Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and ribosomal RNA remodelling events in the nucleolus, nucleoplasm and cytoplasm1,2. Hundreds of assembly factors, organized into sequential functional groups3,4, facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these assembly factors function are largely unknown. Here we use cryo-electron microscopy to characterize the structures of yeast nucleoplasmic pre-60S particles affinity-purified using the epitope-tagged assembly factor Nog2. Our data pinpoint the locations and determine the structures of over 20 assembly factors, which are enriched in two areas: an arc region extending from the central protuberance to the polypeptide tunnel exit, and the domain including the internal transcribed spacer 2 (ITS2) that separates 5.8S and 25S ribosomal RNAs. In particular, two regulatory GTPases, Nog2 and Nog1, act as hub proteins to interact with multiple, distant assembly factors and functional ribosomal RNA elements, manifesting their critical roles in structural remodelling checkpoints and nuclear export. Moreover, our snapshots of compositionally and structurally different pre-60S intermediates provide essential mechanistic details for three major remodelling events before nuclear export: rotation of the 5S ribonucleoprotein, construction of the active centre and ITS2 removal. The rich structural information in our structures provides a framework to dissect molecular roles of diverse assembly factors in eukaryotic ribosome assembly.
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Electron Microscopy Data Bank
Protein Data Bank
The cryo-EM density maps of state 1 and state 2 have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-6615 and EMD-6616, respectively. The atomic model of state 1 has been deposited in the Protein Data Bank (PDB) under accession number 3JCT. The XL–MS data have been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org/) with the dataset identifier PXD003736.
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We thank the National Center for Protein Sciences (Beijing, China) for providing resource for cryo-EM data collection and computation. We also thank members of Woolford laboratory for reading the manuscript. This work was supported by the Ministry of Science and Technology of China (2013CB910404 to N.G. and 2014CB849800 to M.-Q.D.), the National Natural Science Foundation of China (31422016 and 31470722 to N.G., and 21375010 to M.-Q.D.) and National Institutes of Health grant R01GM028301 (to J.L.W.).
The authors declare no competing financial interests.
Extended data figures and tables
a, Mostly non-overlapping assembly factors Nsa1, Nog2 and Nmd3 were used to purify sequential ribosome assembly intermediates. Proteins identified by mass spectrometry analysis were marked on the gel. Orange coloured proteins are only present in Nsa1-TAP particles, green coloured proteins are present both in Nsa1-TAP and in Nog2-TAP particles, light blue coloured proteins are present in all three purified particles to varying levels, dark blue coloured proteins are present only in Nog2-particles, pink coloured proteins are present both in Nog2- and Nmd3-particles in varying levels and yellow coloured proteins are present only in Nmd3-particles. TAP-tagged proteins are indicated by white asterisks. For gel source data, see Supplementary Fig. 1. b, The lifetimes of mostly non-overlapping ribosome assembly intermediates containing assembly factors Nsa1, Nog2 and Nmd3 are indicated. Assembly factors identified in each of Nsa1-TAP, Nog2-TAP and Nmd3-TAP associated samples were colour coded. The colour scheme is identical to that used in a. *Even though this protein was identified in all three intermediates, its levels decreased more than sevenfold from Nsa1-TAP particles to Nog2-TAP particles.
a, Representative 2D class averages of Nog2-particles. b, A flow-chart for 3D classification of Nog2-particles (data batch 8–10, see Methods for details).
a, Representative micrograph of Nog2-particles. b, Local resolution map of the final density map of state 1. c, FSC curve for the final density map (state 1). The nominal resolution is 3.08 Å estimated using the gold-standard (FSC = 0.143) criterion. d, Atomic model cross-validation. Three FSC curves were calculated between the refined model (against Half1 map) and the final map (black), between the refined model with Half1 map (FSCwork, red), and between the refined model with Half2 map (FSCtest, blue) (see Methods for details).
a–l, Cryo-EM densities of representative regions of assembly factors, superimposed with respective atomic models.
