Concerted modification of nucleotides at functional centers of the ribosome revealed by single-molecule RNA modification profiling
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Evaluation Summary:
In this manuscript Bailey et al use single molecule RNA sequencing to dissect the functional relationships between distinct rRNA modification sites. Their method allows for the deconvolution of distinct subpopulations of rRNA and provides new insights in the installment of rRNA modifications, ribosome heterogeneity and ribosome biogenesis. The paper presents a major technological advance in mapping nucleoside modifications across single RNA molecules and identifying factors that influence these modifications.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
Nucleotides in RNA and DNA are chemically modified by numerous enzymes that alter their function. Eukaryotic ribosomal RNA (rRNA) is modified at more than 100 locations, particularly at highly conserved and functionally important nucleotides. During ribosome biogenesis, modifications are added at various stages of assembly. The existence of differently modified classes of ribosomes in normal cells is unknown because no method exists to simultaneously evaluate the modification status at all sites within a single rRNA molecule. Using a combination of yeast genetics and nanopore direct RNA sequencing, we developed a reliable method to track the modification status of single rRNA molecules at 37 sites in 18 S rRNA and 73 sites in 25 S rRNA. We use our method to characterize patterns of modification heterogeneity and identify concerted modification of nucleotides found near functional centers of the ribosome. Distinct, undermodified subpopulations of rRNAs accumulate upon loss of Dbp3 or Prp43 RNA helicases, suggesting overlapping roles in ribosome biogenesis. Modification profiles are surprisingly resistant to change in response to many genetic and acute environmental conditions that affect translation, ribosome biogenesis, and pre-mRNA splicing. The ability to capture single-molecule RNA modification profiles provides new insights into the roles of nucleotide modifications in RNA function.
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Author Response:
Reviewer #1 (Public Review):
The authors describe a single molecule technology to identify RNA modifications. The methodology was validated with yeast ribosomes using depletion of the two major snoRNPs classes. The authors were able to resolve ribosome populations with a a single modification difference and identified which nucleotides are modified in a concerted fashion in the wt ribosome population. Based on the analysis of rRNA from the helicase mutant strains, the authors suggest a hierarchical model for the action of Dbp3 and Prp43/Pxr1, which provides an important insight in the mechanism of ribosome biogenesis. They also found that most annotated modifications do not change much upon stress or in the presence of ribosome inhibitors. These results solve several outstanding questions in understanding how …
Author Response:
Reviewer #1 (Public Review):
The authors describe a single molecule technology to identify RNA modifications. The methodology was validated with yeast ribosomes using depletion of the two major snoRNPs classes. The authors were able to resolve ribosome populations with a a single modification difference and identified which nucleotides are modified in a concerted fashion in the wt ribosome population. Based on the analysis of rRNA from the helicase mutant strains, the authors suggest a hierarchical model for the action of Dbp3 and Prp43/Pxr1, which provides an important insight in the mechanism of ribosome biogenesis. They also found that most annotated modifications do not change much upon stress or in the presence of ribosome inhibitors. These results solve several outstanding questions in understanding how potential ribosome heterogeneity and argue against the proposed involvement of rRNA modifications in stress response. The methodology can be used for other classes of RNA, which is important in view of the current interest in RNA modifications and their role in epitranscriptomic regulation.
We thank the reviewer for their kind words, especially about the potential of this sort of approach for further discovery, and for their specific suggestions for improvement.
Response to the first specific point: We did perform this control in the original manuscript (it was critical for the analysis!) but failed to contrast it to the experimental sample in our presentation. We have now remedied that oversight by providing new supplemental figures (Figure 3 - figure supplements 2 and 3) in which wt with and without cold shift are compared directly with cold shifted prp43-cs, and a control cs splicing mutant prp16-cs. We had previously documented the lack of change in modification in wt upon cold shift in Fig 5, but the new figure shows this directly alongside the two cs helicase mutations used in this study, reinforcing our original conclusion that prp43-cs has a specific defect not generated by either cold shift (WT 18 degrees) or splicing inhibition (prp16-cs).
To the second point: Here we could have been clearer about the motivation and expectations for our experiments. Throughout the study, we used 1 hour as a standard treatment time to represent an acute change in environmental conditions, both for consistency and because many of the treatments are not particularly growth inhibitory. At 30°C for example, cell numbers increase about 1.6 fold per hour (doubling time is ~100 min). We expected ribosomes to accumulate along a similar path, meaning that after 1 hr, as much as 38% or so of the existing ribosomes (to a first approximation) might have been newly synthesized and modified under the new conditions, and thus detectable if the treatment only affected new ribosomes. In that case the loss or gain of a modification as an immediate response to treatment would have appeared in a substantial minority of the ribosomes after 1 hour if such modifications existed. Our inability to detect such modifications does not mean there is no effect of stress on modification over longer time scales, and we have addressed this by clarifying the presentation of the experiment.
