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Structure of the pre-60S ribosomal subunit with nuclear export factor Arx1 bound at the exit tunnel

Nature Structural & Molecular Biology volume 19, pages 1234–1241 (2012)Cite this article

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Abstract

Preribosomal particles evolve in the nucleus through transient interaction with biogenesis factors before export to the cytoplasm. Here, we report the architecture of the late pre-60S particle, purified from Saccharomyces cerevisiae, through Arx1, a nuclear export factor with structural homology to methionine aminopeptidases, or its binding partner Alb1. Cryo-EM reconstruction of the Arx1 particle at 11.9-Å resolution reveals regions of extra density on the pre-60S particle attributed to associated biogenesis factors, confirming the immature state of the nascent subunit. One of these densities could be unambiguously assigned to Arx1. Immunoelectron microscopy and UV cross-linking localize Arx1 close to the ribosomal exit tunnel, in direct contact with ES27, a highly dynamic eukaryotic rRNA expansion segment. The binding of Arx1 at the exit tunnel may position this export factor to prevent premature recruitment of ribosome-associated factors active during translation.

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Figure 1: Arx1 and Alb1 are present on the same pre-60S particles.
Figure 2: Cryo-EM reconstruction of the Arx1 particle.
Figure 3: Immuno-EM reveals the relative position of biogenesis factors and r proteins on the Arx1 particle.
Figure 4: CRAC analysis confirms the location of Arx1 near the exit tunnel.
Figure 5: Arx1 localizes to the knob structure of the Rix1 particle, a precursor of the Arx1 particle.

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References

  1. Kressler, D., Hurt, E. & Bassler, J. Driving ribosome assembly. Biochim. Biophys. Acta 1803, 673–683 (2010).

    Article  CAS  Google Scholar 

  2. Karbstein, K. Inside the 40S ribosome assembly machinery. Curr. Opin. Chem. Biol. 15, 657–663 (2011).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  4. Zemp, I. & Kutay, U. Nuclear export and cytoplasmic maturation of ribosomal subunits. FEBS Lett. 581, 2783–2793 (2007).

    Article  CAS  Google Scholar 

  5. Spahn, C.M. et al. Structure of the 80S ribosome from Saccharomyces cerevisiae–tRNA-ribosome and subunit-subunit interactions. Cell 107, 373–386 (2001).

    Article  CAS  Google Scholar 

  6. Armache, J.P. et al. Cryo-EM structure and rRNA model of a translating eukaryotic 80S ribosome at 5.5-A resolution. Proc. Natl. Acad. Sci. USA 107, 19748–19753 (2010).

    Article  CAS  Google Scholar 

  7. Ben-Shem, A., Jenner, L., Yusupova, G. & Yusupov, M. Crystal structure of the eukaryotic ribosome. Science 330, 1203–1209 (2010).

    Article  CAS  Google Scholar 

  8. Rabl, J., Leibundgut, M., Ataide, S.F., Haag, A. & Ban, N. Crystal structure of the eukaryotic 40S ribosomal subunit in complex with initiation factor 1. Science 331, 730–736 (2011).

    Article  CAS  Google Scholar 

  9. Klinge, S., Voigts-Hoffmann, F., Leibundgut, M., Arpagaus, S. & Ban, N. Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6. Science 334, 941–948 (2011).

    Article  CAS  Google Scholar 

  10. Tschochner, H. & Hurt, E. Pre-ribosomes on the road from the nucleolus to the cytoplasm. Trends Cell Biol. 13, 255–263 (2003).

    Article  CAS  Google Scholar 

  11. Schäfer, T. et al. Hrr25-dependent phosphorylation state regulates organization of the pre-40S subunit. Nature 441, 651–655 (2006).

    Article  Google Scholar 

  12. Strunk, B.S. et al. Ribosome assembly factors prevent premature translation initiation by 40S assembly intermediates. Science 333, 1449–1453 (2011).

    Article  CAS  Google Scholar 

  13. Nissan, T.A. et al. A pre-ribosome with a tadpole-like structure functions in ATP-dependent maturation of 60S subunits. Mol. Cell 15, 295–301 (2004).

    Article  CAS  Google Scholar 

  14. Ulbrich, C. et al. Mechanochemical removal of ribosome biogenesis factors from nascent 60S ribosomal subunits. Cell 138, 911–922 (2009).

    Article  CAS  Google Scholar 

  15. Ho, J.H., Kallstrom, G. & Johnson, A.W. Nmd3p is a Crm1p-dependent adapter protein for nuclear export of the large ribosomal subunit. J. Cell Biol. 151, 1057–1066 (2000).