a–g, Pairwise illustration of binding partners of Nog2 in the pre-60S particle. Residues of Nog2 involved in atomic contacts are coloured red with residue numbers labelled. H and L denote helix and loop, respectively. h, Interactions between rRNA components (H43, H68, H74, H75, H86, H92, H93) and Nog2. For clarification, H69 and H71 are not shown. The N terminus of Nog2 is located in a helical junction composed of H68, H74, H75 and H93.
a, Nsa2, Nog2 and Nog1 collectively stabilize H89 in a distinct conformation. Nog1 interacts with Nog2 and Nsa2 through its GTPase domain and NTD, respectively. b, The CTE of Nog1 interlocks with Rlp24 by wrapping around a long helix at the C-terminal end of Rlp24 (see also Fig. 3). c, d, Comparison of the CTE of Nog1 and the CTE of Rei1 in the polypeptide tunnel. Atomic models of state 1 (c) and 60S-Arx1–Alb1–Rei1 (d) (PDB accession number 5APN)16 are aligned using the 60S subunit. For clarification, only Arx1, Nog1 and Rei1 are shown. e, Superimposition of c and d. Four major places of steric clash between Rei1 and Nog1 are marked by asterisks.
a, An overall view of the ITS2 subcomplex. b–d, L8 interacts with three factors: Nop15 (b), Cic1 (c) and Nop7 (d). e, L27 interacts with Nop53. f–h, L25 interacts with Rlp7 (f), Nop15 (g) and Nop53 (h). Residues involved in atomic interaction sites are labelled with sequence numbers. H, L, S denote helix, loop and strand of respective structures.
Extended Data Figure 8 Restructuring of rRNA helices in the central protuberance region by Nsa2, Rpf2, Rsa4, Rrs1 and Nog2.
a, Conformation of rRNA helices from the central protuberance (H80, H82-H88, 5S rRNA) in the pre-60S particle (state 1). b, Same as a, but for the mature 60S subunit. The mature 60S subunit was aligned to state 1 structure globally. c–g, Pairwise interactions between the central protuberance helices and factors are shown in separate panels.
a, Cryo-EM density maps of three premature states (1–3) and the mature state are displayed in transparent surface representation, superimposed with models of the 5S RNA, H38 and associated central-protuberance-binding factors. b, Zoom-in views of the central protuberance regions in a. For clarification, only atomic models are shown. Comparison of these four states indicates that the 5S RNP rotates to a near-mature state (state 2) after Rpf2–Rrs1 leave, and further release of Rsa4 in state 3 results in a ‘mature-like’ conformation for the 5S RNP. H38 from these four states is in a series of continuous changes coupled with the 5S RNP conformational maturation. c, d, Spatial relationship of the 5S RNP, H38, Rsa4 and Cgr1 in state 1 (c) and state 2 (d). Note that repositioning of H38 from state 1 to state 2 is coupled with a dramatic conformational change on the C-terminal end of Cgr1. e–h, Additional assembly factors identified in the density map of state 2. One piece of additional density between H38 and L1 contains a characteristic HEAT repeat, which contacts the L1 stalk in an inward position (e). The atomic model of Sda1 (PDB accession number 5FL8)14 fits well with the segmented density (f). For clarification, densities immediately above Sda1 are not shown in e and f. A large piece of additional density in the map of state 2, composed of the Rix1 subcomplex and Rea1 (g, h). The density assignment was facilitated by the cryo-EM structure of Rix1–Rea1 particles14. Superimposition of the atomic model of Rea1 (PDB accession number 5FL8)14 with the segmented density map of Rea1 (h).
This file contains the original gel image for Extended Data Fig. 1a. (PDF 171 kb)
This file shows cross-linked peptides identified in CL-MS data. (XLSX 36 kb)
This file shows the analysis results of CL-MS data. (XLS 1144 kb)
The cryo-EM density (sharpened) is first shown in surface representative, with densities of factors individually colored (as in Fig. 1). The rRNA and r-proteins are colored grey and beige, respectively. Groups of factors are then highlighted in zoom-in views. (MP4 29901 kb)
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Wu, S., Tutuncuoglu, B., Yan, K. et al. Diverse roles of assembly factors revealed by structures of late nuclear pre-60S ribosomes. Nature 534, 133–137 (2016). https://doi.org/10.1038/nature17942
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