Inhibition of Ribosome Assembly and Ribosome Translation Has Distinctly Different Effects on Abundance and Paralogue Composition of Ribosomal Protein mRNAs in Saccharomyces cerevisiae

ABSTRACT Many mutations in genes for ribosomal proteins (r-proteins) and assembly factors cause cell stress and altered cell fate, resulting in congenital diseases collectively called ribosomopathies. Even though all such mutations depress the cell’s protein synthesis capacity, they generate many different phenotypes, suggesting that the diseases are not due simply to insufficient protein synthesis capacity. To learn more, we investigated how the global transcriptome in Saccharomyces cerevisiae responds to reduced protein synthesis generated in two different ways: abolishing the assembly of new ribosomes and inhibiting ribosomal function. Our results showed that the mechanism by which protein synthesis is obstructed affects the ribosomal protein transcriptome differentially: ribosomal protein mRNA abundance increases during the abolition of ribosome formation but decreases during the inhibition of ribosome function. Interestingly, the ratio between mRNAs from some, but not all, pairs of paralogous ribosomal protein genes encoding slightly different versions of a given r-protein changed differently during the two types of stress, suggesting that expression of specific ribosomal protein paralogous mRNAs may contribute to the stress response. Unexpectedly, the abundance of transcripts for ribosome assembly factors and translation factors remained relatively unaffected by the stresses. On the other hand, the state of the translation apparatus did affect cell physiology: mRNA levels for some other proteins not directly related to the translation apparatus also changed differentially, though not coordinately with the r-protein genes, in response to the stresses. IMPORTANCE Mutations in genes for ribosomal proteins or assembly factors cause a variety of diseases called ribosomopathies. These diseases are typically ascribed to a reduction in the cell’s capacity for protein synthesis. Paradoxically, ribosomal mutations result in a wide variety of disease phenotypes, even though they all reduce protein synthesis. Here, we show that the transcriptome changes differently depending on how the protein synthesis capacity is reduced. Most strikingly, inhibiting ribosome formation and ribosome function had opposite effects on the abundance of mRNA for ribosomal proteins, while genes for ribosome translation and assembly factors showed no systematic responses. Thus, the process by which the protein synthesis capacity is reduced contributes decisively to global mRNA composition. This emphasis on process is a new concept in understanding ribosomopathies and other stress responses.


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The authors have taken considerable efforts to resubmit a much-improved manuscript. The writing and description of the experiments are much clearer. The analysis is done more clearly, and most conclusions stated are appropriate. In general, the single point assessed, of what happens to overall ribosomal biogenesis if different aspects of translation (ribosome assembly vs translation initiation) are disrupted, is quite clearly answered. It is clear that the overall state of ribosome biogenesis is different, and this is demonstrated.
The last section of results (cell cycle arrest and "ribosome formation and function are central to the 929 regulation of cell growth and resource use.") only state the very obvious, and I would relegate that to two lines in the text, and a supplementary figure if required.
There is no further experimental analysis on what happens to overall translation fidelity or fitness as a consequence of these changes. That would have been ideal, to make stronger conclusions on what this might imply.
But in general, this is a nice story, and clarifies an important concept. The authors do need to proof this manuscript carefully).
Reviewer #2 (Comments for the Author):

Summary
In this revised manuscript, the authors clearly describe their use of a transcriptomic approach to determine whether cells respond similarly to nucleolar stress vs. translation stress generated by reduced expression of a ribosomal protein and a translation elongation factor, respectively. The clarity of the results is greatly improved as the authors leverage their data to (1) validate successful glucose-repression of the two targeted genes, (2) identify changes in mitochondrial gene expression likely caused by the carbon source switch, and (3) demonstrate that nucleolar stress and translation stress typically elicit opposite effects on expression of r-protein genes. Notably, some paralogues of these r-protein genes respond differently to the two stresses. Inclusion of improved heatmaps and deletion of other distracting figures results a streamlined narrative that is better supported by the highlighted data. In the long-run, these findings may inform our understanding of the diverse symptoms associated with various ribosomopathies. Since the eukaryotic microbe S. cerevisiae is used as the model organism for this study, and since the ribosomal machinery is highly conserved, the subject matter of this manuscript is likely to be of significant interest to mSystems readers.

Major Issues
The construction and use of growth curves could be clarified. In the results (lines 142-143), the authors state, "The sampling times were chosen to assure approximately equal growth rates of the two cultures at the time of sampling, estimated from respective growth curves" yet the methods section (lines 413-414) indicates "Cultures were diluted as necessary with prewarmed media to keep the OD 600 <1.0." Which approach was used? Were the yeast cultures allowed to progress through the growth curve or not? Minor: In the figure 2 growth curves, the authors should double-check the y-axis labels, as the values suggest they may have graphed OD rather than logOD. For example, the data for the Pgal-eEF3 strain grown in glucose plateaus around log 2 OD = 8, which would correspond to an actual OD value of 256! It feels odd to dedicate a significant portion of the discussion to changes in expression of cell cycle and septum degradation genes when the corresponding figures are only available in the supplemental data. This part of the discussion could also be clearer. For example, in the first paragraph of cell cycle discussion (Page 12, Lines 358-369), the phrasing used is ambiguous regarding whether there was statistically significant enrichment of the GO term "septum digestion after cytokinesis". If so, the wording should be clarified, and the related heat map (S6A) from the supplemental data might be elevated and featured in the results section of the main paper. If not, this section of discussion might be cut from the paper, as the evidence does not sufficiently support it.

