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Architecture of the Rix1–Rea1 checkpoint machinery during pre-60S-ribosome remodeling

Nature Structural & Molecular Biology volume 23, pages 37–44 (2016)Cite this article

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Abstract

Ribosome synthesis is catalyzed by ∼200 assembly factors, which facilitate efficient production of mature ribosomes. Here, we determined the cryo-EM structure of a Saccharomyces cerevisiae nucleoplasmic pre-60S particle containing the dynein-related 550-kDa Rea1 AAA+ ATPase and the Rix1 subcomplex. This particle differs from its preceding state, the early Arx1 particle, by two massive structural rearrangements: an ∼180° rotation of the 5S ribonucleoprotein complex and the central protuberance (CP) rRNA helices, and the removal of the 'foot' structure from the 3′ end of the 5.8S rRNA. Progression from the Arx1 to the Rix1 particle was blocked by mutational perturbation of the Rix1-Rea1 interaction but not by a dominant-lethal Rea1 AAA+ ATPase-ring mutant. After remodeling, the Rix1 subcomplex and Rea1 become suitably positioned to sense correct structural maturation of the CP, which allows unidirectional progression toward mature ribosomes.

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Figure 1: Interaction of the Rea1 AAA+ ATPase ring with Rix1.
Figure 2: Characterization of the Rix1–Ipi1ΔN50–Ipi3 complex and its interaction with Rea1.
Figure 3: Cryo-EM structure of the Rix1–Rea1 pre-60S particle.
Figure 4: Identification and interactions of biogenesis factors.
Figure 5: Comparison of the Rix1–Rea1 and Arx1 pre-60S particles with the mature 60S subunit.
Figure 6: Cryo-EM structures of pre-60S particles isolated from Rea1 and Rix1 mutants.
Figure 7: Model of 60S biogenesis occurring during the combined remodeling-checkpoint steps on intermediate nucleoplasmic pre-60S particles.

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Acknowledgements

We thank C. Leidig and C. Ungewickell for assistance with cryo-EM and C. Ulbrich (Heidelberg University Biochemistry Center) for providing plasmids. We are grateful to M. Fromont-Racine (Institut Pasteur), M. Remacha (Centro de Biologia Molecular Severo Ochoa), M. Seedorf (Zentrum für Molekulare Biologie der Universität Heidelberg), B. Stillman (Cold Spring Harbor Laboratory), J.R. Warner (Albert Einstein College of Medicine) and D.H. Wolf (University of Stuttgart) for antibodies. This work was supported by grants from the German Research Council (GRK 1721, FOR 1805 and SFB646 to R.B.; HU363/10-5 and HU363/12-1 to E.H.; and BA2316/1-4 to J.B.). R.B. acknowledges support from the Center for Integrated Protein Science (CiPS-M) and the European Research Council (Advanced grant CRYOTRANSLATION). C.B.-G. and M.T. were supported by the Graduiertenkolleg GRK1721 and GRK1188, respectively.

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Author notes

  1. Clara Barrio-Garcia and Matthias Thoms: These authors contributed equally to this work.

Authors and Affiliations

  1. Gene Center, University of Munich, Munich, Germany

    Clara Barrio-Garcia, Lukas Kater, Otto Berninghausen & Roland Beckmann

  2. Heidelberg University Biochemistry Center, Heidelberg, Germany

    Matthias Thoms, Dirk Flemming, Jochen Baßler & Ed Hurt

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  1. Clara Barrio-Garcia
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Contributions

M.T., R.B., E.H. and C.B.-G. designed the study. M.T. generated strains and plasmids, performed experiments and purified samples for negative-stain and cryo-EM. J.B. and M.T. analyzed the XL-MS data. D.F. performed the negative-stain EM of the Rix1–Ipi1ΔN50–Ipi3 complex. O.B. collected the cryo-EM data, and C.B.-G. and L.K. processed the data. C.B.-G. built the models and, together with M.T., E.H. and R.B., analyzed the structures. C.B.-G., M.T., E.H. and R.B. interpreted results and wrote the paper.

Corresponding authors

Correspondence to Roland Beckmann or Ed Hurt.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Interaction of the Rea1 AAA+ ATPase ring with Rix1.

