Concurrent remodelling of nucleolar 60S subunit precursors by the Rea1 ATPase and Spb4 RNA helicase

  1. Valentin Mitterer
  2. Matthias Thoms
  3. Robert Buschauer
  4. Otto Berninghausen
  5. Ed Hurt
  6. Roland Beckmann

  • Curated by eLife

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    eLife assessment

    This fundamental study substantially advances our understanding of the process of ribosome maturation. The authors have purified and determined the structures of several nucleolar ribosome assembly intermediates in yeast using cryo-electron microscopy (cryo-EM). The study combines genetic, biochemical, and structural analysis to provide compelling support for the conclusions the authors wish to draw. The work will be of broad interest to cell biologists, biochemists, and structural biologists.

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Abstract

Biogenesis intermediates of nucleolar ribosomal 60S precursor particles undergo a number of structural maturation steps before they transit to the nucleoplasm and are finally exported into the cytoplasm. The AAA + -ATPase Rea1 participates in the nucleolar exit by releasing the Ytm1–Erb1 heterodimer from the evolving pre-60S particle. Here, we show that the DEAD-box RNA helicase Spb4 with its interacting partner Rrp17 is further integrated into this maturation event. Spb4 binds to a specific class of late nucleolar pre-60S intermediates, whose cryo-EM structure revealed how its helicase activity facilitates melting and restructuring of 25S rRNA helices H62 and H63/H63a prior to Ytm1–Erb1 release. In vitro maturation of such Spb4-enriched pre-60S particles, incubated with purified Rea1 and its associated pentameric Rix1-complex in the presence of ATP, combined with cryo-EM analysis depicted the details of the Rea1-dependent large-scale pre-ribosomal remodeling. Our structural insights unveil how the Rea1 ATPase and Spb4 helicase remodel late nucleolar pre-60S particles by rRNA restructuring and dismantling of a network of several ribosomal assembly factors.

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  1. Version published to 10.7554/elife.84877 on eLife
  2. eLife

    Author Response

    Reviewer #3 (Public Review):

    Over the past decade, Cryo-EM analysis of assembling ribosomes has mapped the major intermediates of the pathway. Our understanding of the mechanisms by which ATPases drive the transitions between states has been slower to develop because of the transient nature of these events. Here, the authors use cryo-EM and biochemical and molecular genetic approaches to examine the function of the DEAD-box ATPase Spb4 and the AAA-ATPase Rea1 in RNP remodeling. Spb4 works on the pre-60S in an early nucleolar state. The authors find that Spb4 acts to remodel the three-way junction of H62/H63/H63a at the base of expansion segment ES27. Interestingly, Spb4 appears to interact stably with a folding intermediate in the ADP rather than ATP-bound form. This work represents one of the few cases in which an RNA helicase of ribosome biogenesis has been captured and engaged with its substrate. The authors then show that the addition of the AAA-ATPase Rea1 to Spb4-purified particles results in the release of Ytm1, a known target of Rea1. However, they did not observe an efficient release of Ytm1 when particles were affinity purified via Ytm1, suggesting that the recruitment of Spb4 is important for this step. Cryo-EM analysis of Spb4-particles treated with Rea1 revealed the previously characterized state NE particles but no additional intermediates. Consequently, this analysis of Rea1 is less informative about its function than is their work on Spb4 helicase activity. In general, the data support the authors' conclusions and the data are well presented.

    Major points

    1. The Erzberger group has recently published work regarding the function of Spb4. They similarly found that Spb4 is necessary for remodeling the 3-way junction at the base of ES27. Although it was posted to Biorxiv in Feb 2022, it was not formally published until Dec 2022. The authors should cite this work and include a brief discussion comparing conclusions.

    We are now citing this study in the introduction and discussion and are briefly comparing the conclusions.

    1. L311. The heading "Coupled pre-60S dissociation of the Ytm1-Erb1 complex and RNA helicase Has1" should be changed. Coupling implies a mechanistic interplay. Although the release of Ytm1 and Has1 both depend on Rea1, the data do not support the conclusion of mechanistic coupling. In fact, the authors write in lines 328-329 "Thus, the Rea1-dependent pre-60S release of the Ytm1-Erb1 complex occurs before and independently of Has1..." Independently cannot also imply coupling.

