural basis for the assembly and nucleic acid binding of the TREX-2 transcription-export complex

Nature Structural & Molecular Biology volume 19, pages 328–336 (2012)Cite this article

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Abstract

The conserved TREX-2 transcription-export complex integrates transcription and processing of many actively transcribed nascent mRNAs with the recruitment of export factors at nuclear pores and also contributes to transcriptional memory and genomic stability. We report the crystal structure of the Sac3–Thp1–Sem1 segment of Saccharomyces cerevisiae TREX-2 that interfaces with the gene expression machinery. Sac3–Thp1–Sem1 forms a previously uncharacterized PCI-domain complex characterized by the juxtaposition of Sac3 and Thp1 winged helix domains, forming a platform that mediates nucleic acid binding. Our structure-guided mutations support the idea that the Thp1-Sac3 interaction is an essential requirement for mRNA binding and for the coupling of transcription and processing to mRNP assembly and export. These results provide insight into how newly synthesized transcripts are efficiently transferred from TREX-2 to the principal mRNA export factor, and they reveal how Sem1 stabilizes PCI domain–containing proteins and promotes complex assembly.

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Figure 1: The structure of the Sac3–Thp1–Sem1 complex.
Figure 2: Structural basis for the Thp1-Sem1 interaction.
Figure 3: Structure-guided mutations that disrupt the Sac3-Thp1 interaction impair mRNA nuclear export and growth.
Figure 4: The juxtaposed winged helix domains of the Sac3–Thp1–Sem1 complex bind nucleic acids.
Figure 5: Structure-guided Thp1 and Sac3 mutants with impaired in vitro nucleic acid binding show cell growth and mRNA export defects, but they do not inhibit TREX-2 assembly.
Figure 6: Schematic illustration of how the TREX-2 complex integrates the formation of an export-competent mRNP adjacent to NPCs.

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References

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

    Google Scholar 

  2. Kelly, S.M. & Corbett, A.H. Messenger RNA export from the nucleus: a series of molecular wardrobe changes. Traffic 10, 1199–1208 (2009).

    Google Scholar 

  3. Stewart, M. Nuclear export of mRNA. Trends Biochem. Sci. 35, 609–617 (2010).

    Google Scholar 

  4. Rodríguez-Navarro, S. & Hurt, E. Linking gene regulation to mRNA production and export. Curr. Opin. Cell Biol. 23, 302–309 (2011).

    Google Scholar 

  5. Chávez, S. et al. A protein complex containing Tho2, Hpr1, Mft1 and a novel protein, Thp2, connects transcription elongation with mitotic recombination in Saccharomyces cerevisiae. EMBO J. 19, 5824–5834 (2000).

    Google Scholar 

  6. Strässer, K. et al. TREX is a conserved complex coupling transcription with messenger RNA export. Nature 417, 304–308 (2002).

    Google Scholar 

  7. Hurt, E., Luo, M.-J., Röther, S., Reed, R. & Strässer, K. Cotranscriptional recruitment of the serine-arginine-rich (SR)-like proteins Gbp2 and Hrb1 to nascent mRNA via the TREX complex. Proc. Natl. Acad. Sci. USA 101, 1858–1862 (2004).

    Google Scholar 

  8. Reed, R. & Hurt, E. A conserved mRNA export machinery coupled to pre-mRNA splicing. Cell 108, 523–531 (2002).

    Google Scholar 

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

    Google Scholar 

  10. Blobel, G. Gene gating: a hypothesis. Proc. Natl. Acad. Sci. USA 82, 8527–8529 (1985).

    Google Scholar 

  11. Rodríguez-Navarro, S. et al. Sus1, a functional component of the SAGA histone acetylase complex and the nuclear pore-associated mRNA export machinery. Cell 116, 75–86 (2004).

    Google Scholar 

  12. Cabal, G.G. et al. SAGA interacting factors confine sub-diffusion of transcribed genes to the nuclear envelope. Nature 441, 770–773 (2006).

    Google Scholar 

  13. Fischer, T. et al. Yeast centrin Cdc31 is linked to the nuclear mRNA export machinery. Nat. Cell Biol. 6, 840–848 (2004).

    Google Scholar 

  14. Light, W.H., Brickner, D.G., Brand, V.R. & Brickner, J.H. Interaction of a DNA zip code with the nuclear pore complex promotes H2A.Z incorporation and INO1 transcriptional memory. Mol. Cell 40, 112–125 (2010).

    Google Scholar 

  15. Ahmed, S. et al. DNA zip codes control an ancient mechanism for gene targeting to the nuclear periphery. Nat. Cell Biol. 12, 111–118 (2010).