(a) Comprehensive yeast 2-hybrid analysis using as bait a Rea1 AAA+ ATPase ring construct (amino acids 1-2372) fused to GAL4 BD (binding domain) and the indicated ribosome assembly factors fused to GAL4 AD (activation domain). Cells were spotted in ten-fold serial dilutions on SDC-Leu-Trp (SDC) and SDC-Leu-Trp-His (SDC-His) plates. Growth was monitored after 3 days at 30°C. (b) Affinity-purification of Rea1 wild-type and Rea1 mutants, either lacking the helix 2 insertion in D2 or D4 (see Fig. 1a, b and c). Final eluates were analyzed by SDS-PAGE and Coomassie staining or western blotting using the indicated antibodies. (c) Growth of the RIX1-HA-aid degron strain on YPD (Rix1 expression) or YPD supplemented with 500 µM auxin (Rix1 degradation). Cells were spotted in ten-fold serial dilutions and grown at 30°C. Western blot analysis of the RIX1-HA-aid strain, revealing efficient Rix1-HA-aid depletion. Auxin was added to the medium (500 μM final concentration) and Rix1 depletion was followed using an anti-HA antibody. Arc1 served as a loading control (see Fig. 2b).

Supplementary Figure 2 Multiple sequence alignment of Rea1.

Multiple sequence alignment of Rea1 showing the helix 2 insertion motifs in D2, D4 and the D6 domain (see also Fig. 1a, b and c). The sequences of Saccharomyces cerevisiae (Sc, Q12019), Schizosaccharomyces pombe (Sp, O94248), Kluyveromyces lactis (Kl, Q6CJB6), Chaetomium thermophilum (Ct, G0SHE6), Arabidopsis thaliana (At, F4HRR8), Drosophila melanogaster (Dm, A8DYB0), Mus musculus (Mm, J3QMC5) and Homo sapiens (Hs, Q9NU22) were aligned using ClustalW2 and Jalview. Abbreviations and Uniprot entries are indicated in brackets. The insertions are highlighted above the alignment.

Supplementary Figure 3 Negative-stain EM and biochemical characterization of the Rix1 subcomplex.

(a) Size exclusion chromatography of purified Rix1-Ipi1-Ipi3 complex (red) and Rix1-Ipi1∆N50-Ipi3 complex (blue). The absorbance at 280 nm was plotted against the elution volume. The grey area marks the fractions analyzed by SDS-PAGE and Coomassie staining (lane 2-17). The asterisk marks an N-terminal truncation of Ipi1. (b) Upper panel: representative electron micrograph of the Rix1-Ipi1∆N50-Ipi3 complex stained with uranyl acetate (100-nm scale bar). Lower panel: selected class averages, reprojections and the corresponding 3D reconstructions (10-nm scale bar). (c) Pre-60S particles affinity-purified via FTpA-tagged Ipi1, Ipi3 or Rix1 from yeast strains either expressing (lane 2, 4, 6) or not expressing (lane 1, 3, 5) an extra copy of the respective plasmid-based GFP-tagged Ipi1, Ipi3 or Rix1. The final eluates were analyzed by SDS-PAGE and Coomassie-staining (upper panel), or western blotting using the indicated antibodies (lower panel). Bait proteins are marked with an asterisk, bands corresponding to Ipi3-GFP and Rix1-GFP are labeled with a dot. A signal in the Anti-GFP blot indicates the presence of a second copy in the affinity-purified complex. Whole cell lysates (WCL) used for the various affinity-purifications were also analyzed by western blotting using the indicated antibodies (right panel). The Anti-pA (ProtA) antibody detects the FTpA-tagged bait protein, and the Anti-GFP antibody shows the expression of the second copy. The Anti-Arc1 antibody detects equal loading of lysates.

Supplementary Figure 4 Analysis of the interaction network within the Rix1–Ipi1–Ipi3 complex.

(a) Rix1-Ipi1∆N50-Ipi3 complex incubated with increasing concentrations of isotopic labeled DSS cross-linker. The cross-linked complex was analyzed by SDS-PAGE, Coomassie staining and mass spectrometry (boxed bands). MS data was analyzed by xProphet and xQuest. Inter protein-protein cross-links (score > 19) are displayed in orange, intra protein cross-links are shown in purple. A representative cross-link of two identical Ipi3 peptides is shown. Proteins are colored according to Fig. 1a and 2e. (b), SEC-MALS analysis of Ipi3 wild-type and Ipi3∆cc lacking the C-terminal 51 amino acids, for which a coiled-coil (cc) helix is predicted. The differential refraction index (dRI) and the molecular weight (Mw) were plotted against the elution volume. The grey area marks fractions analyzed by SDS-PAGE and Coomassie staining. Ipi3 wild-type and Ipi3∆cc are shown in blue and red, respectively. (c) Yeast 2-hybrid interactions between Rix1 and Ipi3. Constructs were N or C-terminal fused to GAL4 DNA-binding domain (GAL4-BD) or GAL4 activation domain (GAL4-AD), the orientation is indicated as e.g. BDC or BDN for C or N-terminal respectively. (d) Multiple sequence alignment of Ipi3 showing the C-terminal region. The sequences of Saccharomyces cerevisiae (Sc, P53877), Kluyveromyces lactis (Kl, Q6CRK4), Yarrowia lipolytica (Yl, Q6C953), Schizosaccharomyces pombe (Sp, Q10272), Chaetomium thermophilum (Ct, G0S1T5), Xenopus laevis (Xl, Q4QR01), Mus musculus (Mm, Q4VBE8) and Homo sapiens (Hs, Q9BV38) were aligned using Clustal W2 and Jalview. Abbreviations and Uniprot entries are shown in brackets. The minimal Rix1-interacting sequence is marked underneath the alignment (see Supplementary Fig. 4c). The predicted coiled-coil helix is indicated in red and the Ipi3Δcc truncation above the alignment.

