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Sequence-specific remodeling of a topologically complex RNP substrate by Spb4

Nature Structural & Molecular Biology volume 29, pages 1228–1238 (2022)Cite this article

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Abstract

DEAD-box ATPases are ubiquitous enzymes essential in all aspects of RNA biology. However, the limited in vitro catalytic activities described for these enzymes are at odds with their complex cellular roles, most notably in driving large-scale RNA remodeling steps during the assembly of ribonucleoproteins (RNPs). We describe cryo-EM structures of 60S ribosomal biogenesis intermediates that reveal how context-specific RNA unwinding by the DEAD-box ATPase Spb4 results in extensive, sequence-specific remodeling of rRNA secondary structure. Multiple cis and trans interactions stabilize Spb4 in a post-catalytic, high-energy intermediate that drives the organization of the three-way junction at the base of rRNA domain IV. This mechanism explains how limited strand separation by DEAD-box ATPases is leveraged to provide non-equilibrium directionality and ensure efficient and accurate RNP assembly.

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Fig. 1: Cryo-EM reconstructions of late nucleolar/early nucleoplasmic pre-60S particles.
Fig. 2: Sequence-specific helix remodeling by Spb4.
Fig. 3: Cis and trans elements modulate Spb4-directed helix remodeling.
Fig. 4: ATP binding and hydrolysis by Spb4 are essential for the formation of the high-energy intermediate.
Fig. 5: The Spb4 high-energy intermediate drives the assembly of the three-way junction at the base of rRNA domain IV.
Fig. 6: Model of DEAD-box ATPase function and kinetic proofreading of ribosome assembly.

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Data availability

The cryo-EM density maps and models have been deposited in EMDB and PDB with accession codes: EMD-24296/PDB-7R7A (E1 overall map/model), EMD-24290/PDB-7R72 (E1 Spb4 local map/model), EMD-24269/PDB-7NAC (E2 Overall map/model), EMD-24270/PDB-7NAD (E2 Spb4 local map/model), EMD-24271/PDB-7NAF (E2 Spb1 local map/model), EMD-24280/PDB-7R6K (E2 Noc2/Noc3 local map/model), EMD-24297/PDB-7R7C (E2 L1 stalk local map/model), EMD-24286/PDB-7R6Q (E2 foot local map/model), EMD-26259/PDB-7U0H (NE1 Overall map/model). Source data are provided with this paper.

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Acknowledgements

We thank M. Rosen, L. Rice, J. Berger and X. Bai for discussions and M. Parker for help with live-cell imaging. We thank D. Stoddard at the UTSW Cryo-Electron Microscopy Facility, funded in part by the CPRIT Core Facility Support Award RP170644, and J. Chen at the UTSW Structural Biology Lab. A portion of this research was supported by NIH grant U24GM129547 and performed at the PNCC at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research, with the assistance of T. Humphreys. F. S. was supported by funding from the German Research Foundation (STE 2517/1 and STE 2517/5-1) and its Collaborative Research Centre 969 (Project A06). J. P. E. was supported by the Cancer Prevention and Research Institute of Texas (RR150074), the Welch Foundation (I-1897), the UTSW Endowed Scholars Fund and the National Institutes of Health (GM135617-01).

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Author notes

  1. Carolin Sailer

    Present address: Department of Biomedical Sciences, University of Copenhagen, København, Denmark

  2. Sahana Balasubramanian

    Present address: Cell Biology & Molecular Physiology Department, University of Pittsburgh, Pittsburgh, PA, USA

Authors and Affiliations

  1. Department of Biophysics, UT Southwestern Medical Center - ND10.124B, Dallas, TX, USA

    Victor Emmanuel Cruz, Kamil Sekulski, Nagesh Peddada, Sahana Balasubramanian, Christine S. Weirich & Jan P. Erzberger

  2. Department of Biology, University of Konstanz, Konstanz, Germany

    Carolin Sailer & Florian Stengel

  3. Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany

    Carolin Sailer & Florian Stengel

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Contributions

V. E. C., C. S. W and J. P. E. conceived the project. V. E. C., K. S., C. S. W., N. P. and S. B. constructed strains. V. E. C. and C. S. W. conducted all growth and microscopy assays. C. S. and F. S. provided key proteomics resources. V. E. C., K. S. and N. P. carried out sample preparations and collected cryo-EM data. V. E. C., K. S. and J. P. E. processed the cryo-EM data. The paper was written by J. P. E. and V. E. C. and edited by V. E. C., C. S. W. and J. P. E. with input from all authors. J. P. E. and F.S. supervised the work.

