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  1. Evaluation Summary:

    This manuscript will interest a large community of molecular biologists studying translation and mRNA decay. The study provides a large-scale comparison of the roles of protein factors in No-Go Decay (NGD) and Codon-Optimality-Mediated Decay (COMD) in the yeast S. cerevisiae. A major strength of the manuscript is the direct comparison between one mRNA with a single strong translational stall and another similar mRNA with many slow translation sites (caused by changes in the genetic code). The analysis of both the factors that cause decay of these mRNAs as well as the ribosome states on the different mRNAs has the potential to reveal the molecular basis for the different mechanisms of mRNA quality control.

    (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. The reviewers remained anonymous to the authors.)

  2. Reviewer #1 (Public Review):

    In this study, the yeast haploid deletion library was screened for altered expression of a GFP reporter containing an array of rare Arg codons known to induce No-Go-Decay (NGD). Mutants lacking Slh1 or components of the RQT machinery (Hel2, Cue3, Rqt3) were found to decrease expression of this "CGA" reporter relative to an OPT reporter lacking the Arg codons, consistent with the prevailing view that increased collided ribosomes in these mutants will accelerate NGD. In contrast, mutants lacking Syh1, Smy2 (yeast orthologs of mammalian NGD factors GIGYF1/2) or Asc1 show elevated CGA/OPT ratios, indicating reduced NGD. Most, if not all of these factors were implicated previously in yeast NGD. After verifying the effects of the deletions of these genes, I believe by re-creating the deletions de novo, and examining reporter expression by flow cytometry and Northern analysis, they move on to examine double or triple mutants of some of these genes, as well as deletion of the CUE2 endonuclease implicated previously in NGD as a backup to the primary Xrn1-mediated pathway that comes into play when Slh1 is missing, using a different CGA-repeat reporter based on the HIS3 gene. These results support a role for Syh1 in NGD, and for Slh1 in reducing collided ribosomes in a manner that diminishes NGD. The most dramatic results involve a strong synthetic effect of deleting SYH1 together with either CUE2 or HEL2. This leads them to propose that Cue2/Hel2 and Syh1/Xrn1 represent two independent pathways for NGD, although they wish to conclude that Syh1 represents the major pathway in WT cells, and that Cue2 functions only when the ability of Slh1 to diminish collided ribosomes is overwhelmed. They go on to show that deleting MBF1 or EAP1 has no detectable effect on the NGD reporter, indicating that these yeast orthologs of EDF1 and (possibly) 4EHP, respectively, do not function in NGD in yeast in the manner observed for the corresponding mammalian factors. The next notable finding is that deletions of HEL2, SYH1, or both genes, which impair NGD, have no effect on a separate reporter (NONOPT), whose rapid turnover in WT cells is driven by a high level of non-optimal codons. Deletion of NOT5 strongly stabilizes the NONOPT promoter, as shown previously, but also stabilizes the CGA reporter to a degree comparable to deleting SYH1, providing the first evidence that Not5 contributes to NGD as well. Finally, they perform 80S and disome ribosome profiling on the two different reporters. In WT cells, they see a large accumulation of both monosomes and disomes at the CGA codons of the CGA reporter, and very low levels of either species downstream of the CGA block, and both monosomes and disomes are tightly packed and thus appear to be in collisions. In agreement with previous results, 80S monosomes escape the CGA block in cells lacking Hel2, and they see a loss of close-packing upstream of the block, from which they conclude that Hel2 helps to stabilize disomes and facilitate their removal from the stall site. On the NONOPT reporter they do not appear to observe a higher than average disome occupancy; however, they see an increased ratio of ribosomes with 21nt vs 28 nt footprints, indicating accumulation of ribosomes with empty A sites. Interestingly, they also observe a very high 21/28 nt ratio within the CGA repeats of the CGA reporter. From these results they conclude that ribosomes collided at a strong barrier to elongation (eg. CGA repeats) trigger both NGD and mRNA turnover elicited by nonoptimal codons (COMD), whereas COMD is triggered by slowly elongating ribosomes with empty A sites. The Not5 contribution to the turnover of the CGA reporter can be rationalized by the high-level of ribosomes with empty A sites that are arrested in the CGA array.

  3. Reviewer #2 (Public Review):

    Veltri and colleagues manuscript, "Distinct ribosome states trigger diverse mRNA quality control pathways" reports on a study designed to compare and contrast the proteins required for NGD and COMD and the ribosomal conformations associated with each pathway in yeast.

