Preprint reviews by James P. B. Lloyd

Widespread translational remodeling during human neuronal differentiation

John D Blair, Dirk Hockemeyer, Jennifer A Doudna, Helen S Bateup, Stephen N Floor

Review posted on 06th July 2017

First off, really great work. It is wonderful to see the relative importance of transcription and translation to be characterized in a developmental context such as this. I guess it should not be a surprise to see that transcription plays a larger role than translation, but translation still plays a major role, as demonstrated here. It was nice to see approaches that allow for gene-level and transcript-level evaluation of translation.

Major comment:

A major finding of this work was linking 3’ UTR length to translational control in a developmental context or neurogenesis. But it was unclear to me what exactly was meant by 3’ UTR length change and how this was determined. I believe that you are using a static, reference, transcriptome annotation (Ensembl GRCh38 v84 transcripts). In Fig 6A, this analysis is comparing transcript isoforms from within the same gene to each other, and this found that longer 3’ UTRs were enriched in the light polysomes in older neurons, is this correct? How comprehensive is the transcriptome used to capture the diversity of 3’ UTR ends and do they reflect the biology of the neurons? By limiting yourself to these reference transcripts and predicted termination sites, you might be limiting your ability to find an effect. This phenomenon might be more prevalent than you can assess with this transcriptome.

Minor comments:
“Equal volumes of samples from the 2-4 ribosomes and the 5-8+ ribosomes fractions were then pooled” – Could this introduce any artifacts into the normalization, simply adding equal volumes of the samples? Did you account for the amount of RNA that was in each sample relative to the others to ensure that you were not over-estimating the amount of (for example) poly5 fraction relative to poly7 fraction?
Fig 2A – it seems strange that in the plot, the largest group, which I think are the no-change (black), appear to have the least dots. I assume this is because they are all stacked on top of each other in the middle. Perhaps another colour might help show how dense this group is?
Fig 2A – it might be worth highlighting in the figure label that the changes are significant changes.
Fig 4A – In MeCP2, there are two differences in the transcripts, as you point out, a cassette exon and a longer 3’ UTR. Do you believe this transcript model to be accurate, as both of these events are too distance for short reads to assemble? Is it clear from your mapping, which event (exon, 3’ UTR or both) is giving the signal for change in abundance across the fractions?
Did you check for contamination of feeder MEFs in the hESC RNA-seq data? Is this a concern? Would it only be a problem for the hESCs and not the other cell types?

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