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Correspondence to Jan P. Erzberger.

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Nature Structural & Molecular Biology thanks Ben Luisi, David Tollervey, John Woolford and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling editor: Carolina Perdigoto, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Position of the domain IV elements engaged by Spb4 within the secondary structure of the mature 25 S rRNA.

Secondary structure diagram of the mature 25S and 5.8S rRNA, showing the major subdomains with their root helices highlighted and labeled. The region in domain IV that is remodeled by Spb4 is boxed and rRNA helices described in the text are labeled and colored as in Fig. 2.

Extended Data Fig. 2 Purified samples of Ytm1∆UBL/Spb4 and of Nop53/Spb1 particles used for Cryo-EM single particle reconstruction.

Coomassie-stained 4−20% SDS−PAGE Gel of purified (a) Ytm1∆UBL/Spb4WT or (b) Nop53/Spb1 pre−ribosomal intermediates. Ytm1∆UBL/Spb4WT is was independently replicated four times while the Nop53/Spb1 was replicated twice. Protein identification was determined based on molecular weight, ambiguous bands were identified by mass spectrometry. Asterisks indicate proteins that have been tagged and therefore run higher than expected.

Source data

Extended Data Fig. 3 Micrographs, 2D classes and Fourier shell correlation (FSC) curves for reconstructions and model validation.

a, Representative electron micrograph from a total of 5122 micrographs and 2D classes of the Ytm1∆UBL/Spb4WT sample that yielded States E1 and E2. b, FSC curves for the overall and Spb4-focused maps corresponding to State E1. c, d, Local resolution filtered maps (left) and model-to-map FSC curves for each half map and full map (right) for the overall and Spb4-focused local map for State E1. e, FSC curves for the Overall, Spb4-focused and Spb1-focused map from State E2 (left). FSC curves for the peripheral maps focused on the foot, Noc2 and the L1-stalk from State E2 (right). f−k, Local resolution filtered maps (left) and model-to-map FSC curves for each half map and full map (right) for the overall and local maps corresponding to State E2. l, Representative electron micrograph from a total of 6978 micrographs and 2D classes of the Spb1/Nop53 sample that yielded State NE1. m, FSC curves for the Overall map corresponding to State NE1. n, Local resolution filtered map (left) and model-to-map FSC curves for each half map and full map (right) for the overall map corresponding to State NE1.

Extended Data Fig. 4 Data processing, classification and sorting scheme for State E1 and State E2.

Workflow of the data processing strategy after 2D classification. Map volumes and masks (red) are shown for all critical steps. Major sorting and classification criteria, particle numbers and percentages (for each 3D classification) as well as final map resolutions are indicated. Mask selection for local refinement were chosen based on boundaries with a perceived map quality drop-off was observed. Some mask boundaries were adjusted by trial and error, allowing for the best local maps to be identified for model building and refinement.

Extended Data Fig. 5 Interactions of the biogenesis factors Loc1, Noc2 and Rrp17 in state E2 and of the newly modeled helix of Nop53 in state NE1.

