Rqc1 and other yeast proteins containing highly positively charged sequences are not targets of the RQC complex
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###Reviewer #3
The work by Barros et al. looks at the role of the Ribosome Quality Control pathway (RQC) in regulating the expression of endogenous messages containing polybasic sequences. Using ribosome profiling and western blotting, the authors show that proteins containing various types of polybasic sequences are not targeted by the RQC. The authors argue that one of the few endogenous RQC substrate, RQC1, is not regulated via the canonical RQC pathway, but by a Ltn1p-dependent post transcriptional mechanism.
The question of whether there are endogenous RQC substrates has previously been explored. With the exception of the few identified substrates, such as RQC1 (Brandman et al, 2012) and SDD1 (Matsuo et al., 2020), these studies largely concluded the RQC has a minimal regulatory role for endogenous messages, and is most likely …
###Reviewer #3
The work by Barros et al. looks at the role of the Ribosome Quality Control pathway (RQC) in regulating the expression of endogenous messages containing polybasic sequences. Using ribosome profiling and western blotting, the authors show that proteins containing various types of polybasic sequences are not targeted by the RQC. The authors argue that one of the few endogenous RQC substrate, RQC1, is not regulated via the canonical RQC pathway, but by a Ltn1p-dependent post transcriptional mechanism.
The question of whether there are endogenous RQC substrates has previously been explored. With the exception of the few identified substrates, such as RQC1 (Brandman et al, 2012) and SDD1 (Matsuo et al., 2020), these studies largely concluded the RQC has a minimal regulatory role for endogenous messages, and is most likely protecting cells from damage and environmental stressors. This idea is further supported by the observation that the RQC is non-essential under standard growth condition, but becomes synthetic lethal with translation inhibitors (Kostova et al, 2017, Choe et al, 2016). The work by Barros et al. comes to the same conclusions, and therefore it is unclear how this work contributes to the already established role of the RQC.
The authors also explore the regulation of RQC1 by the RQC and argue that this gene is regulated by Ltn1p in an RQC-independent way. However, mechanistic understanding of the proposed regulation is lacking, and the data are largely inconsistent with the previously published observations by Brandman et al, 2012.
Major points:
The authors use the dataset published by Pop et al., 2014 for their 27-29 nt no drug ribosome profiling analysis. However, these no-drug samples have been reported to exhibit surprising heterogeneity, and similarities with CHX-pretreated samples (see Hussmann et al., 2015 for detailed analysis). It is unclear how this heterogeneity can affect the analysis in the current manuscript, and whether the authors were aware of these caveats. Have the authors used independent datasets to confirm their observations? Have they excluded replicas that show CHX-like characteristics, such as A-site occupancy bias similar to CHX pretreated samples?
It is not clear what the purpose of the analysis presented in Fig 2 is, and how it is different from the modeling in the Park and Subramaniam 2019 paper? Are the authors using these parameters (TE, Kozak score, etc.) to show adaptations that minimize ribosome collisions?
Fig 3 - some of the selected examples (Dbp3, Yro2, Nop58) lack sufficient coverage in the region of interested highlighted in the right column for the short and/or long footprints. Since the data are insufficient to make conclusions about ribosome stalling and queuing, these examples should be excluded from the analysis.
Fig 4:
-Does ASC1 deletion cause frameshifting? Since the TAP-tag is C-terminal, it is possible that it is now out of frame, and therefore undetectable. Is it possible for the authors to introduce the tag on the N-terminus, and follow simultaneously the stalled nascent polypeptide (upon LTN1 deletion), and the full length protein?
-Is the putative stalling site of Dbp3 too close to the stat codon to cause collisions?
-Can the authors include a positive control, such as TAP-tagged Sdd1 to make sure their assay works and their strains and KOs behave as expected?