    We have changed the heading into “Ytm1–Erb1 release promotes the dissociation of the RNA helicase Has1”.

    1. L339-342 Combining data sets for uniform processing was a great idea! This approach should be used more often in cryo-EM analyses of in vitro maturation reactions.

    We agree with the reviewer that this approach is appropriate to analyse such reactions.

    1. L428 The authors need to amend their comment that this is the first structure of Spb4-bound to the substrate as this has recently been published by the Erzberger group and was first posted as a preprint in early 2022.

    We have removed the statement regarding the first structure of Spb4 and added a citation of the study published by Cruz et al.

  3. eLife

    eLife assessment

    This fundamental study substantially advances our understanding of the process of ribosome maturation. The authors have purified and determined the structures of several nucleolar ribosome assembly intermediates in yeast using cryo-electron microscopy (cryo-EM). The study combines genetic, biochemical, and structural analysis to provide compelling support for the conclusions the authors wish to draw. The work will be of broad interest to cell biologists, biochemists, and structural biologists.

  4. eLife

    Reviewer #1 (Public Review):

    In this study, Mitterer et al continue their comprehensive investigation of the mechanisms underlying the biogenesis of the eukaryotic large, or 60S, ribosomal subunit. Specifically, they elucidate the roles that the DEAD-box helicase Spb4 and its interaction partner, Rrp17, play in the maturation of nucleolar 60S precursor particles. Using cell biology approaches, the authors demonstrate that Spb4 and Rrp17 are associated with late-stage nucleolar 60S precursor particles and that depletion of these factors arrests 60S biogenesis at a step just prior to nucleolar exit. Cryo-EM imaging of particles carrying Spb4 and Rrp17 (purified using affinity-tagged Spb4 or Rrp17) yielded high-quality structures of Spb4- and Rrp17-bound 60S precursor particles. The structures provide novel insights into the roles of Spb4 and Rrp17 in the maturation of nucleolar 60S precursor particles. In addition, the structures provide novel insights into the Spb4 function that may be of interest and importance to the function of other DEAD-box helicases. The authors then establish an in vitro maturation assay that, although unlikely to exactly recapitulate the in vivo maturation process, provides additional insights, particularly when coupled to cryo-EM structures of the in vitro-matured 60S particles.

    A major strength of this work is the combination of cell biology, structural biology, and biochemistry. The cell biology-directed preparation of Spb4- and Rrp17-bound 60S precursor particles is particularly powerful and results in high-quality structures of these precursors. Another strength of the work is the remarkable view of a DEAD-box helicase in action and the interesting finding that the RecA domains of the helicase are in the open conformation while the helicase is likely bound to ADP-this will be an interesting and important observation for researchers working in the broader DEAD-box helicase field. An additional strength of the work is the development and use of an in vitro maturation assay that allowed further details of the activities of Spb4 and Rrp17 in nucleolar maturation of 60S precursor particles to be investigated and visualized.

    A minor weakness of this work is a question about the confidence with which the authors can conclude, using just the structural data presented here, that Spb4 is bound to ADP rather than to ATP or ATP-Pi.

    The considerable strengths of this work far outweigh the minor weakness, and I expect that this work will have a significant impact on the field.

  5. eLife

    Reviewer #2 (Public Review):

    Mitterer et al investigated the role of the essential ATPase Spb4 in the maturation of the large ribosomal subunit precursor in the nucleolus using a combination of genetics, biochemistry, and cryo-EM. They suggest that the helicase Spb4 promotes limited RNA strand separation to drive reconfiguration of helices H62/H63/H63a at the base of domain IV of the 25S rRNA. The study also couples an in vitro pre-ribosome maturation assay with cryo-EM visualisation of pre-60S particles to recapitulate a major structural transition that is dependent on the recruitment of the AAA+ ATPase Rea1 to Spb4-bound particles. This structural transition is important as it promotes nucleolar exit of the 60S precursor from the nucleolus following the release of a limited set of ribosome assembly factors including the Ytm1-Erb1 complex together with the helicase Has1. The quality of the new cryo-EM maps provides a wealth of structural detail on the architecture of late pre-60S nucleolar maturation intermediates.

