PREreview of Newly synthesized mRNA selectively escapes translational repression following acute stress

In this manuscript, the authors use pulse chase analysis and ribosome footprinting to investigate the transcriptional and translational changes in Saccharomyces during starvation stress and ultimately conclude that transcripts produced after the start of stress response escape translational repression. I believe this manuscript includes valuable observations and introduces an interesting starting point to ultimately understand the molecular mechanism by which certain transcripts evade translational repression. However, the way some of the data was displayed makes interpretation difficult and raises questions about the analysis of the data. I will discuss some concerns below and suggest an orthogonal experiment that would greatly improve the manuscript.

Major points -

(1)    I want to make the authors aware of the paper Translational Control during Early Dictyostelium Development: Possible Involvement of Poly(A) Sequences (Palatnik, Wilkins and Jacobson, 1984). This manuscript uses very different methods but reaches similar conclusions to the cited work. Moreover, this work argues the critical issue is the length of the poly(A) tail, which was not examined in this work.  It’s important to recognize previously published papers and these two works in relation to each other will enrich each other and the field. Also, this paper might provide some ideas for the researchers moving forward by showing the likely mechanism of discrimination.

(2)    While ribosome footprinting can be a useful method to indirectly quantify translation, it has the limitation of being dependent on the number of that particular transcript in the cell and does not necessarily quantify production of complete and functional protein. Since the authors have already produced a citrine mRNA reporter (sup. Fig. 2), I suggest quantifying fluorescence as a direct read-out for translation of citrine. This would be an easy experiment that would ultimately solidify the results and improve the overall manuscript.

(3)    I am concerned about the information displayed in Figure 2A. The volcano plots show many more points that have positive fold change in both the total mRNA and footprinting than expected in a situation where almost all translation is completely shut off and transcription is reduced. Can the authors explain this event or use orthogonal methods to confirm that result?

(4)    For many of the experiments presented oligo(dT) is used to enrich mRNAs containing polyA tails. Thus, their analysis excludes any transcripts with very short polyA tracts, which will be shortened once transcription is repressed during starvation stress. This could lead to erroneous interpretations due to the exclusion of a portion of deadenylated mRNAs. Ideally, these experiments should be done without polyA selection for unbiased results.

Minor points –

(1)    I recommend adding transparency to the points in volcano plots so the reader can better see when points are over each other and better interpret how many data points are being presented.

(2)    Some figures that are in direct comparison have y-axis at different scales. They should be at the same scale to facilitate interpretation.

Competing interests

The authors declare that they have no competing interests.