Peer review of: Human NMD ensues independently of stable ribosome stalling (2019)


<b>REVIEWER COMMENTS</B> 

Reviewer #1 (Remarks to the Author): 

The authors have comprehensively responded to my concerns. I just have one small correction: the 
Figure 5 references in the text were not updated to reflect the addition of toe-print quantification in 5c. 
The Fig. 5c reference in the text should be Fig. 5d, etc. 

Reviewer #2 (Remarks to the Author): 

The authors have adequately addressed my concerns with the manuscript via the changes made in the 
revised submission. I commend the authors on a well-conducted study that offers a strong counterpoint 
to the current thinking in the NMD field regarding ribosome stalling at the termination codon in NMD-
sensitive transcripts. 

Reviewer #3 (Remarks to the Author): 

Karousis et al. have submitted a revised draft of a manuscript describing experiments intended to test a 
popular model for mechanistic aspects of nonsense-mediated mRNA decay (NMD). The revisions 
include: a) additional details about the in vitro translation system utilized for some experiments, b) 
quantitative analyses of toeprinting assays, c) a redefinition of the set of NMD substrates utilized for 
bioinformatic analyses of in vivo ribosome pausing, and d) improvements to the visual appearance of 
some figures and some minor issues in the text. 

These revisions have addressed some, but not all of the requests from the three reviewers (further 
comments on those requests below). However, the new information provided has made it possible to 
determine more definitively whether the authors now have a convincing story worthy of publication in 
Nature Communications. I regret to say that this reviewer’s opinion is “no.” The basis for this 
assessment is as follows: 

1. This is fundamentally a negative study, i.e., results were sought but could not be found. Negative 
studies by definition have a burden to prove that the test system(s) could have revealed the “positive 
results” if they were “real.” Three major segments of the revised manuscript fail to meet these criteria: 

a) In the toeprinting experiments of Figures 1 to 4 the authors sought to determine whether an mRNA 
that is targeted by NMD in vivo would manifest ribosomes stalled at premature termination codons in 
vitro, as had been seen in the earlier work of Amrani et al. (2004) and Peixero et al. (2006). A key 
question then is whether the in vitro translation system used for these analyses is capable of recognizing 
an NMD substrate as such, i.e., are there sufficient NMD factors present in the extract and are they 
active. In Amrani et al (2004), those authors showed that NMD was indeed functional in their extracts 

because aberrant premature-termination-dependent toeprints were eliminated in extracts prepared 
from upf1Δ or nmd2Δ (upf2Δ) cells. Karousis et al. have now included details on the characterization of 
their extracts and these details provide no comparable assurance that UPF factors are active in their 
extracts. Accordingly, their inability to detect NMD-dependent paused/stalled ribosomes in vitro can 
easily be interpreted as a shortcoming of the test system, not the underlying NMD model. 

b) In the bioinformatics analyses of Figure 5 the authors employed ribosome profiling to determine 
whether the set of mRNAs defined as endogenous in vivo NMD substrates manifested paused/stalled 
ribosomes at the normal termination codons located at the 3’-ends of their respective ORFs. This test 
should only apply to mRNAs that are NMD substrates because they possess extended 3’-UTRs, and 3’-
UTR extension has been shown to make a normal termination codon behave as if it were a premature 
termination codon (PTC). Nevertheless, the authors carried out their analyses with all 639 NMD 
substrates (defined as the most abundant isoforms of transcripts that accumulate in response to UPF1 
knock down). The complete set of NMD substrates will include mRNAs with extended 3’-UTRs, but also 
mRNAs with actual PTCs generated in different manners, including mRNAs that are transcripts of 
pseudogenes, mRNAs with retained introns, some mRNAs with uORFs, etc. The authors did not explain 
their decision to include all NMD substrates in this analysis, but is clear that doing so makes it highly 
unlikely that statistically significant ribosome pausing at normal termination codons can be detected. 
The latter negative result is precisely what the authors saw. 

c) The concern about negative results also applies to the authors’ failure to detect a role for the mRNA 
poly(A) tail in the in vitro system. There are numerous reports in the literature demonstrating that an 
mRNA poly(A) tail enhances mRNA translational activity in vitro so, for the authors’ negative result to be 
a significant finding they would have had to show that PABP and eRF3 levels (the factors most directly 
influenced by an mRNA poly(A) tail) in the system were normal, and that their synthetic mRNAs retained 
their poly(A) tails during in vitro translation. Otherwise, it’s just an unsubstantiated negative result. 

2. Other: 
a) The authors were asked to provide evidence that their in vitro translation system was highly active, 
but failed to do so. The requested comparison of mRNA specific activities in an additional system was 
carried out in extracts that were IRES-dependent, i.e., extracts incapable of providing the requested test. 
b) The explanation provided for the different toeprint strengths of PTCs A, B, and C should have been 
supported by experimental evidence. 
c) The explanation for seeing +18 termination toeprint bands, as opposed to +15 or +16 seen by others 
using mammalian extracts, was highly speculative and not substantiated by results from the literature. 
d) As noted by another reviewer, the title and conclusions of this paper are much too strong for the 
results obtained.