A co-transcriptional ribosome assembly checkpoint controls nascent large ribosomal subunit maturation

During transcription of eukaryotic ribosomal DNA in the nucleolus, assembly checkpoints exist that guarantee the formation of stable precursors of small and large ribosomal subunits. While the formation of an early large subunit assembly checkpoint precedes the separation of small and large subunit maturation, its mechanism of action and function remain unknown. Here, we report the cryo-electron microscopy structure of the yeast co-transcriptional large ribosomal subunit assembly intermediate that serves as a checkpoint. The structure provides the mechanistic basis for how quality-control pathways are established through co-transcriptional ribosome assembly factors, that structurally interrogate, remodel and, together with ribosomal proteins, cooperatively stabilize correctly folded pre-ribosomal RNA. Our findings thus provide a molecular explanation for quality control during eukaryotic ribosome assembly in the nucleolus.

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Decision Letter, appeal:
Message: 5th Jan 2023 Dear Dr. Klinge, Thank you again for submitting your manuscript "A co-transcriptional ribosome assembly checkpoint controls nascent large ribosomal subunit maturation". We now have comments (below) from the 3 reviewers who evaluated your paper. In light of those reports, we remain interested in your study and would like to see your response to the comments of the referees, in the form of a revised manuscript.
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We look forward to seeing the revised manuscript and thank you for the opportunity to review your work. Reviewer #1: Remarks to the Author: The manuscript by Sanghai et al reports the cryo-EM structure of an early assembly precursor of 60S large ribosomal subunit (pre-60S). The structure contains domains I and II and importantly the Noc1-Noc2 module, which has not been found in the many known structures of pre-60S, and thus represents a novel earlier state. The authors also show biochemically that helix 2, which is bound by Noc1-Noc2, is important for pre-60S assembly and A2 site cleavage. The authors have strongly claimed that the assembly of Noc1-Noc2 to helix 2 serves as a quality control check point for pre-60S assembly. However, this conclusion seems unclear and overstated. The mutational analysis on helix 2 is nice, but there is no evidence whether helix 2 assembly defects occur naturally in cells and are indeed monitored. Overall, the study is technically solid and a nice piece of work that increases our understanding of the early steps of pre-60S assembly. But I feel that the level of advance is not enough to merit publication in NSMB.
Reviewer #2: Remarks to the Author: The manuscript by Sanghai et al. presents cryo-EM structures and complementary evidence that early 25S rRNA species are cotranscriptionally stabilized by a complex consisting of Noc1/Noc2. This is a novel and insightful structure, and it is accompanied by some really elegant genetic experiments, including RNA swaps. I believe it will be of sufficient interest to general readers. I recommend publication in Nature Structural and Molecular Biology, provided the authors address a few points below.
Major points: 1. The manuscript would benefit from a much clearer description of the purification and data processing methods, both briefly in the text and in full in the Methods. It is not sufficiently clear on how each purification is done. The Noc1/2 complex is obtained by tagging Noc1 and Noc2. The Noc3 complex whose structure has been described is indicated as critical evidence that the structures obtained represent on-pathway intermediates. The reader should know how that complex was obtained. How does it differ or is it from one of the other purifications. Also, in the Methods, it states something like grids were made from 'multiple purifications.' Which purification went on which grid? 2. The authors should acknowledge the pioneering work of Miller and colleagues by including a citation such as Miller & Beatty, Science 1969.
3. The authors state that the Noc1-Noc2 complex is evolutionarily related to the Noc2-Noc3 complex and others, but do not provide or point to evidence as such, nor state why this is important. Are the "central loops", which the authors suggest inhibit H2 binding ( Fig  S7 legend), evolutionarily conserved among paralogous proteins?
4. Describe what a 'root helix' is for a general reader.
5. There are no overlays of the EM reconstruction with the corresponding atomic models. This needs to be done. I would recommend in places where the authors are pointing out specific contacts.
Minor point: 1. The authors should describe their statistical analysis in greater detail, e.g. one-or twotailed t-test and whether or not correction for multiple comparisons is required.

