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Modular assembly of the nucleolar pre-60S ribosomal subunit


Early co-transcriptional events during eukaryotic ribosome assembly result in the formation of precursors of the small (40S) and large (60S) ribosomal subunits1. A multitude of transient assembly factors regulate and chaperone the systematic folding of pre-ribosomal RNA subdomains. However, owing to a lack of structural information, the role of these factors during early nucleolar 60S assembly is not fully understood. Here we report cryo-electron microscopy (cryo-EM) reconstructions of the nucleolar pre-60S ribosomal subunit in different conformational states at resolutions of up to 3.4 Å. These reconstructions reveal how steric hindrance and molecular mimicry are used to prevent both premature folding states and binding of later factors. This is accomplished by the concerted activity of 21 ribosome assembly factors that stabilize and remodel pre-ribosomal RNA and ribosomal proteins. Among these factors, three Brix-domain proteins and their binding partners form a ring-like structure at ribosomal RNA (rRNA) domain boundaries to support the architecture of the maturing particle. The existence of mutually exclusive conformations of these pre-60S particles suggests that the formation of the polypeptide exit tunnel is achieved through different folding pathways during subsequent stages of ribosome assembly. These structures rationalize previous genetic and biochemical data and highlight the mechanisms that drive eukaryotic ribosome assembly in a unidirectional manner.

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Figure 1: Structure of the early nucleolar pre-60S particle.
Figure 2: A ring of nucleolar assembly factors prevents premature folding of the 25S rRNA.
Figure 3: Molecular mimicry by Erb1 prevents premature ITS2 processing.
Figure 4: Model of early nucleolar stages of large subunit assembly.

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We thank M. Ebrahim and J. Sotiris for their support with data collection at the Evelyn Gruss Lipper Cryo-EM resource center, M. Tesic-Mark for analysis of mass spectrometry data, C. Cheng for help with the initial manual curation and analysis of the nucleolar pre-60S particles and members of the Walz laboratory for helpful discussions. L.M. is supported in part by NIH T32 GM115327-Tan. J.B. is supported by an EMBO long-term fellowship (ALTF 51-2014) and a Swiss National Science Foundation fellowship (155515). M.C.-M. is supported by a postgraduate scholarship from NSERC. S.K. is supported by the Robertson Foundation, the Irma T. Hirschl Trust, the Alexandrine and Alexander L. Sinsheimer Fund, the Rita Allen Foundation and an NIH New Innovator Award (1DP2GM123459). B.T.C. is supported by National Institute of Health Grant Nos. P41GM103314 and P41GM109824.

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Authors and Affiliations



S.K. and Z.A.S. established purification conditions. Z.A.S., L.M. and S.K. determined the cryo-EM structure of the yeast nucleolar pre-60S particle. K.R.M., J.W. and B.T.C. processed and analysed DSS cross-linking data. Z.A.S., L.M., J.B., M.C.-M., M.H. and S.K. built the model. L.M. performed all RNA work, and Z.A.S., L.M., J.B., M.H., M.C.-M. and S.K. interpreted the results and wrote the manuscript.

Corresponding author

Correspondence to Sebastian Klinge.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks A. Amunts, D. Tollervey and J. Woolford for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Purification of tagged Nsa1–Nop2 nucleolar pre-60S particles and analysis of RNA components.

a, Schematic of tandem-affinity purification of the nucleolar pre-60S particle with tagged proteins Nsa1 and Nop2. b, Coomassie-blue stained SDS–PAGE of pre-60S particles purified as in a. Protein labels are based on in-solution mass spectrometry analysis of purified pre-60S particles and the approximate molecular weight. c, Schematic processing of the large ribosomal subunit rRNAs in yeast. The locations of the previously published pre-60S particles (the Nog2 particles2, the Arx1 particle35, the Rix1–Rea1 particle3 and the Nmd3 particle4) are represented by blue bars. Binding sites of northern blot probes are indicated on the 35S and pre-5S transcript. d, e, Pre-rRNA was visualized on an agarose gel and stained using SYBR Green II. Northern blot analysis was performed for the 25S, ITS2, ITS1 and 3′ ETS RNAs (d) and for the 5S rRNA (e).

Extended Data Figure 2 Cryo-EM data-processing workflow.