Supplementary Figure 5 In silico sorting scheme of the pre-60S particles.

The particles obtained for the Rix1-Rea1 data (a), Rix1-Rea1-K1089A mutant (b), and Rix1∆C mutant (c), were 3D classified using iterative multi-reference projection alignment. The Rix1-Rea1 and Rix1-Rea1-K1089A mutant data gave rise to classes mostly differing on factor presence. The classes enriched for the Rix1 subcomplex and Rea1 (10% of the data in the case of the Rix1-Rea1 particle and 8% in the case of the Rix1-Rea1-K1089A) were refined to the final volume. In the case of the Rix1∆C mutant, particles were sorted into 3 sub-populations, 2 of them containing an unrotated 5S RNP (left and middle), and the third subpopulation, representing 8% of the initial amount of particles, showing a rotated 5S RNP (right). The most populated class (39% of the data) was refined to the final volume.

Supplementary Figure 6 Characterization of the Rsa4 UBL domain–Rea1 interaction and depletion of the heat-repeat protein Sda1 from yeast cells.

(a) Growth analysis of a GAL1-3xHA-SDA1 depletion strain on YPD (glucose; repressed) and YPG (galactose; induced) plates. Cells were spotted in ten-fold serial dilutions on the indicated plates and growth was monitored after 2 days at 30°C. Western blot analysis of the 3xHA tagged Sda1 in glucose and galactose medium at the indicated time points. Antibody against Arc1 was used to verify equal loading (lower panel). (b) GAL1-3xHA SDA1 strains with either TAP-Flag tagged Rsa4 or FTpA tagged Rix1 were grown in YPG (galactose) or shifted for 6h to YPD (glucose) medium. Eluates from the indicated affinity-purifications were analyzed by SDS-PAGE and Coomassie staining or western blotting using the indicated antibodies. (c) Rix1-FTpA affinity-purifications using either endogenous Rsa4 wild-type (lane1) or overexpressed GFP tagged Rsa4 wild-type, Rsa4ΔUBL or Rsa4 E114D (lane 2-4). Eluates were analyzed with SDS-PAGE and Coomassie staining or western blotting using the indicated antibodies. Protein lanes corresponding to overexpressed GFP-tagged Rsa4 alleles are marked with a black dot. (d) Wild-type yeast strain transformed with plasmids overexpressing the N-terminal GFP tagged RSA4 wild-type, the lethal rsa4 ΔUBL or rsa4 E114D alleles under control of the GAL1-10 promoter. Cells were spotted in 10-fold serial dilutions and growth was monitored on SDC-Trp (glucose) and SGC-Trp (galactose) plates.

Supplementary Figure 7 Pre-60S particles isolated from mutant yeast strains and used for cryo-EM.

(a, b) Affinity-purified pre-60S particles: Rix1-Rea1-K1089A mutant (a) and Rix1∆C mutant (b) were analyzed by SDS-PAGE/Coomassie staining (major bands are indicated) and used for cryo-EM analysis (left). Cryo-EM reconstruction particles (middle) and the same maps colored according to their local resolution (right) are shown.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1–4 (PDF 2480 kb)

Supplementary Data Set 1

Uncropped gels and western blots (PDF 7204 kb)

Central protuberance maturation mechanism

Remodeling steps occurring between the Arx1 and Rix1–Rea1 particle, related to Figure 7 (MP4 24786 kb)

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Barrio-Garcia, C., Thoms, M., Flemming, D. et al. Architecture of the Rix1–Rea1 checkpoint machinery during pre-60S-ribosome remodeling. Nat Struct Mol Biol 23, 37–44 (2016). https://doi.org/10.1038/nsmb.3132

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