No Ramp Needed: Spandrels, Statistics, and a Slippery Slope

Reviewer #1 (Public Review):

The manuscript by Sejour et al. is testing "translational ramp" model described previously by Tuller et al. in S. cerevisiae. Authors are using bioinformatics and reporter based experimental approaches to test whether "rare codons" in the first 40 codons of the gene coding sequences increase translation efficiency and regulate abundance of translation products in yeast cells. Authors conclude that "translation ramp" model does not have support using new set of reporters and bioinformatics analyses. The strength of bioinformatic evidence and experimental analyses of the rare codons insertion in the reporter make compelling case for authors claims. However major weakness of the manuscript is that authors do not take in account other confounding effects in their analyses as well as multiple previous studies that argue with "translation ramp" model. The existence of the early elongation ramp with "rare codons" was previously contested with local mRNA structure at the start codon, peptidyl-tRNA drop-off or interactions of the nascent peptide chain with exit channel of the ribosome models. All of these effects are not considered or discussed in the manuscript at this point. Such an authors approach makes the manuscript rather biased and short on discussing multiple other possible conclusions on reasons of slow translation elongation at the beginning of the protein synthesis.

Reviewer #2 (Public Review):

Tuller et al. first made the curious observation, that the first ∼30-50 codons in most organisms are encoded by scarce tRNAs and appear to be translated slower than the rest of the coding sequences (CDS). They speculated that this has evolved to pace ribosomes on CDS and prevent ribosome collisions during elongation - the "Ramp" hypothesis. Various aspects of this hypothesis, both factual and in terms of interpretating the results, have been challenged ever since. Sejour et al. present compelling results confirming the slower translation of the first ~40 codons in S. cerevisiae but providing alternative explanation for this phenomenon. Specifically, they show that the higher amino acid sequence divergence of N-terminal ends of proteins and accompanying lower purifying selection (perhaps the result of de novo evolution) is sufficient to explain the prevalence of rare slow codons in these regions. These results are an important contribution in understanding how aspects of evolution of protein coding regions can affect translation efficiency on these sequences and directly challenge the "Ramp" hypothesis proposed by Tuller et al.

I believe the data is presented clearly and the results generally justify the conclusions. I do have one specific concern related to interpretating the data. The authors show that the conservation score of the last 40 codons is not dissimilar to the conservation score of the first 40 (Fig. 4 A & C). They also show that the calculated translational speed of the first 40 codons is significantly lower than the rest of the CDS. At the same time, they show lack of statistically significant decrease of calculated translational speed for the last 40 codons (Figure S1). If the poor conservation of the first 40 codon explains the slower speed of their translation what is the authors' explanation for the absence of statistically significant reduction of calculated translational speed for the last 40 codons?

"Although the reporter is GFP, the N- terminal region of this particular protein is derived from yeast HIS3, not GFP, and has little if any effect on the fluorescence of the GFP fused downstream."

The statement above is logical and reasonable; however, it is not supported by any reference or control experiments. At the very least this fact should be explicitly acknowledged. Also, the RNA levels of reporters were not measured, which means it cannot be categorically concluded that the observed effect is due to changes of translational efficiency. This is an important caveat.

Author Response

We thank the reviewers for their comments, and their evident close reading of the manuscript. Generally, we agree with the reviewers on the strengths and weaknesses of our manuscript. We plan to submit a revised version which has a more extensive discussion of alternative explanations for initial high ribosome density as seen by ribosome profiling, and which more specifically points out the limitations of our work.

As a preface to specific responses to the reviewers, we will say that we could divide observations of slow initial translation into two categories, which we will call “encoded slow codons”, and “increased ribosome density”. With respect to the first category, Tuller et al. documented initial “encoded slow codons”, that is, there is a statistical excess of rare, slowly-translated codons at the 5’ ends of genes. Although the size of this effect is small, statistical significance is extremely high, and the existence of this enrichment is not in any doubt. At first sight, this appears to be a strong indication of a preference for slow initial translation. In our opinion, our main contribution is to show that there is an alternative explanation for this initial enrichment of rare, slow codons—that they are a spandrel, a consequence of sequence plasticity at the 5’ (and 3’) ends of genes. The reviewers seem to generally agree with this, and we are not aware that any other work has provided an explanation for the 5’ enrichment of rare codons.