Reviewer #2 (Public Review):
rRNA modifications have been proposed to be a main source of ribosome heterogeneity, and there has been much speculation of how co-occurrence of modification defects could both further exacerbate the heterogeneity, as well as amplify functional differences. Moreoever, there has been speculation about changes in the modification in response to different cellular states. Bailey et al directly address these questions by sequencing entire rRNA molecules using nanopore sequencing. The data not only show that most residues are modified to very high extent, but also demonstrate that most sites are independent of each other. Nevertheless, the authors do demonstrate some modification sites that are dependent on others. Some of these are readily explained by a shared snoRNA guide, but others are not. E.g., modification of the exit tunnel is concerted. Whether this is due to concerted modification, or preferential stabilization of fully modified RNA is not explored, and to this reviewer this is not necessary.
Importantly, the authors do not find any evidence for a dynamic regulation of the modifications, which to this reviewer makes sense, because rRNAs are just too long lived for this to make sense as a way to respond to cellular stress.
Overall, the claims in the manuscript are supported by data, and they are interesting and novel. I have only very minor concerns, although I am not an expert in the nanopore technology, the computational analysis, or the machine learning part.
We thank the reviewer for their enthusiasm and constructive feedback. Their second comment in particular sent us back to the data for deeper inspection and revealed an additional relationship between separate modifications that may be worthy of future experimentation.
To the first specific point, unfortunately, to our knowledge, no published data exist in which subclasses of partly modified fragments containing closely spaced modifications have been described. The mass spectrometry study from Taoka et al. 2016 identified and quantified several fragments with multiple modifications but only reported individual modification frequencies, usually >95%, suggesting that residual, partly modified fragments were not detected or could not be analyzed for modification status at the nearby site. Modification correlation analysis was also not done in HPLC modification papers (Yang et al. 2016), RiboMeth-Seq papers (Birkedal et al. 2014; Marchand et al. 2016), or primer extension-based papers. We did confirm the concerted loss of modification pattern by comparing the nanopore signal means directly. We fear this is the best that can be done without significant new investment in experimentation.
We are excited that the reviewer believes as we do that there is much in the data that has not fully been explored! The reviewer notes a scenario in which binding of a snoRNA has structural consequences on partly assembled ribosomes that influence rRNA folding or ribosomal protein binding at distant locations, that then affects the access of a second snoRNP to its substrate. As the reviewer knows, there are numerous situations during ribosome biogenesis where this sort of dependency or collision could occur, including the example we detailed concerning dbp3 and prp43 in Fig 6. Prompted by the reviewer’s suggestion, we searched for correlation changes between snoRNA knockouts in our data and discovered a relationship between snR83 and snR4 and their targets. This is now described in a new figure (Figure 2-figure supplement 3) and with a short additional section of text. To pursue more thoroughly in the future, we hope to test a comprehensive set of single snoRNA knockout strains. We thank the reviewer for their insight and enthusiasm.
Reviewer #3 (Public Review):
In this study the authors developed a novel strategy to map nucleoside modifications by using Nanopore sequencing of the 25S and 18S rRNAs in yeast. By comparing Nanopore sequencing reads on in vitro transcribed RNAs and RNAs extracted from cells, the authors were able to identify all 110 annotated modifications in single, full-length ribosomal RNAs.
Overall, this is an impactful manuscript that informs the field on a new technique to detect rRNA modifications and offers important insights into subpopulations of ribosomes that are lacking certain modifications. The main highlights of this paper are (1) the single molecule, direct RNA sequencing approach to detect individual modifications along an entire rRNA molecule, (2) rRNA modification is coordinated at certain positions, , and (3) subpopulations of ribosomes accumulate that are missing one or more modifications. This manuscript is relevant from the perspective of ribosome assembly, in that it informs on the order and dependencies of rRNA modifications, as well as other factors (Dbp3 and Pxr1) that are necessary for proper modification. It is also relevant in the context of the "specialized ribosome" hypothesis by showing that ribosomes are heterogenous in modification status. Most of the nucleoside modifications analyzed are promoted by guide snoRNAs, and genetic depletion of protein components of the guide snoRNPs or knockout of guide snoRNAs result in the expected decrease in the modification profiles for most positions. Interestingly they show that 2'-O-methylation is largely independent from Pseudouridylation. Another important finding of the study is the correlation of modifications at distant sites that correspond to functionally important regions of the ribosome.
