Profiling of terminating ribosomes reveals translational control at stop codons

Reviewer #1 (Public review):

Summary:

The authors use high-resolution ribosome profiling (Ezra-seq) and eRF1 pulldown-based ribosome profiling (eRF1-seq) developed in their lab to identify a GA rich sequence motif located upstream of the stop codon responsible for translation termination pausing. They then perform a massively parallel assay with randomly generated sequences to further characterize this motif. Using mouse tissues, they show that termination pausing signatures can be tissue-specific. They use a series of published ribosome structures and 18S rRNA mutants, and eS26 knockdown experiments to propose that the GA rich sequence interacts with the 3′-end of the 18S rRNA.

Strengths:

(1) Robust ribosome profiling data and clear analyses clarify the subtle behavior of terminating ribosomes near the stop codon.

(2) Novel termination or "false termination" sites revealed by eRF1-seq in the 5′-UTR, 3′-UTR, and CDS highlight a previously underappreciated facet of translation dynamics.

Weakness:

(1) Modest effects seen in ABCE1 knockdown do not seem to add up to the level of regulation. The authors state "ABCE1 regulates terminating ribosomes independent of the sequence context" on pg 9, and "ABCE1 modulates termination pausing independent of the mRNA sequence context" in the figure caption for Figure S4. Given the modest effect of the knockdown, such phrasing is most likely not supported. Further clarification of "ABCE1 plays a generic role in translation termination" is necessary.

(2) The authors propose that the GA rich sequence element upstream of the stop codon on the mRNA could potentially base pair with the 3′-end of the 18S rRNA. In the PDBs the authors reference in their paper and also in 3JAG, 3JAH, 3JAI (structures of terminating ribosomes with the stop codon in the A-site and eRF1), the mRNA exiting the ribosome and the 3′-end of the 18S rRNA are about 25-30 A apart. In addition, a segment of eS26 is wedged in between these two RNA segments. This reviewer noted this arrangement in a random sampling of 5 other PDBs of mammalian and human ribosome 80S structures. How do the authors anticipate the base pairing they have proposed to occur in light of these steric hindrances? RpsS26 is known to be released by Tsr2 in yeast during very specific stresses. Is it their expectation that termination pausing in human/mammalian cells happens during stressful conditions only?

(3) The authors say, "It is thus likely that mRNA undergoes post-decoding scanning by 18S rRNA." (pg. 10). It is unclear what the authors mean by "scanning." Do they mean that the mRNA gets scanned in a manner similar to scanning during initiation? There is no evidence presented to support that particular conclusion.

(4) Role of termination pausing in the testis is highly speculative. The authors state: "It is thus conceivable that the wide range of ribosome density at stop codons in testis facilitates functional division of ribosome occupancy beyond the coding region." It is unclear what type of functional division they are referring to.

Reviewer #2 (Public review):

Summary:

This paper presents results interpreted to indicate that sequences upstream of stop codons capable of base-pairing with the 3' end of 18S rRNA prolong the dwell time of 80S ribosomes at stop codons in a manner impeded by Rps26 in the 40S subunit exit channel, which leads to the proper completion of termination and ribosome recycling and prevents spurious translation of 3'UTR sequences by one or more unconventional mechanisms.

Strengths:

The standard 80S and selective eRF1 80S ribosome profiling data obtained using EZRA-Seq are of high quality, allowing the authors to detect an enrichment for purine-rich sequences upstream of stop codons at sites where termination is relatively slow and ribosomal complexes are paused with eRF1 still engaged in the A site.

Weaknesses:

There are many weaknesses in the experimental design, interpretation of results, and description of assay design and assumptions, the data obtained, and the interpretation of results, all of which detract from the scientific quality and significance of this work. In fact, a large proportion of paragraphs in the text and figure panels present some difficulty either in understanding how the experiment or data analysis was conducted or what the authors wish to conclude from the results, or that stem from an overinterpretation of findings or failure to consider other equally likely explanations.

Reviewer #3 (Public review):

Summary:

This study from Jia et al carried out a variety of analyses of terminating ribosomes, including the development of eRF1-seq to map termination sites, identification of a GA-rich motif that promotes ribosome pausing, characterization of tissue-specific termination dynamics, and elucidation of the regulatory roles of 18S rRNA and RPS26. Overall, the study is thoughtfully designed, and its biological conclusions are well supported by complementary experiments. The tools and datasets generated provide valuable resources for researchers investigating the mechanisms of RNA translation.