    Google Scholar 

  16. Abruzzi, K.C., Belostotsky, D.A., Chekanova, J.A., Dower, K. & Rosbash, M. 3′-end formation signals modulate the association of genes with the nuclear periphery as well as mRNP dot formation. EMBO J. 25, 4253–4262 (2006).

    Google Scholar 

  17. Chekanova, J.A., Abruzzi, K.C., Rosbash, M. & Belostotsky, D.A. Sus1, Sac3, and Thp1 mediate post-transcriptional tethering of active genes to the nuclear rim as well as to non-nascent mRNP. RNA 14, 66–77 (2008).

    Google Scholar 

  18. Egecioglu, D. & Brickner, J.H. Gene positioning and expression. Curr. Opin. Cell Biol. 23, 338–345 (2011).

    Google Scholar 

  19. González-Aguilera, C. et al. The THP1–SAC3-SUS1–CDC31 complex works in transcription elongation-mRNA export preventing RNA-mediated genome instability. Mol. Biol. Cell 19, 4310–4318 (2008).

    Google Scholar 

  20. Tous, C. et al. A novel assay identifies transcript elongation roles for the Nup84 complex and RNA processing factors. EMBO J. 30, 1953–1964 (2011).

    Google Scholar 

  21. Fischer, T. et al. The mRNA export machinery requires the novel Sac3p-Thp1p complex to dock at the nucleoplasmic entrance of the nuclear pores. EMBO J. 21, 5843–5852 (2002).

    Google Scholar 

  22. Jani, D. et al. Sus1, Cdc31, and the Sac3 CID region form a conserved interaction platform that promotes nuclear pore association and mRNA export. Mol. Cell 33, 727–737 (2009).

    Google Scholar 

  23. Wilmes, G.M. et al. A genetic interaction map of RNA-processing factors reveals links between Sem1/Dss1-containing complexes and mRNA export and splicing. Mol. Cell 32, 735–746 (2008).

    Google Scholar 

  24. Faza, M.B. et al. Sem1 is a functional component of the nuclear pore complex-associated messenger RNA export machinery. J. Cell Biol. 184, 833–846 (2009).

    Google Scholar 

  25. Pick, E., Hofmann, K. & Glickman, M.H. PCI complexes: Beyond the proteasome, CSN, and eIF3 Troika. Mol. Cell 35, 260–264 (2009).

    Google Scholar 

  26. Brickner, D.G. et al. H2A.Z-mediated localization of genes at the nuclear periphery confers epigenetic memory of previous transcriptional state. PLoS Biol. 5, e81 (2007).

    Google Scholar 

  27. Wickramasinghe, V.O. et al. mRNA export from mammalian cell nuclei is dependent on GANP. Curr. Biol. 20, 25–31 (2010).

    Google Scholar 

  28. Dessau, M. et al. The Arabidopsis COP9 signalosome subunit 7 is a model PCI domain protein with subdomains involved in COP9 signalosome assembly. Plant Cell 20, 2815–2834 (2008).

    Google Scholar 

  29. Wei, Z. et al. Crystal structure of human eIF3k, the first structure of eIF3 subunits. J. Biol. Chem. 279, 34983–34990 (2004).

    Google Scholar 

  30. Gajiwala, K.S. & Burley, S.K. Winged helix proteins. Curr. Opin. Struct. Biol. 10, 110–116 (2000).

    Google Scholar 

  31. Gallardo, M., Luna, R., Erdjument-Bromage, H., Tempst, P. & Aguilera, A. Nab2p and the Thp1p-Sac3p complex functionally interact at the interface between transcription and mRNA metabolism. J. Biol. Chem. 278, 24225–24232 (2003).

    Google Scholar 

  32. Faza, M.B., Kemmler, S. & Panse, V.G. Sem1: A versatile “molecular glue”? Nucleus 1, 12–17 (2010).

    Google Scholar 

  33. Sharon, M., Taverner, T., Ambroggio, X.I., Deshaies, R.J. & Robinson, C.V. Structural organization of the 19S proteasome lid: insights from MS of intact complexes. PLoS Biol. 4, e267 (2006).

    Google Scholar 

  34. Isono, E., Saeki, Y., Yokosawa, H. & Toh-e, A. Rpn7 Is required for the structural integrity of the 26 S proteasome of Saccharomyces cerevisiae. J. Biol. Chem. 279, 27168–27176 (2004).

    Google Scholar 

  35. Funakoshi, M., Li, X., Velichutina, I., Hochstrasser, M. & Kobayashi, H. Sem1, the yeast ortholog of a human BRCA2-binding protein, is a component of the proteasome regulatory particle that enhances proteasome stability. J. Cell Sci. 117, 6447–6454 (2004).