- Fig 5:
-What is causing the inconsistency with the Brandman et al., 2012 data about RQC-dependent regulation of RQC1? In the original paper, Rqc1p has an N-terminal FLAG tag, so the authors primarily follow the stalled nascent polypeptide, whereas the current study focuses on the full length protein. Can the authors compare the same construct (FLAG-tagged Rqc1p) in their strains, so it is an "apples to apples" comparison?
-Fig 5c bottom panel - the read coverage is too sparse to make a conclusion. This analysis should be removed.
-5 d, e. The comparison between the GFP-12R-RFP stalling reporter and RQC2-TAP is not fair. The GFP construct reports on the fate of the stalled nascent polypeptide, whereas the RQC1-TAP looks at the full-length protein, and remains blind to the putative stalling product. Can the authors change the location of the tag, and repeat the experiment now looking at the stalled nascent polypeptide for RQC1? In addition, the signal in Fig. 5e look saturated. Is it possible that no effect is observed simply because the TAP signal is out of the dynamic range for the assay?
Minor Comments:
- The introduction presents an overly simplistic view of ribosome stalling, arguing that stalling can be caused by polybasic stretches. We now know that stalling is much more complex, and there are many other factors, including the presence of non-optimal codon pairs, that cause ribosome collisions. Although the authors discuss these factors in their discussion, they should also be emphasized in the introductory paragraph.
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###Reviewer #2 In this manuscript, Barros et al. examine published ribosome profiling data in an effort to identify possible targets for ribosome-quality-control (RQC) process in yeast. They found that although many of the obvious mRNA features, such as polybasic sequences, appear to stall the ribosome, they in fact are not targets of RQC. The authors then went on to confirm these observations by western-blot analysis of a few candidate genes and observe that deletion of the RQC factors Ltn1 and Asc1 has no effect on the levels of the full-length protein products. The authors conclude that RQC has little to no endogenous targets in yeast. While I have no doubt about the authors' conclusions and most of their analyses, I have major issues with the originality of the manuscript.
The argument that RQC has little to no endogenous targets is …
###Reviewer #2 In this manuscript, Barros et al. examine published ribosome profiling data in an effort to identify possible targets for ribosome-quality-control (RQC) process in yeast. They found that although many of the obvious mRNA features, such as polybasic sequences, appear to stall the ribosome, they in fact are not targets of RQC. The authors then went on to confirm these observations by western-blot analysis of a few candidate genes and observe that deletion of the RQC factors Ltn1 and Asc1 has no effect on the levels of the full-length protein products. The authors conclude that RQC has little to no endogenous targets in yeast. While I have no doubt about the authors' conclusions and most of their analyses, I have major issues with the originality of the manuscript.
The argument that RQC has little to no endogenous targets is not new. Many groups, including the authors' one, made the same arguments before. The authors recently published a paper in the Biochemical Journal "Influence of nascent polypeptide positive charges on translation dynamics". In particular, the analysis in that paper appears similar to the one carried out here. Furthermore, the Guydosh group made similar arguments in their recent paper (Meyden and Guydosh, Mol Cell).
The authors conclude their abstract by stating that "our results suggest that RQC should not be regarded as a general regulatory pathway for gene expression". To the best of my knowledge, RQC has not been regarded as such and instead the consensus has been that the process is a quality control one (as the name suggests).
The authors use LTN1 and ASC1 deletions to determine whether certain sequences are RQC targets or not. But for the ltn1D, instead of looking at the stabilized shorter products, the authors only looked at the full-length one. Ltn1 has no effect on readthrough on stalling sequences. A better deletion should have been that of HEL2.