The second category of observations pertaining to slow initial translation is “increased ribosome density”. Early ribosome profiling studies used cycloheximide, and these showed a much greater density of ribosomes near the 5’ end of genes than elsewhere. This high initial ribosome density helped motivate the paper of Tuller et al., though their finding of “encoded slow codons” could explain only a very small part of the increased ribosome density. More modern ribosome profiling studies do not use cycloheximide as the first step in arresting translation, and in these studies, the density of ribosomes near the 5’ end of genes is greatly reduced. And yet, there remains, even in the absence of cycloheximide at the first step, a significantly increased density of ribosomes near the 5’ end (e.g., Weinberg et al., 2016). (However, at least some of these studies do use cycloheximide at later steps in the protocol, and the possibility of a cycloheximide artefact is difficult to exclude.) It appears to us that some of the reviewer’s main concerns are that we do not explain the increased 5’ ribosome density seen by ribosome profiling. We agree; but we feel it is not the main point of our manuscript. In revision, we will more extensively discuss other work on increased ribosome density, and more explicitly point out the limitations of our manuscript in this regard. We also note, though, that increased ribosome density is not a direct measure of translation speed—it can have other causes.

Specific Responses.

Reviewer 1 was concerned that we did not more fully discuss other work on possible reasons for slow initial translation. We will discuss such work more extensively in our revision. However, as far as we know, none of this work proposes a reason for the 5’ enrichment of rare, slow codons.

Reviewer 1 was also concerned about confounding effects in our reporter gene analysis of the effects of different codons on efficiency of translation. We have two comments. First, it is important to remember that although we changed codons in our reporters, we did not change any amino acids. We changed codons only to synonymous codons. Thus at least one of the reviewer’s possible confounding effects—interactions of the nascent peptide chain with the exit channel of the ribosome—does not apply. However, of course, the mRNA nucleotide sequence is altered, and this would cause a change in mRNA structure or abundance, which could matter. We agree this is a limitation to our approach. However, to fully address it, we feel it would be necessary to examine a really large number of quite different sequences, which is beyond the scope of this work.

Reviewer 2 was concerned that the conservation scores for the 5’ 40 amino acids, and the 3’ 40 amino acids were similar, but slow translation was only statistically significant for the 5’ 40 amino acids. As we say in the manuscript, we are also puzzled by this. We note that 3’ translation is statistically slow, if one looks over the last 100 amino acids. Our best effort at an explanation is a sort of reverse-Tuller explanation: that in the last 40 amino acids, the new slow codons created by genome plasticity are fairly quickly removed by purifying selection, but that in the first 40 amino acids, for genes that need to be expressed at low levels, purifying selection against slow codons is reduced, because poor translation is actually advantageous for these genes. To expand on this a bit, we feel that the 5000 or so proteins of the proteome have to be expressed in the correct stoichiometric ratios, and that poor translation can be a useful tool to help achieve this. In this explanation, slow translation at the 5’ end is bad for translation (in agreement with our reporter experiments), but good for the organism, whereas in Tuller, slow translation at the 5’ end is good for translation.

Reviewer 2 wondered whether the N-terminal fusion peptide affects GFP fluorescence in our reporter. This specific reporter, with this N-terminus, has been characterized by Dean and Grayhack (2012), and by Gamble et al. (2016), and the idea that a super-folder GFP reporter is not greatly affected by N-terminal fusions is based on the work of Pedelacq (2006). None of these papers show whether this N-terminal fusion might have some effect, but together, they provide good reason to think that any effect would be small. We will add these citations to the revision.