Four data collections were performed, resulting in 14,201 micrographs. These micrographs were aligned using MotionCor2 (ref. 21) with dose weighting, and imported into Relion2.1 (ref. 23) for further processing. Autopicking followed by manual cleaning, 2D and 3D classification produced a total of 514,746 ‘good’ particles. These particles were refined to produce the core map. Further 3D classification without and with alignment was used to obtain the state 1, state 2, state 2A and state 3 maps. Density regions corresponding to domain III (red), domain VI (blue) and Mak11 (green) are highlighted.

Extended Data Figure 3 Overall and local resolution estimates for core, state 2 and state 3 cryo-EM maps.

ad, Overall and local resolution of core map at 3.4 Å (a), state 2 map at 3.7 Å (b), state 2A map with additional density for Mak11 at 4.2 Å (c) and state 3 map at 4.6 Å (d). Fourier Shell Correlation (FSC) curves for the unmasked (dashed black line), phase-randomized (solid grey line), masked (dashed grey line) and the corrected map (solid black line) are also shown. A thin black line indicates an FSC value of 0.143. A clipped view is shown next to two views of the obtained cryo-EM maps. The density volumes are coloured according to local resolution using Resmap25.

Extended Data Figure 4 Cryo-EM density fit of selected nucleolar pre-60S ribosomal subunit proteins and RNA models.

a, Near-atomic models of assembly factors and their cryo-EM density. b, Selected regions of the 25S rRNA and 5.8S rRNA models and their cryo-EM density. Images generated in PyMOL or Chimera.

Extended Data Figure 5 rRNA domains of state 2, state 3 and the mature 60S ribosomal subunit.

The 5.8S rRNA, the 5S rRNA and the domains of the 25S rRNA are colour-coded and displayed in the crown and back view for each structure.

Extended Data Figure 6 Ribosomal proteins associated with Diamond–Blackfan anaemia are positioned at rRNA domain junctions in the nucleolar pre-60S particle.

a, Two views of the nucleolar pre-60S particle state 2 model, with Diamond–Blackfan anaemia-associated ribosomal proteins shown in surface representation. b, Rpl15 is located between the 5.8S-domain I duplex and a domain I–domain II interface. c, Rpl33 (Rpl35 in Homo sapiens) binds at the junction of domains I, II and VI in the 25S rRNA. d, Rpl26 associates with the domain I–5.8S rRNA interface and additionally inserts its N terminus (N) between domain I and domain II.

Extended Data Figure 7 Intermediates of nucleolar pre-60S assembly.

The structural data presented in this paper (states 1, 2, and 3) are complemented by recent data on pre-60S assembly: states C (EMD-3893) and E (EMD-3891)16. State 1 is highly similar to state A (EMD-3888)16. States 2 and 2A correspond closely to state B (EMD-3889)16, but state B lacks the Ssf1–Rrp15–Rrp14 module and Mak11. Built assembly factors that become ordered or leave in subsequent particles are indicated with arrows. Two possible pathways are shown that result in the final incorporation of the DEAD-box helicase Spb4 (previously unidentified16).

Extended Data Figure 8 Steric hindrance during nucleolar pre-60S assembly.

a, b, Comparative views of state 2 (a) and state E (PDB 6ELZ)16 (b), highlighting that the binding of the Brx1 CTD to Rrp1 prevents premature incorporation of Rpl28. c, d, Comparative views of state E (c) and the Nog2 particle (PDB 3JCT) (d), highlighting that the presence of Spb1 prevents the binding of Bud20.

Extended Data Table 1 Molecular models of the nucleolar pre-60S ribosomal subunit

Supplementary information

Life Sciences Reporting Summary (PDF 74 kb)

Supplementary Figure 1

Source data for the RNA gel and northern blots showing the uncropped images for Extended Data Figure 1d and e. (PDF 882 kb)

Supplementary Data 1

DSS cross-links for the nucleolar pre-60S ribosomal subunit. (CSV 35 kb)

Supplementary Data 2

PyMOL session file for the structural analysis of the nucleolar pre-60S ribosomal subunit. (ZIP 5170 kb)

360° view of the cryo-EM reconstruction of the S. cerevisiae nucleolar pre-60S particle

A 360° rotation of a composite cryo-EM map consisting of the 3.4 Å state 1 and the 3.7 Å state 2 maps. Densities for nucleolar pre-60S assembly factors and rRNA domains are colour-coded as in Figure 1 and ribosomal proteins are colored in grey. The rotation is paused at front and back view as shown in Figure 1. (MP4 20291 kb)

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Sanghai, Z., Miller, L., Molloy, K. et al. Modular assembly of the nucleolar pre-60S ribosomal subunit. Nature 556, 126–129 (2018).

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