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

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

The nonsense-mediated mRNA decay pathway has been intensively studied, but basic mechanistic 
questions about its execution remain unanswered. A model popular in the field for about 15 years has 
been that the NMD machinery targets mRNAs undergoing inefficient translation termination. This model 
is based on the idea that termination events at “premature” stop codons are slower than termination 
events at “normal” stop codons. One way in which termination efficiency is thought to be modulated 
and sensed in NMD is through competition between the NMD factor UPF1 and poly-A binding protein. 
PABPs have been described to enhance termination through interactions with the eukaryotic release 
factors; thus, in the absence of this interaction, the termination kinetics model posits that UPF1 is able 
to interact with eRFs and induce decay. Much of the evidence supporting this model is derived from in 
vitro assays of translation termination on NMD-sensitive and -insensitive mRNAs. In this important 
paper, Karousis and colleagues in the Muhlemann lab revisit the evidence for inefficient termination at 
NMD-inducing stop codons. They first describe the careful establishment of an in vitro ribosome 
toeprinting assay capable of distinguishing differential ribosome association at stop codons. With this 
system, they do not observe a difference in ribosome occupancy due to changes in 3’UTR length or the 
presence of a pA tail, contrary to prior models. Further, they show that ribosome toeprinting 
experiments in rabbit reticulocyte lysates may be confounded by NMD-independent RNA cleavage. In 
addition to the in vitro experiments presented, the authors also analyze ribosome profiling data, finding 
no evidence for differential termination efficiency at NMD-sensitive and -insensitive stop codons. While 
largely based on negative data, this work is a significant contribution to the ongoing debate over NMD 
mechanisms. Please see below for specific comments: 

Major comments: 

1. Figure 3B and Figure 5B: The authors use ABCE1 depletion in 3B to show that their assay is capable of 
detecting ribosomes stalled at termination codons, and then present data in 5B illustrating no difference 
in ribosome toeprints on NMD target and non-target mRNAs with and without polyA tails. The 
performance of the assay and impact of the negative result in 5B would be enhanced by quantification 
of the toeprinting assays in both figures. 
2. Figure 5D shows that a previously defined set of NMD target transcripts and non-NMD target mRNAs 
have similar ribosome occupancy at their termination codons. This result is a little difficult to interpret 
without more complete information about how the transcripts were chosen for each set and how 
annotation ambiguity was dealt with. Were all transcripts arising from the NMD-sensitive genes 
identified in the Colombo et al paper used in the “NMD” set, or were genes divided into “NMD” and 
“non-NMD” transcript isoforms? If the former, the analysis may be confounded by multiple-counting of 
the same termination event or by assigning many normal termination events from non-NMD isoforms to 
the “NMD” category. If the latter, how were the annotations parsed to select which termination codon 
was used for analysis? 
3. In general, the authors are careful to match their claims to the available data, but I think the title of 

the paper overstates the findings somewhat. A title that conveys that the message of the paper is a lack 
of evidence for stalling at NMD-sensitive stop codons, rather than affirmative evidence that stalling does 
not occur would be preferable. Similarly, the last sentence of the abstract should be softened. 

Minor comments: 

1. The methods are generally thorough, but information about the quantity of in vitro transcribed mRNA 
added to the in vitro translation reactions should be included. 
2. The description of the approach used to distinguish footprints from translation-dependent RNA 
cleavage could be clearer. I’m not sure whether the text at the bottom of page 3 is only meant to refer 
to the +18 band throughout or all of the bands observed, some of which still partially remain in the PC-
extracted samples. 
3. The blue and green colored dots used in Figure 1D and E are difficult to distinguish 
4. Figure 2A legend: the sentence beginning, “The red arrow…” is prematurely terminated. 
5. Figure 5C would benefit from a clearer illustration of the distribution of NMD targets across the Y axis. 

Reviewer #2 (Remarks to the Author): 

SUMMARY: In this study, Karousis et al. conduct a careful comparison of ribosome occupancy at the 
termination codon of NMD-sensitive versus insensitive substrates using in vitro and in vivo approaches. 
First, they optimize a toeprinting assay using translation-competent HeLa cell lysate to detect ribosome 
occupancy at the termination codon of an in vitro translated mRNA. Using this assay, they demonstrate 
an increased ribosome occupancy at the termination codon upon adding a hCMV uORF2 stalling peptide 
to their reporter, as well as upon depletion of the ribosome splitting factor ABCE1. They then use this 
assay to ask if an NMD-sensitive reporter mRNA (with a long 3’ UTR) shows higher occupancy of 
ribosomes at the termination codon compared to the NMD-insensitive mRNA, and find this not to be the 
case. They complement this result with an analysis of a ribosome footprinting dataset, focusing on 
footprints at the termination codon, and show that the there is no statistically significant difference in 
ribosome occupancy at the termination codon of NMD targets versus non-targets. 

Taken together, the observations of Karousis et al. challenge the current view of NMD wherein stable 
ribosome stalling at the termination codon is a key trigger for NMD. While it is possible that there is 
transient pausing of the ribosome at the termination codon of NMD substrates at a time scale that is 
indistinguishable from normal termination by the assays used in this study, this is still an important 
distinction to be made from stable stalling of the terminating ribosomes on NMD substrates. 

