Structural basis for the final steps of human 40s ribosome maturation

ABSTRACT Eukaryotic ribosomes consist of a small 40S and a large 60S subunit that are assembled in a highly coordinated manner. More than 200 factors ensure correct modification, processing

and folding of ribosomal RNA and the timely incorporation of ribosomal proteins1,2. Small subunit maturation ends in the cytosol, when the final rRNA precursor, 18S-E, is cleaved at site 3

by the endonuclease NOB13. Previous structures of human 40S precursors have shown that NOB1 is kept in an inactive state by its partner PNO14. The final maturation events, including the

activation of NOB1 for the decisive rRNA-cleavage step and the mechanisms driving the dissociation of the last biogenesis factors have, however, remained unresolved. Here we report five

cryo-electron microscopy structures of human 40S subunit precursors, which describe the compositional and conformational progression during the final steps of 40S assembly. Our structures

explain the central role of RIOK1 in the displacement and dissociation of PNO1, which in turn allows conformational changes and activation of the endonuclease NOB1. In addition, we observe

two factors, eukaryotic translation initiation factor 1A domain-containing protein (EIF1AD) and leucine-rich repeat-containing protein 47 (LRRC47), which bind to late pre-40S particles near

RIOK1 and the central rRNA helix 44. Finally, functional data shows that EIF1AD is required for efficient assembly factor recycling and 18S-E processing. Our results thus enable a detailed

understanding of the last steps in 40S formation in human cells and, in addition, provide evidence for principal differences in small ribosomal subunit formation between humans and the model

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support SIMILAR CONTENT BEING VIEWED BY OTHERS STRUCTURAL SNAPSHOTS OF HUMAN PRE-60S RIBOSOMAL PARTICLES BEFORE AND AFTER NUCLEAR EXPORT Article Open access 15 July 2020 STRUCTURAL INSIGHTS

INTO ASSEMBLY OF THE RIBOSOMAL NASCENT POLYPEPTIDE EXIT TUNNEL Article Open access 09 October 2020 A DISTINCT ASSEMBLY PATHWAY OF THE HUMAN 39S LATE PRE-MITORIBOSOME Article Open access 27

July 2021 DATA AVAILABILITY Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under the accession codes EMD-11517, EMD-11518, EMD-11519, EMD-11520 and EMD-11521.

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references ACKNOWLEDGEMENTS We thank S. Rieder, C. Ungewickell, H. Sieber and A. Gilmozzi for technical assistance, T. Fröhlich for mass-spectrometry analysis and L. Kater, J. Cheng and T.

Becker for discussions and critical comments on the manuscript. This project has received funding from the Deutsche Forschungsgemeinschaft (DFG) through the GRK1721 to R.B., a DFG fellowship

through the QBM (Quantitative Biosciences Munich) graduate school to M.A., from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme

(Grant agreement No. 885711—Human-Ribogenesis) to R.B., and the Swiss National Science Foundation (the NCCR ‘RNA and disease’ and grant 31003A_166565) to U.K. AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Gene Center Munich, Department of Biochemistry, University of Munich, Munich, Germany Michael Ameismeier, Matthias Thoms, Otto Berninghausen & Roland Beckmann * Institute

of Biochemistry, Department of Biology, ETH Zurich, Zurich, Switzerland Ivo Zemp, Jasmin van den Heuvel & Ulrike Kutay * Molecular Life Sciences Ph.D. Program, Zurich, Switzerland

Jasmin van den Heuvel Authors * Michael Ameismeier View author publications You can also search for this author inPubMed Google Scholar * Ivo Zemp View author publications You can also

search for this author inPubMed Google Scholar * Jasmin van den Heuvel View author publications You can also search for this author inPubMed Google Scholar * Matthias Thoms View author

publications You can also search for this author inPubMed Google Scholar * Otto Berninghausen View author publications You can also search for this author inPubMed Google Scholar * Ulrike