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###Reviewer #1 In this manuscript the authors use existing high throughput data sets and perform some new experiments to explore in yeast potential physiological substrates of RQC. In a first step, they use bioinformatics to identify genes with features previously implicated in RQC (usually with reporter assays) including inhibitory codon pairs, poly-basic stretches, and poly-A tracts. With these genes in hand, they characterized various features of "translatability", using existing ribosome profiling data sets, and concluded that with the exception of the ICPs, that there were no strong signatures indicative of reduced ribosome density that might have evolved to deal with problematic ribosome queueing. The authors then looked at the RP data at higher resolution, looking for characteristic patterns of RPF distribution around the pausing …
###Reviewer #1 In this manuscript the authors use existing high throughput data sets and perform some new experiments to explore in yeast potential physiological substrates of RQC. In a first step, they use bioinformatics to identify genes with features previously implicated in RQC (usually with reporter assays) including inhibitory codon pairs, poly-basic stretches, and poly-A tracts. With these genes in hand, they characterized various features of "translatability", using existing ribosome profiling data sets, and concluded that with the exception of the ICPs, that there were no strong signatures indicative of reduced ribosome density that might have evolved to deal with problematic ribosome queueing. The authors then looked at the RP data at higher resolution, looking for characteristic patterns of RPF distribution around the pausing site, and found that the striking patterns seen previously for Sdd1 (and for reporter analysis in D'Orazio et al. eLife) were not recapitulated for any of the top candidates in their list. In a final set of experiments, the authors took advantage of TAP-tagged variants of their proteins of interest and asked whether deletion of Asc1 or Ltn1 impacted protein levels - and found that there were no discernible effects (though validation with TAP-tagged Sdd1 is an important missing control). Importantly, expression of full length Rqc1 (previously argued to be a direct target of the RQC) was unaffected by RQC components including Asc1, Hel2 and Rqc2, but was strongly impacted by Ltn1. These data together argue for an RQC-independent role for Ltn1 in regulating Rqc1 expression.
Overall, the manuscript was thought provoking for consideration of what might be natural targets of RQC, and in the end, one would conclude that natural targets of RQC are not encoded in the genome, but may instead be predominantly either prematurely polyadenylated mRNA substrates that escape nuclear QC, or instead, ubiquitous damaged mRNAs in the cell. In general, the discussion of the analysis of RP data indicated naivete about the identity of different RPF sizes and their relevance to mechanism (this could be corrected easily in a revised version). In the end, this manuscript brings important questions to the table, and provides some reasonable evidence to suggest that natural poly-basic stretches, including the one found in Rqc1, are not targets for the RQC under normal conditions. Moreover, the data support a non-canonical role for Ltn1 in regulating expression of Rqc1 which needs to be more fully explored. Importantly, however, what is critical to support the negative results surrounding Rqc1 is a demonstration of a role for RQC for Sdd1, around which the narrative is constructed (this gene exhibits characteristics by RP of being a target and is reported previously to be impacted by the relevant genes Asc1 etc.).
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##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 4 of the manuscript.
###Summary:
There were substantial concerns about the novelty of the study, the choice of RP libraries, coverage depth, and analysis of the ribosome profiling data. Previous studies have argued that there are very few endogenous targets (so far Rqc1 and Sdd1) of the RQC pathway, and that this is rather a QC pathway for damaged mRNAs. While we appreciate that your studies were inconsistent with these earlier studies, it will be critical for you to replicate those experiments, using protein tags that allow you to follow …
##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 4 of the manuscript.
###Summary:
There were substantial concerns about the novelty of the study, the choice of RP libraries, coverage depth, and analysis of the ribosome profiling data. Previous studies have argued that there are very few endogenous targets (so far Rqc1 and Sdd1) of the RQC pathway, and that this is rather a QC pathway for damaged mRNAs. While we appreciate that your studies were inconsistent with these earlier studies, it will be critical for you to replicate those experiments, using protein tags that allow you to follow the fate of both the full length and truncated species. Additionally, it will be important to validate using your own approaches and reagents that Sdd1 is indeed a substrate for RQC, given that your data suggest that Rqc1 itself is not. Finally, the novel Ltn1-dependent, RQC-independent pathway proposed to regulate Rqc1 expression requires further mechanistic work.
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