I found that most of the conclusions made by the authors are supported by the data, with the exception of a few experiments described below. This manuscript will represent a major resource for the community, as it provides a new standard and approach to map ribosomal RNA modifications on single rRNA transcripts, and I anticipate that it will become a widely used tool for the scientific community. Besides the technological innovation, the information obtained on the correlation of modification at specific positions is an important finding for the fields of ribosomes and translation. In terms of specificity of identification of modifications,
The only weakness of the manuscript lies in some of the genetic experiments used to assess the impact of the inactivation of specific factors or environmental conditions on modification patterns as described below - I found three specific issues.
The first issue is the use of the prp44 cold-sensitive (cs) mutant. The authors compare the modification patterns obtained for this cs mutant after a shift to non-permissive temperature. However, there is no control experiment done with a wild-type strain shifted to the same cold temperature, which is problematic as a basic control is missing. So it would be necessary to perform a control experiment with a wild-type strain shifted to a similar temperature. Also the dbp knockout analysis is performed at steady state while prp43-cs is a cold shift so it is quite difficult to compared the result directly.
Another issue that may need to be considered is the level of depletion of individual snoRNAs after depletion of the snoRNP proteins. It is possible that some snoRNAs are depleted more rapidly than others, and that this may affect the modification patterns. The authors should perform RNA sequencing of RNA samples used after depletion of Cbf5 or Nop58 such that they can directly correlate snoRNA levels to modification levels. Unless the authors provide these data, it is difficult to conclude whether specific sites are more or less resilient to genetic depletion of snoRNP proteins.
Finally, the title of the last section of the results is also misleading in terms of its conclusions ("Resilience of rRNA modification profiles to splicing perturbations and environmental treatments"). Regarding splicing perturbations, and with the exception of the dbr1 knockout, the mutants used in the study do not result in a major depletion of intron encoded snoRNAs so it is quite expected that there is no loss of modification at these positions. Similarly, the environmental stresses used are short, and are not expected to affect modification patterns in a major way considering the stability of ribosomes. Unless the authors perform sequencing on rRNAs synthesized after a shift into stress conditions, it is misleading to state that rRNA modification profiles are unaffected by environmental treatments. My feeling is that the paper is significant enough without the studies presented in the last paragraph, and that this paragraph and the data within should be removed from the manuscript because they are inconclusive, and the title is misleading.
I spent the last few paragraphs highlighting some of the issues that need to be addressed, but overall, I found that the article presents a major advance in the field and that it provides a landmark study in our understanding of nucleoside modifications in rRNA.
Thanks very much to the reviewer for their kind assessment of the significance of our efforts, and for the detailed analysis they put into their review.
To the first section of comments, the initial observation that we failed to adequately compare the cold-shifted wild type cells to the cold shifted mutants was also raised by reviewer #1 and we addressed those above.
The second comment refers to the wholesale depletion of the Nop58 or Cbf5 proteins of snoRNPs and the relationship those dynamics may have on both snoRNA levels and snoRNP function. We are concerned that this endpoint depletion experiment is too complex to obtain reliable information about the relationships between snoRNA levels and modification efficiency across hundreds of modified sites. Steady state levels of snoRNAs appear to vary by more than 10-fold in wt cells despite resulting in equivalent levels of modification, suggesting that snoRNA level per se may not be strictly coupled to modification efficiency. In the two decades old Nop58 and Cbf5 depletion experiments we reproduced from Lafontaine, Tollervey and colleagues, snoRNAs are also likely competing for increasingly smaller amounts of protein, and relative amounts of residual snoRNA may not be assembled, obscuring the connection between snoRNA level and functional snoRNP level. Ultimately, we do not believe and did not claim that the experiment provides scaled quantitative information about the relative activities of snoRNPs. Still, the reviewer raises several important questions about the relationship between snoRNA levels and snoRNP modification activity that deserve future attention.
In their third comment, reviewer #3 raises concerns about the conclusion concerning the resilience of modification pattern to splicing changes, as possibly generated by potential impacts of splicing inhibition on snoRNP function. We clarified our motivations in these short (we now call these “acute” changes throughout the revised manuscript) above in response to reviewer #1’s second point. Reviewer #3 focuses on the splicing tests with an eye toward their effect on snoRNA levels. We looked for effects of splicing-related mutations because of known connections between splicing and ribosome biogenesis: (1) 90% of the splicing done in vegetatively growing yeast is devoted to the translation apparatus, (2) some snoRNAs are intron-encoded, and (3) Prp43 has roles in both ribosome biogenesis and splicing. Our idea was to test this broadly without necessarily expecting reduction of snoRNA levels that might or might not be expected in a given splicing mutant. As we suggest above, the relationship between snoRNA level and modification efficiency under partial snoRNA expression is unknown for nearly all snoRNAs, and snoRNA level may not be the only possible mechanism for loss of modification. We have clarified this by adding: “Loss of rRNA modifications in response to splicing, environment or stress conditions could occur through at least two mechanisms: 1) enzymatic removal of pre-existing modifications or 2) synthesis of nascent rRNAs that lack snoRNA-guided rRNA modifications.” in the revised manuscript.