Kutay View author publications You can also search for this author inPubMed Google Scholar * Roland Beckmann View author publications You can also search for this author inPubMed Google

Scholar CONTRIBUTIONS M.A., R.B., I.Z., J.v.d.H. and U.K designed the study. M.A. and M.T. generated stable cell lines and purified native complexes. M.A. and M.T. prepared the cryo-EM

samples and O.B. collected cryo-EM data. M.A. processed the cryo-EM data and built the molecular models. For the functional analysis of novel RBFs, I.Z. and J.v.d.H. performed cellular

analyses. I.Z., J.v.d.H. and U.K. analysed the data and interpreted results. All authors interpreted the combined results and wrote the manuscript. CORRESPONDING AUTHOR Correspondence to

Roland Beckmann. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature_ thanks Sebastian Klinge, David

Tollervey and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims

in published maps and institutional affiliations. EXTENDED DATA FIGURES AND TABLES EXTENDED DATA FIG. 1 SAMPLE PREPARATION AND CRYO-EM DATA ANALYSIS. A, SDS–PAGE analysis of native pre-40S

complexes purified with RIOK1(D324) and NOB1(D10N). Identified protein bands are labelled. For gel source data, see Supplementary Fig. 1. B, Representative micrographs from the three data

sets. Scale bar 50 nm C, Subset of 2D averages of extracted particles after initial 2D classification. D, E, Summarized classification scheme of RIOK1(D324A) (D) and NOB1(D10N) (E).

Particles of final states, marked in orange, were subjected to CTF parameter refinement and Bayesian polishing before the last 3D refinement. F, Particles of state G from both NOB1(D10N)

data sets were combined and classified. Final volume is marked in orange. G, Summarized classification scheme of LRRC47. EXTENDED DATA FIG. 2 LOCAL RESOLUTION, REFINEMENT AND MODEL

STATISTICS. A, Local resolution distribution of states F1 – H2 with their respective colour grading scheme as estimated by Relion. B, Fourier shell correlation (FSC) curve for all states.

Average resolution values as stated in Fig. 1 are calculated according to the ‘gold standard’ at FSC = 0.143. C, FSC plot of the models against their volume as provided by Phenix. D, Local

resolution estimation of the ‘head’ region of state F2 after focused refinement. E, F, Model and cryo-EM density of state H1 around the post-transcriptionally modified rRNA residues C1337,

G1490 (E), and A1832 (see ref. 43.) (F). EXTENDED DATA FIG. 3 DETAILS ON RIOK1 BINDING TO THE PRE-40S PARTICLE. A, Superposition of models of RIOK1 and RIOK2 after alignment of state D

(PDB-6G51) and H1 highlight the overlapping binding site at the decoding centre and the rotation of their central RIO domain by approximately 15°. B, Cartoon representation of eL41

(PDB-6G5H), as well as the C terminus of LTV1 (PDB-6G51) and RIOK1 relative to the top of the matured h44 in state H1. Overlaps in binding sites highlight mutually exclusive binding. C,

Models of state F1, F2 and H1 with focus on RIOK1 position. Unaccounted density within the mRNA entry tunnel in states F1 and F2 (yellow) is likely the flexible N terminus of RIOK1, which

overlaps with the two helices of factor X present in state E (marked with a red cross, see ref. 4.). D, Surface representation of state D (PDB-6G51) with RBF TSR1 in cartoon representation.

Models of RIOK1 (left) and LRRC47 (right) of state F1 after alignment of the particles emphasize the overlaps in binding sites around h44. E, Post-transcriptional modification of U1248 leads

to formation of 1-methyl-3-α-amino-α-carboxypropyl pseudouridine (m1acp3Ψ). Modifying enzymes and their contribution to the structure are indicated by colours. F, Cartoon representation

showing the coordination of m1acp3Ψ1248 by RIOK1. EXTENDED DATA FIG. 4 STRUCTURAL DETAILS OF NOVEL FACTORS LRRC47 AND EIF1AD. A, Overall structure of the two domains of LRRC47. Leucine

residues, secondary structure elements and position of h44 are highlighted. B, Low-resolution cryo-EM reconstruction of a sample using N-terminally tagged LRRC47 (left) and state H1 filtered

at 7 Å (right). The leucine-rich domain of LRRC47 (blue) binds simultaneously with TSR1, while the C-terminal domain remains delocalized and is therefore not visible. C, Models of LRRC47,

h44 and parts of TSR1 show the conformational changes in h44 that accompany the transition between the states. A central part of h44 moves after release of TSR1, enabled by the lack of its