Strengths:

(1) The study introduces eRF1-seq, a novel approach for mapping translation termination sites, providing a methodological advance for studying ribosome termination.

(2) Through integrative bioinformatic analyses and complementary MPRA experiments, the authors demonstrate that GA-rich motifs promote ribosome pausing at termination sites and reveal possible regulatory roles of 18S rRNA in this process.

(3) The study characterizes tissue-specific ribosome termination dynamics, showing that the testis exhibits stronger ribosome pausing at stop codons compared to other tissues. Follow-up experiments suggest that RPS26 may contribute to this tissue specificity.

Weaknesses:

The biological significance of ribosome pausing regulation at translation termination sites or of translational readthrough, for example, across different tissue types, remains unclear. Nevertheless, this question lies beyond the primary scope of the current study.

Reviewer #4 (Public review):

Summary:

This manuscript by Qian and colleagues utilizes ribosome profiling, and reporter assays to dissect translation termination. Unfortunately, the data do not support the conclusions of the paper, controls are missing and several assays are not well validated and do not reproduce previous findings from others.

Specific comments:

• Translation termination has been studied in several organisms including mammalian cells and yeast. In those cases what is analyzed is not the peak height at the stop codon, but rather the difference in the ribosome density before and after the stop. Thus, analyzing peak height is not validated. I understand that this is relevant only for the ribosome profiling experiments (and Ezra-seq) not the RF1 profiling. But much of the data was acquired that way.

• Moreover, the data do not reproduce previous findings and no effort is made to connect them to previous data. Previous data has shown that stop codon efficacy varies. This is not reproduced (S1C). Similarly, an effect from the +1 residue is not reproduced. The data isn't even stratified by different stop codons as previous work has shown that different surrounding residues have different effects in the context of different stop codons. Thus, none of the sequencing data is validated or trusted and does not reproduce previous findings.

• The GA-rich sequence identified by Ezra-Seq and RF1 seq is not the same and it differs from previous sequences (Wangen &Green).

• The authors claim that the majority of Rf1 peaks is at stop codons, but that is not true. It is only about 30% of the peaks. Also, not all mRNAs have peaks at the stop codons. That is at best problematic. Finally, there are mRNAs that are known to "suffer" from NMD, what do these look like in the Ezra-Seq and RF1-Seq? How about mRNAs that have programmed frameshifts? This raises questions on the validity of the eRF1 data.

• Figure 4: First, instead of M/P ratio, one should analyze M/M+P, to normalize out differences in the loading and effects from collisions, which are guaranteed to occur here, but not considered or analyzed. Second, the data are analyzed as if what matters are codons in the P and E site (and beyond, where there are definitely NOT recognized codons). While there is evidence for some interactions, one would think that an additional analysis based on sequence would be helpful. Also, the supplemental data indicates that very rarely are there reciprocal changes (as should be the case), and as seen for stop codons.

• Regarding the HiBit reporter assay: The two sequecnes clearly have effects on translation without considering stop codon context (Figure 4C), which need to be taken into account. Also, the effect from the sequences varies in the context of the assay in 4C and 4D (2-fold vs .5 fold), further questioning the assay. Moreover, the authors claim that re-initiation cannot account for Hibit levels, but that is clearly incorrect. The western in Figure 4E does not reproduce the data in 4D. While Hibit goes up (as in 4D, the putative GFP-fusion goes down. Finally, while the second reading frame should be more efficient is not explained and further argues for an artifact. Previous work (and work herein) suggests that read-through occurs equally in each reading frame. No controls for these assays are presented: e.g. stimulation by antibiotics, ABCE1 depletion, etc.

• Figure 5 has similar problems. I don't understand how the Figure in 5A is made, but when you overlay the cited structures on Rps26, the molecules are identical. I guess the authors used some fantasy to build non-existing sequences differently into the structure. There is no basis for that. In panel C and the same in Figure 7, the number of analyzed mRNAs varies. This could influence the outcome and the EXACT same set of mRNAs should be analyzed. But the main problem here is that the authors need to analyze readthrough and not peak height as detailed above. Essential controls are missing that show what fraction of the 18S rRNA is mutated. Previous work has shown that 2 nt truncated 18S rRNA is actively degraded. It is hard to believe how 15% of altered ribosomes can abolish 100% of the effect from the C-rich sequences. Important validation is missing: the authors should analyze rRNA sequences in their ribo-seq dataset to demonstrate that they have the mutated rRNAs, and that these enrich and de-enrich as predicted.