    Article  CAS  Google Scholar 

  16. Gadal, O. et al. Nuclear export of 60s ribosomal subunits depends on Xpo1p and requires a nuclear export sequence-containing factor, Nmd3p, that associates with the large subunit protein Rpl10p. Mol. Cell. Biol. 21, 3405–3415 (2001).

    Article  CAS  Google Scholar 

  17. Sengupta, J. et al. Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S ribosomal subunit. J. Cell Biol. 189, 1079–1086 (2010).

    Article  CAS  Google Scholar 

  18. Yao, W. et al. Nuclear export of ribosomal 60S subunits by the general mRNA export receptor Mex67-Mtr2. Mol. Cell 26, 51–62 (2007).

    Article  CAS  Google Scholar 

  19. Yao, W., Lutzmann, M. & Hurt, E. A versatile interaction platform on the Mex67-Mtr2 receptor creates an overlap between mRNA and ribosome export. EMBO J. 27, 6–16 (2008).

    Article  CAS  Google Scholar 

  20. Bradatsch, B. et al. Arx1 functions as an unorthodox nuclear export receptor for the 60S preribosomal subunit. Mol. Cell 27, 767–779 (2007).

    Article  CAS  Google Scholar 

  21. Hung, N.J., Lo, K.Y., Patel, S.S., Helmke, K. & Johnson, A.W. Arx1 is a nuclear export receptor for the 60S ribosomal subunit in yeast. Mol. Biol. Cell 19, 735–744 (2008).

    Article  CAS  Google Scholar 

  22. Yao, Y. et al. Ecm1 is a new pre-ribosomal factor involved in pre-60S particle export. RNA 16, 1007–1017 (2010).

    Article  CAS  Google Scholar 

  23. Oeffinger, M., Dlakic, M. & Tollervey, D. A pre-ribosome-associated HEAT-repeat protein is required for export of both ribosomal subunits. Genes Dev. 18, 196–209 (2004).

    Article  CAS  Google Scholar 

  24. Hackmann, A., Gross, T., Baierlein, C. & Krebber, H. The mRNA export factor Npl3 mediates the nuclear export of large ribosomal subunits. EMBO Rep. 12, 1024–1031 (2011).

    Article  CAS  Google Scholar 

  25. Lebreton, A. et al. A functional network involved in the recycling of nucleocytoplasmic pre-60S factors. J. Cell Biol. 173, 349–360 (2006).

    Article  CAS  Google Scholar 

  26. Nissan, T.A., Bassler, J., Petfalski, E., Tollervey, D. & Hurt, E. 60S pre-ribosome formation viewed from assembly in the nucleolus until export to the cytoplasm. EMBO J. 21, 5539–5547 (2002).

    Article  CAS  Google Scholar 

  27. Ben-Shem, A. et al. The structure of the eukaryotic ribosome at 3.0 A resolution. Science 334, 1524–1529 (2011).

    Article  CAS  Google Scholar 

  28. Kowalinski, E. et al. The crystal structure of Ebp1 reveals a methionine aminopeptidase fold as binding platform for multiple interactions. FEBS Lett. 581, 4450–4454 (2007).

    Article  CAS  Google Scholar 

  29. Monie, T.P. et al. Structural insights into the transcriptional and translational roles of Ebp1. EMBO J. 26, 3936–3944 (2007).

    Article  CAS  Google Scholar 

  30. Gartmann, M. et al. Mechanism of eIF6-mediated inhibition of ribosomal subunit joining. J. Biol. Chem. 285, 14848–14851 (2010).

    Article  CAS  Google Scholar 

  31. Groft, C.M., Beckmann, R., Sali, A. & Burley, S.K. Crystal structures of ribosome anti-association factor IF6. Nat. Struct. Biol. 7, 1156–1164 (2000).

    Article  CAS  Google Scholar 

  32. Lebreton, A., Saveanu, C., Decourty, L., Jacquier, A. & Fromont-Racine, M. Nsa2 is an unstable, conserved factor required for the maturation of 27 SB pre-rRNAs. J. Biol. Chem. 281, 27099–27108 (2006).

    Article  CAS  Google Scholar 

  33. Granneman, S., Petfalski, E. & Tollervey, D. A cluster of ribosome synthesis factors regulate pre-rRNA folding and 5.8S rRNA maturation by the Rat1 exonuclease. EMBO J. 30, 4006–4019 (2011).