N-terminal helix (left). The B3/4 domain of LRRC47 would clash with both displayed helices of TSR1. LRRC47 continues to bind in an almost unchanged position after maturation of h44 (right).

D, Structure of EIF1AD with its N-terminal helix and residues N36, R58, K59 and W62 labelled. E, Cartoon representation of rRNA segments and ribosomal proteins surrounding EIF1AD in state

H1. Model of yeast eIF1A (PDB-6GSN) after alignment of a pre-48S translation initiation complex to the pre-40S particle shows a shifted binding location. F, Sequence alignment of human

EIF1AD and eIF1A. Conserved residues that bind to rRNA are coloured blue and the IDDI motif of eIF1A in red. The conserved C-terminal stretch that binds to uS13 and uS19 is marked with a

blue box. G, Model of state H1 with cryo-EM volume of EIF1AD and eS25 Gaussian filtered at 1.5 standard deviations. Additional density extends from well resolved parts of the C terminus of

EIF1AD. Detailed views on eS25 N terminus and EIF1AD C terminus are shown in boxes A and B. EXTENDED DATA FIG. 5 EIF1AD BUT NOT LRRC47 DEPLETION AFFECTS LATE 40S SUBUNIT MATURATION. A,

Western blot analysis of the experiment shown in Fig. 4 confirming the effectiveness of siRNA treatments for EIF1AD and RIOK2. B, Western blot analysis for the experiment in C confirming the

depletion of LRRC47 or RIOK2 upon siRNA treatment. For gel source data of A and B, see Supplementary Fig. 1. C, Immunofluorescence analysis of HeLa cells treated with siRNAs against LRRC47

or RIOK2 using antibodies against the indicated RBFs. For immunofluorescence analysis of NOB1, cells were treated with 20 nM leptomycin B (LMB) for 90 min. Note that only RIOK2 but not

LRRC47 depletion leads to cytoplasmic recycling defects of the tested RBFs. Scale bar, 20 μm. D, FISH analysis of experiment in B, revealing cytoplasmic accumulation of 18S-E pre-rRNA upon

RIOK2 but not LRRC47 depletion. FISH pictures were processed in parallel, using a gamma correction of 1.5. All experiments were done in triplicates (_n_ = 3). EXTENDED DATA FIG. 6 STRUCTURAL

DETAILS OF RRNA 3′END MATURATION. A, Cartoon representation of the 3′ end of 18S-E rRNA with PNO1, NOB1 and eS26 throughout the maturation process in states F2, G and H1. Panel of eS26 with

18S rRNA in state H1 has been shifted slightly upwards as indicated by the line to the right. B, Detailed view of NOB1(D10N) active site with its substrate. Dashed circle marks site 3

cleavage site. Electron density around ITS1 and the 3′end shown in blue. C, Stick representation of NOB1 residues that interact with the ITS1 and 3′ end with electron density shown in blue.

SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE Supplementary Figure 1 This file contains a figure with the uncropped SDS-PAGE and western blot images. Related to Extended Data Figs. 1 and 5.

REPORTING SUMMARY RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Ameismeier, M., Zemp, I., van den Heuvel, J. _et al._ Structural basis for the final

steps of human 40S ribosome maturation. _Nature_ 587, 683–687 (2020). https://doi.org/10.1038/s41586-020-2929-x Download citation * Received: 14 October 2019 * Accepted: 28 August 2020 *

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