    The study has many notable strengths, including a large-scale screen to quantitate the effects of deleting yeast genes on NGD and COMD. These screens use fluorescent reporters designed to trigger NGD (due to CGA and AAA repeat stall sequences) and COMD (due to a high frequency of rare codons). Positive hits from this screen were validated independently. In addition, the authors used careful Northern blot analysis to quantitate reporter RNA levels and turnover rates. Finally, the use of high-resolution, conformation-specific ribosome profiling on reporters correlated ribosome configurations to specific decay pathways. These powerful methods were combined with thorough and careful analyses. There are also weaknesses to the manuscript, including some sections that are not completely clear. There are also limitations in the genetic screen, which covered ~80% of viable gene deletions, and failed to identify many previously characterized COMD factors or any novel factors in either NGD or COMD.

    i. Overall, the experiments described do provide a broad comparison of the proteins required for NGD and COMD and correlate ribosomal conformations with these decay pathways on specific reporter mRNAs. The authors conclude that the two pathways are largely independent, at least in yeast, and find that some of the components of human NGD do not have functional orthologs in yeast. These conclusions are well supported by their results.

    i. The experiments are well executed and I expect this manuscript should have a moderate to strong impact on the fields of translation and mRNA decay. On the one hand, the conclusions are largely consistent with recent work. For example, Not5 has been reported to be the key link between rare codons and mRNA turnover in yeast COMD (Buschauer et al., 2020). Buschauer et al. also revealed that ribosome structures consistent with open A and E sites create the Not5 binding site. Similarly, the importance of Syh1 in NGD was reported by Hickey et al., by some of the authors of this manuscript, and previous work has shown the structures formed by ribosome collisions which induce NGD. The main impact of this manuscript is that it suggests the NGD and COMD pathways are largely separate, at least in yeast under rich media growth conditions, and identifies Syhl as a primary effector of NGD. This is important because both NGD and COMD are involved in coupling translation to mRNA decay. The authors also report that some components that contribute to human NGD are not involved in yeast NGD. The data used in the study also have the potential to be useful for future work, as they include high-resolution ribosome profiling data (21-mer and 28-mer monsomes and disome footprints) from wildtype and mutant yeast.

  4. Reviewer #3 (Public Review):

    The molecular basis for distinct mechanisms of mRNA quality control is poorly understood and an important problem in gene expression. In particular, the molecular mechanisms that act in response to a strong translation stall appear to differ from the mechanisms that act in response to continued slow translation. In this manuscript, a genetic screen revealed a quantitatively significant role for Syh1 in decay of an mRNA with a strong translation stall; similar effects of Syh1 on the same reporter had been reported in a 2020 publication from this laboratory. Here, they find that Syh1 function in mRNA decay does not require function of the major NGD regulator Hel2, of the NGD endonuclease Cue2 or of the ribosome disassociation factor Slh1. By contrast, none of these factors affect mRNA stability of a reporter in which translation elongation is likely uniformly slowed by suboptimal codons, but this reporter is a target of COMD (codon optimality mediated decay) and is stabilized by deletion of NOT5. The surprising result is that the strong stalling reporter is also regulated by Not5, in a manner quantitatively similar that of Syh1. Thus, mRNA stability of the strong stalling reporter is regulated by both NGD and COMD. To understand the molecular basis for recruitment of distinct decay factors, the authors investigate the ribosome states on these reporters using ribosome profiling. In the reporters without a strong stall site, single ribosomes and collided ribosomes are uniformly positioned across the coding sequence, while in the reporter with strong stall (due to CGA codons), ribosomes are absent from the region downstream of the CGA repeats and collided ribosome are substantially increased and stacked at the CGA repeat (compared to the OPT reporter). In addition, ribosomes lacking tRNA from the A site are enriched on the slowly translated reporter. The authors infer that the distinct ribosome signals of collided ribosomes or ribosomes with empty A sites are strong determinants of the factors that lead to mRNA decay.

    A major strength of the manuscript is the direct comparison between one mRNA with a single strong translational stall and another similar mRNA with many slow translation sites (caused by changes in the genetic code). The analysis of both the factors that cause decay of these mRNAs as well as the ribosome states on the different mRNAs has the potential to reveal the molecular basis for the different mechanisms of mRNA quality control.

    The genetic analysis is complicated and not completely consistent with the claim in the abstract that "Syh1 acts as the primary link to mRNA decay in NGD". While deletion of SYH1 does stabilize mRNA with a strong stalling site, the deletion of both SYH1 and genes encoding other factors known to be in the NGD pathway results in much greater stabilization of the mRNA with the strong stalling site. In the discussion, the authors correctly interpret this result as evidence of two independent mechanisms by which NGD is triggered. This is one of the most novel results presented in this manuscript, but is not followed up and leaves some questions about the primary role of Syh1 in NGD. Subsequent findings that neither Syh1 nor Hel2 are involved in decay of an mRNA encoded with non-optimal codons, that Not5 is involved in decay of mRNAs with non-optimal codons or strong stalls are convincing. The analysis of ribosome states on the same reporters in wild type and mutant strains provides clear and convincing differences in the ribosome states and distribution across the different reporters, which are likely to provide mechanistic insights into the distinct pathways. However, in the absence of any evidence of a causal relationship between these differences in ribosomal state and the difference in mRNA decay, the paper lacks sufficient support for the title "distinct ribosome states trigger diverse mRNA quality control pathways."