    Article  CAS  Google Scholar 

  34. Granneman, S., Petfalski, E., Swiatkowska, A. & Tollervey, D. Cracking pre-40S ribosomal subunit structure by systematic analyses of RNA-protein cross-linking. EMBO J. 29, 2026–2036 (2010).

    Article  CAS  Google Scholar 

  35. Granneman, S., Kudla, G., Petfalski, E. & Tollervey, D. Identification of protein binding sites on U3 snoRNA and pre-rRNA by UV cross-linking and high-throughput analysis of cDNAs. Proc. Natl. Acad. Sci. USA 106, 9613–9618 (2009).

    Article  CAS  Google Scholar 

  36. Beckmann, R. et al. Architecture of the protein-conducting channel associated with the translating 80S ribosome. Cell 107, 361–372 (2001).

    Article  CAS  Google Scholar 

  37. Sweeney, R., Chen, L. & Yao, M.C. An rRNA variable region has an evolutionarily conserved essential role despite sequence divergence. Mol. Cell. Biol. 14, 4203–4215 (1994).

    Article  CAS  Google Scholar 

  38. Wilson, D.N. & Nierhaus, K.H. Ribosomal proteins in the spotlight. Crit. Rev. Biochem. Mol. Biol. 40, 243–267 (2005).

    Article  CAS  Google Scholar 

  39. Kemmler, S., Occhipinti, L., Veisu, M. & Panse, V.G. Yvh1 is required for a late maturation step in the 60S biogenesis pathway. J. Cell Biol. 186, 863–880 (2009).

    Article  CAS  Google Scholar 

  40. Lo, K.Y., Li, Z., Wang, F., Marcotte, E.M. & Johnson, A.W. Ribosome stalk assembly requires the dual-specificity phosphatase Yvh1 for the exchange of Mrt4 with P0. J. Cell Biol. 186, 849–862 (2009).

    Article  CAS  Google Scholar 

  41. Kruiswijk, T., Planta, R.J. & Krop, J.M. The course of the assembly of ribosomal subunits in yeast. Biochim. Biophys. Acta 517, 378–389 (1978).

    Article  CAS  Google Scholar 

  42. Bassler, J., Kallas, M. & Hurt, E. The NUG1 GTPase reveals an N-terminal RNA-binding domain that is essential for association with 60 S pre-ribosomal particles. J. Biol. Chem. 281, 24737–24744 (2006).

    Article  CAS  Google Scholar 

  43. Hung, N.J. & Johnson, A.W. Nuclear recycling of the pre-60S ribosomal subunit-associated factor Arx1 depends on Rei1 in Saccharomyces cerevisiae. Mol. Cell. Biol. 26, 3718–3727 (2006).

    Article  CAS  Google Scholar 

  44. Hedges, J., West, M. & Johnson, A.W. Release of the export adapter, Nmd3p, from the 60S ribosomal subunit requires Rpl10p and the cytoplasmic GTPase Lsg1p. EMBO J. 24, 567–579 (2005).

    Article  CAS  Google Scholar 

  45. Strunk, B.S., Novak, M.N., Young, C.L. & Karbstein, K. A translation-like cycle is a quality control checkpoint for maturing 40S ribosome subunits. Cell 150, 111–121 (2012).

    Article  CAS  Google Scholar 

  46. Thomson, E. & Tollervey, D. The final step in 5.8S rRNA processing is cytoplasmic in Saccharomyces cerevisiae. Mol. Cell. Biol. 30, 976–984 (2010).

    Article  CAS  Google Scholar 

  47. Becker, T. et al. Structure of monomeric yeast and mammalian Sec61 complexes interacting with the translating ribosome. Science 326, 1369–1373 (2009).

    Article  CAS  Google Scholar 

  48. DeLano, W.L. The PyMOL Molecular Graphics System. http://www.pymol.org/ (2002).

  49. Puig, O. et al. The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218–229 (2001).

    Article  CAS  Google Scholar 

  50. 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).

    Article  CAS  Google Scholar 

  51. Ludtke, S.J., Baldwin, P.R. & Chiu, W. EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999).

    Article  CAS  Google Scholar 

  52. van Heel, M., Harauz, G., Orlova, E.V., Schmidt, R. & Schatz, M. A new generation of the IMAGIC image processing system. J. Struct. Biol. 116, 17–24 (1996).