David Taylor Remarks to the Author:
This is an interesting, high-quality study that reports the purification and structure determination by cryo-EM of an early co-transcriptional pre-60S Noc1-Noc2 RNP complex using a physiologically relevant system in which a pre-rRNA mimic is expressed from a plasmid in vivo in S. cerevisiae. The authors also purify a known late pre-60S assembly intermediate (State E) that contains Noc2-Noc3. The significance of the work lies in showing that the Noc1-Noc2 complex controls post transcriptional ribosome assembly at the level of 1) RNA topology, 2) RNA processing, 3) the incorporation of ribosomal proteins, and 4) the chronology of ribosome assembly factor association. A further strength of the work is that the authors functionally test the idea that the Noc1-Noc2 RNP forms a physiologically relevant assembly checkpoint using an elegant combination of yeast genetics, Northern blotting and comparative mass spectrometry. The data are summarized nicely in a model that highlights how the atomic models presented in the current work relate to the historic Miller spread EM images of co-transcriptional ribosome assembly intermediates. Interestingly, the study identifies a role for three Diamond-Blackfan anemia-related proteins in stabilizing the interfaces between domains I, II and the 5.8S rRNA. The links to human ribosomopathy will broaden the appeal of the work to a wide readership beyond the ribosome assembly community. The manuscript is well written with informative, clear figures. A wealth of supporting technical detail is provided in the extensive supplementary methods and figures that appropriately discuss the cryo-EM data collection, processing and classification strategies used and robustly support the conclusions that the authors wish to draw.
I have only a few minor comments: 1. All the work presented in the study is performed in yeast. Presumably the proposed checkpoint is likely to be conserved in mammalian cells, but are there any data to support this? Perhaps consider adding a statement in the text to address this point or add "in yeast" to the title? Interestingly, a very recent study (https://doi.org/10.1242/jcs.260110) shows that Noc1 is critical for pre-rRNA processing, growth and cell competition in Drosophila and would be worth citing.
2. I find some of the same-color labels difficult to read eg Extended data  Table 2 uses lower case and the new nomenclature. Extended Fig 9 has Rpl17 in the legend, but upper case RPL17 in the Figure. On p7, the authors refer to DBA ribosomal proteins RPL17, RPL26 and RPL35A (but RPL35A is the human nomenclature?). Do they mean uL33 using the new nomenclature? Please unify and rationalize the nomenclature throughout the text.
5. The inclusion of Rpl17/RPL17 as a DBA protein appears to be based on the identification of variants in 2 members of a single Swiss family reported in the cited reference 23. It is important to note that although supported by functional analysis in zebrafish, the pathogenicity of this variant and therefore its formal assignment as a DBA protein, needs further validation. Author Rebuttal, first revision: We thank the three reviewers for their helpful comments, which we address below.

Referee expertise:
Referee #1: Cryo-EM, translation Referee #2: Cryo-EM, ribosome biogenesis Referee #3: Ribosome biogenesis Reviewers' Comments: Reviewer #1: Remarks to the Author: The manuscript by Sanghai et al reports the cryo-EM structure of an early assembly precursor of 60S large ribosomal subunit (pre-60S). The structure contains domains I and II and importantly the Noc1-Noc2 module, which has not been found in the many known structures of pre-60S, and thus represents a novel earlier state. The authors also show biochemically that helix 2, which is bound by Noc1-Noc2, is important for pre-60S assembly and A2 site cleavage. The authors have strongly claimed that the assembly of Noc1-Noc2 to helix 2 serves as a quality control check point for pre-60S assembly. However, this conclusion seems unclear and overstated. The mutational analysis on helix 2 is nice, but there is no evidence whether helix 2 assembly defects occur naturally in cells and are indeed monitored. Overall, the study is technically solid and a nice piece of work that increases our understanding of the early steps of pre-60S assembly.
We thank reviewer 1 for the appreciating the impact of our work. But I feel that the level of advance is not enough to merit publication in NSMB.
Here we respectfully disagree and refer to the assessment of reviewers 2 and 3.