• In Figure 5-7 the authors develop a model that the sequence selectivity arises from base pairing between 18S rRNA and the mRNA. If so, then they should really stratify the data by number of WC pairs that can be formed. And only WC pairs, as GU pairs have a totally different geometry that will likely be discriminated against in this context. Also, the mutation is in a part of the helix that has no effect (Figure S3G). Thus, the data within the manuscript are inconsistent.

• Figure 6 does not agree with published data (Li et al., Nature 2022). Previous work did not show testis-depletion of Rps26 in purified ribosomes. This is the critical difference as the authors here did not purify ribosomes. Also, another Rps is an essential control, even if purified ribosomes are used. The validity of this dataset is thus questionable . Depletion from polysomes is hard to believe, as overall there is less signal in the polysomes.

• Figure 7 has similar problems as figure 5. Different pools of mRNAs are analyzed; peak height is not validated. Overexpression of Rps26 is not shown, as only Myc is shown, not Rps26. Beyond that, increased occupancy in ribosomes needs to be shown for the effect to come from ribosomes. Given how sick the cells are it is most likely that all effects are secondary and arise from whatever else is going on in the overexpression or depletion of Rps26. No controls are presented to show specific effects from Rps26.

• The authors need to check Rli1/ABCE levels in their cells. Their data have features that are indicative of low ABCE1 levels. These include a very small effect from ABCE1 depletion. These could be responsible for some of the effects they observe.

Author response:

We thank the editor and reviewers for their thoughtful feedback. We agree with eLife’s overall assessment that, while profiling terminating ribosomes is informative in revealing termination dynamics, the underlying mechanisms require more evidence. Our revision will focus on three conceptual points.

(1) We will tone down the statement that putative mRNA:rRNA interaction contributes to sequence-specific termination pausing.

(2) We will clarify the potential role of Rps26 in regulating translation termination.

(3) We will expand the discussion of tissue-specific termination pausing.

Reviewer #1 (Public Review):

(1) We admit that the modest effects of ABCE1 were partly due to the incomplete ABCE1 knockdown in HEK293 cells. Since the elevated ribosome density occurred at all stop codons, we argue that the action of ABCE1 is likely independent of the sequence context. We will rephrase relevant statements in the revised manuscript.

(2) In terms of Rps26 structures, we agree the structural rearrangement in the absence of Rps26 is highly speculative. However, we do not believe the Rps26 stoichiometry is solely dependent on stress. We will clarify this important point in the revised manuscript.

(3) We apologize for the confusion about 18S rRNA “scanning” and will revise the sentence in the main text.

(4) We agree that functional significance of testis-specific termination dynamics is unclear. Since other reviewers raised similar concern, we will expand the discussion of tissue-specific termination pausing in the revised manuscript.

Reviewer #2 (Public Review):

We appreciate the Reviewer’s time and efforts in reviewing our manuscript. We are grateful for the insightful comments and many recommendations made by the reviewer to improve our manuscript. We feel that the reviewer may have some misunderstanding in terms of the sequence motif associated with the termination pausing, partly because of the lack of clarity in our original description of the results from MPRA and reporter assays. We will ensure that the reviewer’s points are fully addressed in the revised manuscript.

Reviewer #3 (Public Review):

We thank the reviewer’s positive comment on our manuscript. We agree that the tissue-specific termination differences were poorly described in the main text. Notably, other reviewers raised similar concerns. We will expand the relevant discussion in the revised manuscript, outlining this as a limitation and a future direction.

Reviewer #4 (Public Review):

We believe the reviewer mixed xthe public view with recommendation comments. The reviewer appears to be preoccupied by previous studies and questioned some inconsistency in our results. With the development of new technology such as eRF1-seq, we are encouraged to present “new” and “different” findings. All other reviewers appreciate the development of eRF1-seq to profile terminating ribosomes. In fact, we do not believe our data is fundamentally different from the established principles. Rather, our data provides new perspectives to further our understanding of ribosome dynamics at stop codons. We thank the reviewer for understanding.

The reviewer is quite confused by our sequencing analysis based on peak height, or read density, which is commonly used to infer ribosome dynamics such as pausing. Regarding the sequencing analysis and reporter assays in cells expressing 18S mutant (Figure 5) and Rps26 (Figure 7), we feel that the reviewer has some misunderstanding. In the revised manuscript, we will do our best to clarify those relevant issues. Finally, the reviewer’s comment on base pairing is well-received and we will thoroughly revise the main text and discussion in the revised manuscript.