a, Cartoon representation of the interaction between Rrp17 (red), Spb1-MTD (teal) and H64 (blue). A plot of residue conservation scores as implemented in Jalview66 from on an alignment of 15 representative fungal Rrp17 homologs is shown below the model. Residues with an arbitrarily chosen conservation score >7 are highlighted and shown as sticks in the structural model. b, Cartoon representation of the interaction between Rrp17 (red), eL30 (green), Spb4-D1 (yellow), the Spb1 helical element (teal) and H34 (gray). Conserved residues (identified in plot) are shown as sticks (same criteria as in panel a). c, Cartoon representation of the interaction between the C-terminal helix of Rrp17 (red), Spb1-MTD (teal) and H64 (blue). Adjacent rRNA helices H90 (green) and H92 (orange) are also shown. Conserved residues (identified in plot) are shown as sticks (same criteria as in panel A). d, Detail of the position and interactions of Loc1 within the state E2 60S particle. The cartoon representation shows the interaction of Loc1 (purple) with various RBFs and rRNA elements. Model building and validation was aided by a crosslink between Loc1 and Brx1 (green connected spheres)31. e, Cartoon representation of the Noc2 (green)/Noc3(blue) interaction highlighting the conserved Noc module (orange) of Noc3 and the extended N-terminus of Noc2, which extends into the 60 S core (inset). Positions of crosslinks between Noc2 and Noc3 and Noc2 and Nip7 are shown as green connected spheres31. The interaction between Noc2/Noc3 is analogous to the interaction between Noc4 and Nop14, revealing Noc2 and Nop14 to be structural paralogs67. f, State NE1 with Nop53 colored maroon for emphasis. The newly modeled Nop53 helix is boxed and expanded in the inset. Surface view of the helix is shown for clarity.

Extended Data Fig. 6 Data processing, classification and sorting scheme for State NE1.

Workflow of the data processing strategy after 2D classification. Map volumes and masks (red) are shown for all critical steps. Major sorting and classification criteria, particle numbers and percentages (for each 3D classification) as well as final map resolutions are indicated. Masking of the L1 stalk region allowed us to classify a small subset of pre-rotation Nog2 particles, which are a contaminant in our prep as this intermediate does not contain Spb1. The final sorting was performed to select for a subset with improved density for the Spb1-AD and Spb1-CTD regions.

Extended Data Fig. 7 Visualization of global SHAPE analysis39 of helices H62 and H63 in early and late pre-60S particles.

a. Nucleotides in the H62/H63 region (helices are outlined and labeled) were colored according to their SHAPE reactivity values derived from experiments on early (Prp5-TAP) nucleolar pre-60S intermediates39. Nucleotides without reactivity values are shown in solid grey. b. Nucleotides in the H62/H63 region were colored according to the difference in SHAPE reactivity values between early (Prp5-TAP) and late (Nsa2-TAP) pre-60S intermediates39. The similarity in the pattern and the stability of H63 in the earliest nucleolar 60S particles indicates that H62 and H63 form a duplex structure before and after Spb4 engagement.

Extended Data Fig. 8 Structural homology to the Spb4-CTE defines a subset of DEAD-box ATPases involved in ribosome biogenesis.

a, Comparison of the CTE of Spb4, Has1 (pdb 6C0F)34, Dbp4 (Alphafold model) and Dbp7 (Alphafold model)68. Red spheres represent conserved residues among DEAD-box ATPases. b, Multiple sequence alignment of the CTE of Spb4, Has1, Dbp7 and Dbp4. The helix boundaries of Spb4 and Has1 are shown as red tubes and conserved residues, also shown in the structure cartoons, are marked with red dots. Residues within the CTD that are implicated in base specific interactions and conserved in Spb4 homologues, but not other DEAD-box proteins are indicated by green arrows and green boxes.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2

Reporting Summary

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Supplementary Video 1

Animated morph between state E2 and state NE1 highlighting the rearrangement of H62 and H63 upon Spb4 release.

Source data

Source Data Fig. 3

Uncropped plates from spotting assays and fluorescent images.

Source Data Fig. 4

Unprocessed plates from spotting assays.

Source Data Extended Data Fig. 2

Uncropped Coomassie stained SDS–PAGE.

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Cruz, V.E., Sekulski, K., Peddada, N. et al. Sequence-specific remodeling of a topologically complex RNP substrate by Spb4. Nat Struct Mol Biol 29, 1228–1238 (2022). https://doi.org/10.1038/s41594-022-00874-9

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  • DOI: https://doi.org/10.1038/s41594-022-00874-9

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