Reviewer #2:
Remarks to the Author: The manuscript by Sanghai et al. presents cryo-EM structures and complementary evidence that early 25S rRNA species are cotranscriptionally stabilized by a complex consisting of Noc1/Noc2. This is a novel and insightful structure, and it is accompanied by some really elegant genetic experiments, including RNA swaps. I believe it will be of sufficient interest to general readers. I recommend publication in Nature Structural and Molecular Biology, provided the authors address a few points below.
We thank reviewer 2 for the positive assessment of our work and have addressed the individual points below.
Major points: 1. The manuscript would benefit from a much clearer description of the purification and data processing methods, both briefly in the text and in full in the Methods. It is not sufficiently clear on how each purification is done. The Noc1/2 complex is obtained by tagging Noc1 and Noc2. The Noc3 complex whose structure has been described is indicated as critical evidence that the structures obtained represent on-pathway intermediates. The reader should know how that complex was obtained. How does it differ or is it from one of the other purifications.
We agree with reviewer 2 that the purification of these complexes should be described in more detail in the text and have therefore clarified the text on page 4 as follows: "By using the pre-rRNA mimic with MS2 RNA aptamers and tagged Noc1 as baits for affinity purification, we were able to isolate an early co-transcriptional assembly intermediate (hereafter referred to as the Noc1-Noc2 RNP) (Extended Data Fig. 1). Similarly, by using the same prerRNA mimic with MS2 RNA aptamers and tagged Noc2 we were able to isolate the late nucleolar pre-60S assembly intermediate (State E, containing Noc2-Noc3) 16 , indicating our pre-rRNA mimic proceeds along a physiologically relevant assembly pathway (Extended Data Fig. 2)." In addition to the indicated changes in the main text, we have expanded the methods section with the following sentences on page 23: "The resulting strains were used for the purification of the Noc1-Noc2 RNP (MS2-3C-GFP; Noc1sbp), State E (MS2-3C-GFP; Noc2-sbp) and pre-60S particles for comparative mass spectrometry (MS2-3C-GFP; Cic1-sbp). These strains were subsequently transformed with the pESC_URA plasmids coding for pre-60S rRNA mimics for transient expression." Also, in the Methods, it states something like grids were made from 'multiple purifications.' Which purification went on which grid?
To further clarify how the eight separate cryo-EM datasets were obtained for the Noc1Noc2 RNP, we expanded the methods section on page 25 with the following statement: "Eight separate purifications of the Noc1-Noc2 RNP were performed to prepare eight separate cryo-EM grids that were used to collect eight data sets (DS1-DS8)." 2. The authors should acknowledge the pioneering work of Miller and colleagues by including a citation such as Miller & Beatty, Science 1969.
We thank reviewers 2 and 3 for spotting this oversight and have introduced this reference (new reference 2).
3. The authors state that the Noc1-Noc2 complex is evolutionarily related to the Noc2-Noc3 complex and others, but do not provide or point to evidence as such, nor state why this is important. Are the "central loops", which the authors suggest inhibit H2 binding (Fig S7 legend), evolutionarily conserved among paralogous proteins?
The Noc1-Noc2, Noc2-Noc3 and Nop14-Noc4 complexes adopt similar shapes, and have previously been shown to be evolutionarily related, which we have now referenced on page 6 (reference 22). The loops displayed in Extended Data Fig. 7 only show the available structural data in yeast and we are not aware of a conservation of these elements. To clarify this further, we have edited the figure legend title to the following: "Extended Data Fig. 7. Comparison of Noc1-Noc2, Noc2-Noc3 and Noc4-Nop14 heterodimers in yeast."

Describe what a 'root helix' is for a general reader.
We thank reviewer 2 for this point and have added the definition at the earliest possible instance, on page 5: "Since root helices form the base of each of the 25S rRNA subdomains (I-VI) and hence the architectural core of the LSU 20,21 , their early recognition by the Noc1-Noc2 complex indicates a key point of quality control." 5. There are no overlays of the EM reconstruction with the corresponding atomic models. This needs to be done. I would recommend in places where the authors are pointing out specific contacts.
We agree with reviewer 2 that this is important and in addition to detailed views of atomic models and density maps which we previously showed in Extended Data Fig. 6, we have now also included a view that highlights the essence of the structural data, namely the recognition of root helices by Noc1-Noc2 in Extended Data Fig. 6a. Given the intermediate resolution of our composite map ~4 Ångstroms, we have been cautious in our interpretation and avoided drawing conclusions about interactions at the side-chain level in the manuscript.
Minor point: 1. The authors should describe their statistical analysis in greater detail, e.g. one-or two-tailed ttest and whether or not correction for multiple comparisons is required.

David Taylor
We agree with reviewer 2 that this should be more clearly explained and now mention that a two-tailed student t-test was carried out.
As outlined in the methods (page 30), an FDR correction was applied for proteins and peptides at 1% and 2% respectively. We further note that in the Volcano plots shown in Figs. 3f,g only proteins with p-values of 10 -4 are displayed. We are therefore highlighting only proteins with a cut-off that is two orders of magnitude more stringent than the typical p-value of 0.01. In addition, calculated Q-values, which are a result of FDR-correction are listed in the supplementary mass spectrometry data.