    Google Scholar 

  36. Wei, S.-J. Identification of a specific motif of the DSS1 protein required for proteasome interaction and p53 protein degradation. J. Mol. Biol. 383, 693–712 (2008).

    Google Scholar 

  37. Scheufler, C. et al. Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine. Cell 101, 199–210 (2000).

    Google Scholar 

  38. Scheel, H. & Hofmann, K. Prediction of a common structural scaffold for proteasome lid, COP9-signalosome and eIF3 complexes. BMC Bioinformatics 6, 71 (2005).

    Google Scholar 

  39. Jimeno, S. et al. New suppressors of THO mutations identify Thp3 (Ypr045c)-Csn12 as a protein complex involved in transcription elongation. Mol. Cell. Biol. 31, 674–685 (2011).

    Google Scholar 

  40. Matsuura, Y. & Stewart, M. Structural basis for the assembly of a nuclear export complex. Nature 432, 872–877 (2004).

    Google Scholar 

  41. Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2007).

    Google Scholar 

  42. de La Fortelle, E. & Bricogne, G. Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472–494 (1997).

    Google Scholar 

  43. Abrahams, J.P. & Leslie, A.G.W. Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Crystallogr. D Biol. Crystallogr. 52, 30–42 (1996).

    Google Scholar 

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

    Google Scholar 

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

    Google Scholar 

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

  47. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Google Scholar 

  48. Longtine, M.S. et al. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961 (1998).

    Google Scholar 

  49. Edelheit, O., Hanukoglu, A. & Hanukoglu, I. Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies. BMC Biotechnol. 9, 61 (2009).

    Google Scholar 

  50. Amberg, D.C., Goldstein, A.L. & Cole, C.N. Isolation and characterization of RAT1: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mRNA. Genes Dev. 6, 1173–1189 (1992).

    Google Scholar 

  51. Rigaut, G. et al. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17, 1030–1032 (1999).

    Google Scholar 

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Acknowledgements

We are most grateful to our colleagues in Cambridge and Heidelberg, especially D. Jani, S. Kuhlmann, N. Marshall, E. Valkov and M. Halls for their helpful comments and assistance. We also thank T. Sorensen and M. Williams at the Diamond Light Source. We thank J. Jäntti (University of Helsinki) and R. Kölling (University of Hohenheim) for providing the anti-Sem1 and anti-Sac3 antibodies. A.M.E. was supported by a Marie Curie Fellowship and a European Molecular Biology Organization (EMBO) Long-term Fellowship. This work was supported in part by grants from the Medical Research Council (U105178939, to M.S.) and the Wellcome Trust (080522, to M.S.) and the Deutsche Forschungsgemeinschaft (SFB 638/B3, to E.H.).

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Authors and Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Cambridge, UK

    Andrew M Ellisdon & Murray Stewart

  2. Biochemie-Zentrum der Universität Heidelberg, Heidelberg, Germany

    Lyudmila Dimitrova & Ed Hurt

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Contributions

A.M.E. and M.S. determined structures and conducted in vitro experiments, and L.D. and E.H. carried out in vivo studies. All authors contributed to the interpretation of the results and helped write the manuscript.

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Correspondence to Murray Stewart.

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Supplementary information

Supplementary Text and Figures

Supplementary Discussion, Supplementary Methods, Supplementary Tables 1–3 and Supplementary Figures 1–4 (PDF 601 kb)

Supplementary Movie 1

Structure of the Sac3-Thp1-Sem1 complex. Overview of the Sac3(blue)-Thp1(gray)-Sem1(yellow) complex with α-helices represented as cylinders and Sem1 in worm format. (AVI 4665 kb)

Supplementary Movie 2

Structural basis for the Thp1-Sem1 interaction. Sem1 (yellow) makes extensive contacts with Thp1 (grey), burying 2741 Å2 of surface area, but makes only minor contact to Sac3 (blue), burying 293 Å2. Sem1 is represented in worm format and the surface of both Sac3 and Thp1 is shown. (AVI 4657 kb)

Supplementary Movie 3

Structure of the PCID2:DSS1 complex. Overview of the PCID2(green):DSS1(orange) structure with PCID2 α-helices represented as cylinders and DSS1 in worm format. Important DSS1 sidechains are shown as sticks. (AVI 3705 kb)

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Ellisdon, A., Dimitrova, L., Hurt, E. et al. Structural basis for the assembly and nucleic acid binding of the TREX-2 transcription-export complex. Nat Struct Mol Biol 19, 328–336 (2012). https://doi.org/10.1038/nsmb.2235

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