Again, we thank all the reviewers for their very helpful suggestions. Their efforts have improved our presentation and sharpened our thinking on numerous points, as well as helped shape our vision of the future experimentation that may be possible using single molecule modification profiling.
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Evaluation Summary:
In this manuscript Bailey et al use single molecule RNA sequencing to dissect the functional relationships between distinct rRNA modification sites. Their method allows for the deconvolution of distinct subpopulations of rRNA and provides new insights in the installment of rRNA modifications, ribosome heterogeneity and ribosome biogenesis. The paper presents a major technological advance in mapping nucleoside modifications across single RNA molecules and identifying factors that influence these modifications.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
The authors describe a single molecule technology to identify RNA modifications. The methodology was validated with yeast ribosomes using depletion of the two major snoRNPs classes. The authors were able to resolve ribosome populations with a a single modification difference and identified which nucleotides are modified in a concerted fashion in the wt ribosome population. Based on the analysis of rRNA from the helicase mutant strains, the authors suggest a hierarchical model for the action of Dbp3 and Prp43/Pxr1, which provides an important insight in the mechanism of ribosome biogenesis. They also found that most annotated modifications do not change much upon stress or in the presence of ribosome inhibitors. These results solve several outstanding questions in understanding how potential ribosome …
Reviewer #1 (Public Review):
The authors describe a single molecule technology to identify RNA modifications. The methodology was validated with yeast ribosomes using depletion of the two major snoRNPs classes. The authors were able to resolve ribosome populations with a a single modification difference and identified which nucleotides are modified in a concerted fashion in the wt ribosome population. Based on the analysis of rRNA from the helicase mutant strains, the authors suggest a hierarchical model for the action of Dbp3 and Prp43/Pxr1, which provides an important insight in the mechanism of ribosome biogenesis. They also found that most annotated modifications do not change much upon stress or in the presence of ribosome inhibitors. These results solve several outstanding questions in understanding how potential ribosome heterogeneity and argue against the proposed involvement of rRNA modifications in stress response. The methodology can be used for other classes of RNA, which is important in view of the current interest in RNA modifications and their role in epitranscriptomic regulation.
-
Reviewer #2 (Public Review):
rRNA modifications have been proposed to be a main source of ribosome heterogeneity, and there has been much speculation of how co-occurrence of modification defects could both further exacerbate the heterogeneity, as well as amplify functional differences. Moreoever, there has been speculation about changes in the modification in response to different cellular states. Bailey et al directly address these questions by sequencing entire rRNA molecules using nanopore sequencing. The data not only show that most residues are modified to very high extent, but also demonstrate that most sites are independent of each other. Nevertheless, the authors do demonstrate some modification sites that are dependent on others. Some of these are readily explained by a shared snoRNA guide, but others are not. E.g., …
Reviewer #2 (Public Review):
rRNA modifications have been proposed to be a main source of ribosome heterogeneity, and there has been much speculation of how co-occurrence of modification defects could both further exacerbate the heterogeneity, as well as amplify functional differences. Moreoever, there has been speculation about changes in the modification in response to different cellular states. Bailey et al directly address these questions by sequencing entire rRNA molecules using nanopore sequencing. The data not only show that most residues are modified to very high extent, but also demonstrate that most sites are independent of each other. Nevertheless, the authors do demonstrate some modification sites that are dependent on others. Some of these are readily explained by a shared snoRNA guide, but others are not. E.g., modification of the exit tunnel is concerted. Whether this is due to concerted modification, or preferential stabilization of fully modified RNA is not explored, and to this reviewer this is not necessary.
Importantly, the authors do not find any evidence for a dynamic regulation of the modifications, which to this reviewer makes sense, because rRNAs are just too long lived for this to make sense as a way to respond to cellular stress.
Overall, the claims in the manuscript are supported by data, and they are interesting and novel. I have only very minor concerns, although I am not an expert in the nanopore technology, the computational analysis, or the machine learning part.