    The paper is of high quality and clearly written with appropriately detailed methods. The figures are generally well-presented and informative. A strength of the study is that it provides insight into the function and mechanism of action of a poorly understood class of DEAD-box RNA helicases. The study reports the utility of in vitro pre-ribosome maturation combined with cryo-EM analysis to capture additional ribosome maturation intermediates, an approach that may become more widely adopted in the future among the ribosome synthesis community. The biochemical, genetic, and structural analyses strongly support the proposed mechanism for Spb4 function in reconfiguring helices H62/H63/H63a following induced RNA strand separation prior to the release of the Ytm1-Erb1 complex.

    The authors suggest that Spb4 "induces" bending and strand separation of the rRNA at the base of ES27. They also suggest that the C-terminal domain of Spb4 "induces" substrate RNA strand disruption. However, an alternative possibility could be that the rRNA is sampling multiple conformations and that Spb4 stabilises one of these conformers. No direct experimental evidence for "induced" bending and strand separation by Spb4 is provided to support the claims.

    The findings in the manuscript are generally consistent with a very recently published study on Spb4 function (Cruz et al., https://doi.org/10.1038/s41594-022-00874-9). However, the authors should cite this work and update the text to take account of this report.

  6. eLife

    Reviewer #3 (Public Review):

    Over the past decade, Cryo-EM analysis of assembling ribosomes has mapped the major intermediates of the pathway. Our understanding of the mechanisms by which ATPases drive the transitions between states has been slower to develop because of the transient nature of these events. Here, the authors use cryo-EM and biochemical and molecular genetic approaches to examine the function of the DEAD-box ATPase Spb4 and the AAA-ATPase Rea1 in RNP remodeling. Spb4 works on the pre-60S in an early nucleolar state. The authors find that Spb4 acts to remodel the three-way junction of H62/H63/H63a at the base of expansion segment ES27. Interestingly, Spb4 appears to interact stably with a folding intermediate in the ADP rather than ATP-bound form. This work represents one of the few cases in which an RNA helicase of ribosome biogenesis has been captured and engaged with its substrate. The authors then show that the addition of the AAA-ATPase Rea1 to Spb4-purified particles results in the release of Ytm1, a known target of Rea1. However, they did not observe an efficient release of Ytm1 when particles were affinity purified via Ytm1, suggesting that the recruitment of Spb4 is important for this step. Cryo-EM analysis of Spb4-particles treated with Rea1 revealed the previously characterized state NE particles but no additional intermediates. Consequently, this analysis of Rea1 is less informative about its function than is their work on Spb4 helicase activity. In general, the data support the authors' conclusions and the data are well presented.

    Major points
    1. The Erzberger group has recently published work regarding the function of Spb4. They similarly found that Spb4 is necessary for remodeling the 3-way junction at the base of ES27. Although it was posted to Biorxiv in Feb 2022, it was not formally published until Dec 2022. The authors should cite this work and include a brief discussion comparing conclusions.
    2. L311. The heading "Coupled pre-60S dissociation of the Ytm1-Erb1 complex and RNA helicase Has1" should be changed. Coupling implies a mechanistic interplay. Although the release of Ytm1 and Has1 both depend on Rea1, the data do not support the conclusion of mechanistic coupling. In fact, the authors write in lines 328-329 "Thus, the Rea1-dependent pre-60S release of the Ytm1-Erb1 complex occurs before and independently of Has1..." Independently cannot also imply coupling.
    3. L339-342 Combining data sets for uniform processing was a great idea! This approach should be used more often in cryo-EM analyses of in vitro maturation reactions.
    4. L428 The authors need to amend their comment that this is the first structure of Spb4-bound to the substrate as this has recently been published by the Erzberger group and was first posted as a preprint in early 2022.

  7. Version published to 10.1101/2022.11.14.516466v1 on bioRxiv