Reviewer #3: Remarks to the Author:
This is an interesting, high-quality study that reports the purification and structure determination by cryo-EM of an early co-transcriptional pre-60S Noc1-Noc2 RNP complex using a physiologically relevant system in which a pre-rRNA mimic is expressed from a plasmid in vivo in S. cerevisiae. The authors also purify a known late pre-60S assembly intermediate (State E) that contains Noc2-Noc3. The significance of the work lies in showing that the Noc1-Noc2 complex controls post transcriptional ribosome assembly at the level of 1) RNA topology, 2) RNA processing, 3) the incorporation of ribosomal proteins, and 4) the chronology of ribosome assembly factor association. A further strength of the work is that the authors functionally test the idea that the Noc1-Noc2 RNP forms a physiologically relevant assembly checkpoint using an elegant combination of yeast genetics, Northern blotting and comparative mass spectrometry. The data are summarized nicely in a model that highlights how the atomic models presented in the current work relate to the historic Miller spread EM images of co-transcriptional ribosome assembly intermediates. Interestingly, the study identifies a role for three Diamond-Blackfan anemia-related proteins in stabilizing the interfaces between domains I, II and the 5.8S rRNA. The links to human ribosomopathy will broaden the appeal of the work to a wide readership beyond the ribosome assembly community. The manuscript is well written with informative, clear figures.
A wealth of supporting technical detail is provided in the extensive supplementary methods and figures that appropriately discuss the cryo-EM data collection, processing and classification strategies used and robustly support the conclusions that the authors wish to draw.
We thank reviewer 3 for the positive assessment of our work.
I have only a few minor comments: 1.
All the work presented in the study is performed in yeast. Presumably the proposed checkpoint is likely to be conserved in mammalian cells, but are there any data to support this? Perhaps consider adding a statement in the text to address this point or add "in yeast" to the title? Interestingly, a very recent study (https://doi.org/10.1242/jcs.260110) shows that Noc1 is critical for pre-rRNA processing, growth and cell competition in Drosophila and would be worth citing.
We agree with reviewer 3 that all our data is from Saccharomyces cerevisiae. However, given the highly conserved nature of early eukaryotic ribosome assembly, we believe that the observed principles are generally applicable to other species and have edited the conclusion of our manuscript to include the following statement, which includes the suggested reference (here reference 6): "Our structural and functional data in yeast shed light on the earliest events during cotranscriptional large ribosomal subunit formation, which are highly conserved in eukaryotes 26 ."

2.
I find some of the same-color labels difficult to read eg Extended data Fig 8. Consider changing to black font.
To address this point, we have increased the font size and added a black outline so that the color coding of the labels remains.

3.
Some of the proteins identified in the mass spec analysis in Extended Data Fig. 1e are not seen in the final reconstruction (eg Rex4, Nop12, Nop4, Nop16). Please comment.
As observed for many ribosome assembly intermediates, cryo-EM reconstructions only capture the most ordered regions of a given particle. In our case, relative to the two wellordered domains I and II, we believe that several proteins (such as Rex4, Nop12, Nop4 and Nop16) and RNA segments are present as confirmed by mass spectrometry but not visualized by cryo-EM.

4.
RP nomenclature is confusing and inconsistent. Why are the some of the yeast ribosomal proteins shown in upper case, but assembly factors in lower case in the Figures? Supp Table 2 uses lower case and the new nomenclature. Extended Fig 9 has Rpl17 in the legend, but upper case RPL17 in the Figure. On p7, the authors refer to DBA ribosomal proteins RPL17, RPL26 and RPL35A (but RPL35A is the human nomenclature?). Do they mean uL33 using the new nomenclature? Please unify and rationalize the nomenclature throughout the text.
We thank reviewer 3 for spotting this and have therefore used the yeast nomenclature throughout the manuscript. The label RPL35A has now been corrected to Rpl35 (yeast nomenclature; uL29 universal nomenclature).

5.
The inclusion of Rpl17/RPL17 as a DBA protein appears to be based on the identification of variants in 2 members of a single Swiss family reported in the cited reference 23. It is important to note that although supported by functional analysis in zebrafish, the pathogenicity of this variant and therefore its formal assignment as a DBA protein, needs further validation.
We agree that a formal assignment as a DBA protein is still missing for Rpl17/RPL17 and have amended the relevant sentence as follows: "The importance of critical interfaces between domains I, II and the 5.8S rRNA is further highlighted by the finding that key stabilizers of these sites include Rpl17(RPL17) and DiamondBlackfan anemia proteins Rpl26(RPL26) and Rpl33A(RPL35A) (Extended Data Fig. 9c Thank you for submitting your revised manuscript "A co-transcriptional ribosome assembly checkpoint controls nascent large ribosomal subunit maturation" (NSMB-A46944B). It has now been assessed editorially, and we find the paper has improved in revision, and therefore we'll be happy in principle to publish it in Nature Structural & Molecular Biology, pending minor revisions to comply with our editorial and formatting guidelines.
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