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

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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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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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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