COMMENTS: 
• Major: In some of the toe-printing gels (eg. Fig 5B), it looks as if the loading of RNA across the lanes is 
inconsistent. This paper would be strengthened by a more quantitative treatment of the toe-printing 
data including intensity measurement of the toe-print signal as well as normalizing this signal to that of 
the full-length mRNA. Given that every experiment was done in at least triplicates (as per Materials and 
Methods), a bar graph of the quantified signal could also be provided. 
• Minor: The black and white color scheme of Figure 5C makes the plot hard to read. A more contrasting 
color or a different scaling that improves contrast would be preferable. 
• Minor: A legend needs to be provided for Figure 5D that indicates what the different data points (filled 
square, empty square, and x’s) are. 

Reviewer #3 (Remarks to the Author): 

Karousis et al. describe experiments intended to test a popular model for events occurring during 
translation of mRNAs that are targeted by the nonsense-mediated mRNA decay (NMD) pathway. Earlier 
work from Amrani et al. (Nature, 2004) and Peixeiro et al. (NAR, 2012) demonstrated, respectively, that 
yeast and human mRNAs harboring premature termination codons (PTCs) are not only in vivo substrates 
for NMD but, when translated in vitro, manifest ribosome pausing at their PTCs. The latter ribosome 
pausing events were detected in both papers by toeprinting, a method also known as primer extension 
inhibition. The earlier results were significant because they implied that termination at PTCs differed 
from normal termination and thus may have created a kinetic “window” for ribosomal association of 
factors such as Upf1 that are known to promote NMD. Here, Karousis et al. use a similar methodology, 
as well as ribosome profiling, to argue that the conclusions of the earlier studies cannot be confirmed 
and may, therefore, be wrong. While the subject of this study is important (because the mechanism 
underlying NMD remains unknown), the experimental evidence presented by the authors is quite thin 
and unconvincing. The following points address the shortcomings of the manuscript: 

1. A major focus of this study is a set of in vitro translation experiments in HeLa cell lysates. As such, it is 
imperative that the authors characterize the translation activity of their lysates extensively and 
demonstrate that they are operating at peak efficiency. Hence, it was surprising that the authors chose 
very specific translation conditions without data supporting those choices. There was: a) no justification 
for the use of micrococcal nuclease treatment, b) no demonstration of a bona fide Mg2+ optimum, c) no 
mRNA concentration curve (actually no precise mRNA amount specified), d) no time course of protein 
synthesis, e) no demonstration that the synthetic mRNAs are fully capped or stable, and f) no 
assessment of the lysate’s response to poly(A)+ or poly(A)- mRNAs. There is an experiment showing that 
the authors’ synthetic Rluc mRNA produces considerable luminescence when translated (Fig. 1B), but 
this data is meaningless in the absence of a comparison of that mRNA’s specific activity in these lysates 
vs. others (e.g., purchased lysates). These points are not trivial because the work of Amrani et al. 
showed that the termination toeprints observed in yeast extracts prepared from wild-type cells could 
only be seen at PTCs and not at normal termination codons (NTCs). They argued that termination at 

PTCs must be slower than that at NTCs, a point they substantiated by demonstrating that only extracts 
prepared from cells expressing a mutant release factor (eRF1) could yield toeprints at NTCs. The latter 
point is especially relevant here because the authors of this paper only look at toeprints from NTCs. The 
fact that they see such toeprints from their standard HeLa lysates implies that those lysates are most 
likely operating at low efficiency (i.e., the equivalent of the yeast lysates with defective eRF1). 
2. The authors need to explain why: 
a) All of their Rluc mRNA NTC toeprints are at +18, i.e., toeprints 18 nt 3’ to the first nt of the nonsense 
codon. Comparable experiments with mammalian extracts (e.g., Pisarev et al., Mol Cell 2010) usually 
yield +15 toeprints. 
b) The TC2 and TC3 mRNAs of Figure 1E have such diminished +18 toeprints. It would be expected that 
their intensities would be almost identical to that of TC1. 
c) There was no toeprint from the CTR+pA mRNA (Fig. 2B). That mRNA should have yielded results 
comparable to their standard Rluc mRNA. 
d) They considered the +18 band in the ABCE1 knock-down experiment of Fig. 3B to be of “higher 
intensity.” There is so much variation in the +18 toeprints presented in this paper that a claim of “higher 
intensity” can only be justified by multiple repeat experiments and statistical assessments. 
3. Fig. 4 demonstrates that the authors’ standard mRNA (200 + pA) is “normal,” i.e., not a substrate for 
NMD. Following on point #1 (above), this means that no toeprint should be detected from this mRNA in 
a normal extract, and that its presence implies that the extracts are translating inefficiently. 
4. The experiments presented in Fig. 5C and D illustrate that the authors are missing key points of the 
model they hope to be testing. Why would they expect an accumulation of ribosomes at the normal 
termination codons of mRNAs known to be NMD substrates? These mRNAs are known to become NMD 
substrates by virtue of a variety of PTCs originating from uORFs, included introns, frameshift signals, 
etc., and if any ribosome pausing were to occur it would most likely happen at those PTCs, not at the 
NTCs. Further, the experiment suffers from a lack of statistical details, i.e., how many mRNAs were 
analyzed in the NMD “target” vs “other” groups, and what is the rationale for the specific use of Anova 
(vs. other tests)? 
In short, this manuscript has created false tests of a model it seeks to refute and its negative results are 
thus not surprising or convincing.