Minor Issues
There is no mention of Figure 7 in the results text. Since Figure 7 presents data on nuclear pore genes, it would make sense to reference that figure in the corresponding section of the results (Page 9, Lines 256-259). Also Page 12 line 354 contains a reference to Figure 6, but should it instead refer to Figure 7, which contains the nuclear pore gene expression data?
The discussion of possible explanations for changes in r-protein mRNA abundance could be clearer. For example, is one hypothesis that the cell would compensate for proteasome-mediated degradation of r-proteins by upregulating transcription of rprotein genes? And regarding ribosome collisions and mRNA degradation, it would be helpful to briefly clarify (or speculate on) how/why this would alter mRNA abundance of specific subsets of genes as opposed to resulting in a global reduction of total mRNA.
Page 12 line 371: Should delta lfc be greater than or equal to 1 as opposed to less than or equal to 1? If the authors did conduct GO term enrichment analysis on genes with delta lfc less than or equal to 1, this choice of cut off should be better justified.
Under Implications, the statement that "We suggest that a given mutation simultaneously affects both the synthesis of ribosomes and the ribosome function" (Page 13, Lines 397-398) could be rephrased to more clearly connect to and support adjacent sentences.
We thank the reviewers for their very helpful comments. We are pleased with the reviewers' positive reactions to the revised manuscript.

Reviewer #1 (Comments for the Author):
The authors have taken considerable efforts to resubmit a much-improved manuscript. The writing and description of the experiments are much clearer. The analysis is done more clearly, and most conclusions stated are appropriate. In general, the single point assessed, of what happens to overall ribosomal biogenesis if different aspects of translation(ribosome assembly vs translation initiation) are disrupted, is quite clearly answered. It is clear that the overall state of ribosome biogenesis is different, and this is demonstrated. The last section of results (cell cycle arrest and "ribosome formation and function are central to the 929 regulation of cell growth and resource use.") only state the very obvious, and I would relegate that to two lines in the text, and a supplementary figure if required. There is no further experimental analysis on what happens to overall translation fidelity or fitness as a consequence of these changes. That would have been ideal, to make stronger conclusions on what this might imply.
Both reviewers are critical of our discussion of the cell cycle. In general, we agree that this section does not live up to the general standard of the manuscript. Thus, we have eliminated the narrative section on "Cell Cycle" and Table S6. We also condensed the narrative of "Other mRNAs ….", but do not think that it should be eliminated completely, since it completes the analysis.
But in general, this is a nice story, and clarifies an important concept. The authors do need to proof this manuscript carefully).
Reviewer #2 (Comments for the Author): Summary In this revised manuscript, the authors clearly describe their use of a transcriptomic approach to determine whether cells respond similarly to nucleolar stress vs. translation stress generated by reduced expression of a ribosomal protein and a translation elongation factor, respectively. The clarity of the results is greatly improved as the authors leverage their data to (1) validate successful glucoserepression of the two targeted genes, (2) identify changes in mitochondrial gene expression likely caused by the carbon source switch, and (3) demonstrate that nucleolar stress and translation stress typically elicit opposite effects on expression of r-protein genes. Notably, some paralogues of these r-protein genes respond differently to the two stresses. Inclusion of improved heatmaps and deletion of other distracting figures results a streamlined narrative that is better supported by the highlighted data. In the long-run, these findings may inform our understanding of the diverse symptoms associated with various ribosomopathies. Since the eukaryotic microbe S. cerevisiae is used as the model organism for this study, and since the ribosomal machinery is highly conserved, the subject matter of this manuscript is likely to be of significant interest to mSystems readers.

Major Issues
The construction and use of growth curves could be clarified. In the results (lines 142-143), the authors state, "The sampling times were chosen to assure approximately equal growth rates of the two cultures at the time of sampling, estimated from respective growth curves" yet the methods section (lines 413-414) indicates "Cultures were diluted as necessary with pre-warmed media to keep the OD600<1.0." Which approach was used? Were the yeast cultures allowed to progress through the growth curve or not? Minor: In the figure 2 growth curves, the authors should double-check the y-axis labels, as the values suggest they may have graphed OD rather than logOD. For example, the data for the Pgal-eEF3 strain grown in glucose plateaus around log 2 OD = 8, which would correspond to an actual OD value of 256! We have clarified the recording of the growth curve data and that the culture dilutions are necessary to keep the culture from entering stationary phase which would prevent the identification of the specific stress-induced changes to the transcriptome.
It feels odd to dedicate a significant portion of the discussion to changes in expression of cell cycle and septum degradation genes when the corresponding figures are only available in the supplemental data. This part of the discussion could also be clearer. For example, in the first paragraph of cell cycle discussion (Page 12, Lines 358-369), the phrasing used is ambiguous regarding whether there was statistically significant enrichment of the GO term "septum digestion after cytokinesis". If so, the wording should be clarified, and the related heat map (S6A) from the supplemental data might be elevated and featured in the results section of the main paper. If not, this section of discussion might be cut from the paper, as the evidence does not sufficiently support it.
We have eliminated the discussion of the cell cycle; see our response to Reviewer 1's comments.

Minor Issues
There is no mention of Figure 7 in the results text. Since Figure 7 presents data on nuclear pore genes, it would make sense to reference that figure in the corresponding section of the results (Page 9, Lines 256-259). Also Page 12 line354 contains a reference to Figure 6, but should it instead refer to Figure 7, which contains the nuclear pore gene expression data?
We apologize for the error. It has been corrected and the Figure numbering corrected to follow the narrative.
The discussion of possible explanations for changes in r-protein mRNA abundance could be clearer. For example, is one hypothesis that the cell would compensate for proteasome-mediated degradation of r-proteins by upregulating transcription of rprotein genes?
We have added specificity to this hypothesis.