    Article  CAS  Google Scholar 

  53. Radermacher, M., Wagenknecht, T., Verschoor, A. & Frank, J. Three-dimensional reconstruction from a single-exposure, random conical tilt series applied to the 50S ribosomal subunit of Escherichia coli. J. Microsc. 146, 113–136 (1987).

    Article  CAS  Google Scholar 

  54. Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199 (1996).

    Article  CAS  Google Scholar 

  55. Pettersen, E.F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

  56. Wagenknecht, T., Grassucci, R. & Frank, J. Electron microscopy and computer image averaging of ice-embedded large ribosomal subunits from Escherichia coli. J. Mol. Biol. 199, 137–147 (1988).

    Article  CAS  Google Scholar 

  57. Penczek, P.A., Frank, J. & Spahn, C.M. A method of focused classification, based on the bootstrap 3D variance analysis, and its application to EF-G-dependent translocation. J. Struct. Biol. 154, 184–194 (2006).

    Article  CAS  Google Scholar 

  58. Ho, J.H., Kallstrom, G. & Johnson, A.W. Nascent 60S ribosomal subunits enter the free pool bound by Nmd3p. RNA 6, 1625–1634 (2000).

    Article  CAS  Google Scholar 

  59. Santos-Rosa, H. et al. Nuclear mRNA export requires complex formation between Mex67p and Mtr2p at the nuclear pores. Mol. Cell. Biol. 18, 6826–6838 (1998).

    Article  CAS  Google Scholar 

  60. Vilardell, J. & Warner, J.R. Ribosomal protein L32 of Saccharomyces cerevisiae influences both the splicing of its own transcript and the processing of rRNA. Mol. Cell. Biol. 17, 1959–1965 (1997).

    Article  CAS  Google Scholar 

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Acknowledgements

We are very thankful to the European Molecular Biology Laboratory–Heidelberg for providing the electron microscopy facility and computational infrastructure, without which this work would not have been possible, and especially M. Diepholz, C. Blachiere-Batisse, J. Briggs, F. Thommen and M. Wahlers for advice and technical support. The plasmid pFA6a-HTpA-HIS3MX4 was a kind gift of D. Kressler (Unit of Biochemistry, Department of Biology, University of Fribourg, Fribourg, Switzerland). We thank C. Dargemont (Institut Jacques Monod, Universités Paris VI and VII, Centre National de la Recherche Scientifique, Paris, France), A.W. Johnson (Molecular Genetics & Microbiology, University of Texas at Austin, Austin, Texas, USA), A. Lebreton (Unité des Interactions Bactéries-Cellules, Institut Pasteur, Paris, France) and J.R. Warner (Department of Cell Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York, USA) for providing antibodies. This work was supported by grants from the Wellcome Trust (to D.T.) and the Deutsche Forschungsgemeinschaft (DFG Hu363/10-4 to E.H.).

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  1. Sander Granneman & Bettina Böttcher

    Present address: Present addresses: Centre for Synthetic and Systems Biology, University of Edinburgh, Edinburgh, UK (S.G.); School of Biological Sciences, University of Edinburgh, Edinburgh, UK (B. Böttcher).,

  2. Bettina Bradatsch and Christoph Leidig: These authors contributed equally to this work.

Authors and Affiliations

  1. Biochemistry Center, Universität Heidelberg, Heidelberg, Germany

    Bettina Bradatsch, Marén Gnädig & Ed Hurt

  2. Department of Biochemistry, Gene Center and Center for Integrated Protein Science, Ludwig-Maximilians-Universität München, Munich, Germany

    Christoph Leidig & Roland Beckmann

  3. Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, UK

    Sander Granneman & David Tollervey

  4. European Molecular Biology Laboratory, Heidelberg, Germany

    Bettina Böttcher

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Contributions

B. Bradatsch designed and performed the experiments and wrote the manuscript; C.L. performed the cryo-EM analysis, supervised by R.B.; S.G. performed the CRAC experiments and the computational analyses in the laboratory of D.T.; M.G. performed some of the biochemical purifications and growth analysis; B. Böttcher provided know-how and supplied the electron microscopy facility for the negative-stain electron microscopy; E.H. directed the project, designed experiments and wrote the manuscript; all authors contributed to the interpretation of the results and helped write the manuscript.

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Correspondence to Ed Hurt.

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Bradatsch, B., Leidig, C., Granneman, S. et al. Structure of the pre-60S ribosomal subunit with nuclear export factor Arx1 bound at the exit tunnel. Nat Struct Mol Biol 19, 1234–1241 (2012). https://doi.org/10.1038/nsmb.2438

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