-
Reviewer #3 (Public Review):
In this study the authors developed a novel strategy to map nucleoside modifications by using Nanopore sequencing of the 25S and 18S rRNAs in yeast. By comparing Nanopore sequencing reads on in vitro transcribed RNAs and RNAs extracted from cells, the authors were able to identify all 110 annotated modifications in single, full-length ribosomal RNAs.
Overall, this is an impactful manuscript that informs the field on a new technique to detect rRNA modifications and offers important insights into subpopulations of ribosomes that are lacking certain modifications. The main highlights of this paper are (1) the single molecule, direct RNA sequencing approach to detect individual modifications along an entire rRNA molecule, (2) rRNA modification is coordinated at certain positions, , and (3) subpopulations of …
Reviewer #3 (Public Review):
In this study the authors developed a novel strategy to map nucleoside modifications by using Nanopore sequencing of the 25S and 18S rRNAs in yeast. By comparing Nanopore sequencing reads on in vitro transcribed RNAs and RNAs extracted from cells, the authors were able to identify all 110 annotated modifications in single, full-length ribosomal RNAs.
Overall, this is an impactful manuscript that informs the field on a new technique to detect rRNA modifications and offers important insights into subpopulations of ribosomes that are lacking certain modifications. The main highlights of this paper are (1) the single molecule, direct RNA sequencing approach to detect individual modifications along an entire rRNA molecule, (2) rRNA modification is coordinated at certain positions, , and (3) subpopulations of ribosomes accumulate that are missing one or more modifications. This manuscript is relevant from the perspective of ribosome assembly, in that it informs on the order and dependencies of rRNA modifications, as well as other factors (Dbp3 and Pxr1) that are necessary for proper modification. It is also relevant in the context of the "specialized ribosome" hypothesis by showing that ribosomes are heterogenous in modification status. Most of the nucleoside modifications analyzed are promoted by guide snoRNAs, and genetic depletion of protein components of the guide snoRNPs or knockout of guide snoRNAs result in the expected decrease in the modification profiles for most positions. Interestingly they show that 2'-O-methylation is largely independent from Pseudouridylation. Another important finding of the study is the correlation of modifications at distant sites that correspond to functionally important regions of the ribosome.
I found that most of the conclusions made by the authors are supported by the data, with the exception of a few experiments described below. This manuscript will represent a major resource for the community, as it provides a new standard and approach to map ribosomal RNA modifications on single rRNA transcripts, and I anticipate that it will become a widely used tool for the scientific community. Besides the technological innovation, the information obtained on the correlation of modification at specific positions is an important finding for the fields of ribosomes and translation. In terms of specificity of identification of modifications,
The only weakness of the manuscript lies in some of the genetic experiments used to assess the impact of the inactivation of specific factors or environmental conditions on modification patterns as described below - I found three specific issues.
The first issue is the use of the prp44 cold-sensitive (cs) mutant. The authors compare the modification patterns obtained for this cs mutant after a shift to non-permissive temperature. However, there is no control experiment done with a wild-type strain shifted to the same cold temperature, which is problematic as a basic control is missing. So it would be necessary to perform a control experiment with a wild-type strain shifted to a similar temperature. Also the dbp knockout analysis is performed at steady state while prp43-cs is a cold shift so it is quite difficult to compared the result directly.
Another issue that may need to be considered is the level of depletion of individual snoRNAs after depletion of the snoRNP proteins. It is possible that some snoRNAs are depleted more rapidly than others, and that this may affect the modification patterns. The authors should perform RNA sequencing of RNA samples used after depletion of Cbf5 or Nop58 such that they can directly correlate snoRNA levels to modification levels. Unless the authors provide these data, it is difficult to conclude whether specific sites are more or less resilient to genetic depletion of snoRNP proteins.
Finally, the title of the last section of the results is also misleading in terms of its conclusions ("Resilience of rRNA modification profiles to splicing perturbations and environmental treatments"). Regarding splicing perturbations, and with the exception of the dbr1 knockout, the mutants used in the study do not result in a major depletion of intron encoded snoRNAs so it is quite expected that there is no loss of modification at these positions. Similarly, the environmental stresses used are short, and are not expected to affect modification patterns in a major way considering the stability of ribosomes. Unless the authors perform sequencing on rRNAs synthesized after a shift into stress conditions, it is misleading to state that rRNA modification profiles are unaffected by environmental treatments. My feeling is that the paper is significant enough without the studies presented in the last paragraph, and that this paragraph and the data within should be removed from the manuscript because they are inconclusive, and the title is misleading.
I spent the last few paragraphs highlighting some of the issues that need to be addressed, but overall, I found that the article presents a major advance in the field and that it provides a landmark study in our understanding of nucleoside modifications in rRNA.
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