Peer review of: The Perlman syndrome DIS3L2 exoribonuclease safeguards endoplasmic reticulum-targeted (2020)



REVIEWERS' COMMENTS: 

Reviewer #1 (Remarks to the Author): 

In their revised manuscript, Pirouz and colleagues have adequately addressed all concerns that I had 
after reading the initial version of the paper. Specifically, they verified histone mRNA expression in 
mESCs, showing that steady-state levels of these transcripts are not significantly deregulated in this 
particular cell type, as opposed to other cell lines. Moreover, the authors have now eliminated several 
technical shortcomings pointed out in the referee’s report. They also provided explanations which 
rationalize the use of scintillation instead of phosphorimaging in the analysis of secreted newly 
synthesized proteins. Additional experiments performed during revision argue against the link between 
rRNA or tRNA misprocessing caused by DIS3L2 knock-out and altered mRNA translation. Instead, data 
from experiments performed using reporters convincingly support authors’ conclusions pointing to 7SL 
RNA quality control deficiency and SRP malfunction as the major cause of associated molecular 
phenotypes. Although experiments with Osr1-GFP reporter in DIS3L2 knock-out vs wt cells have not 
been done, the authors’ attempts to further demonstrate the utility of shRNA-mediated approach have 
to be appreciated. Furthermore, new data for knock-out cells are now provided to demonstrate up-
regulation of renal-specific markers and synergistic effect of overexpression of truncated 7SL RNA 
specific to these cells, but not observed in the wt control. Together with additional experiments carried 
out in response to other reviewers’ reports, amendments and clarifications in the text, the manuscript 
by Pirouz et al. has gained a lot of quality and I recommend accepting it for publication in Nature 
Communications without further hesitation. 

Reviewer #2 (Remarks to the Author): 

The authors have generally done a good job of responding to reviewer comments. However, I still have a 
hard time accepting the central theme of the paper that the accumulation of uridylated 7SL is the 
underlying mechanism connecting the loss of DIS3L2 with the various downstream phenotypes (defects 
in ER associated mRNA translation, ER Ca2+ retention , and stem cell differentiation). The reason is that 
the fraction of uridylated 7SL relative to non-uridylated 7SL appears to be remarkably low (as shown in 
their new figure 3F). It is unclear to me how this very small fraction could have such a large downstream 
impact. In addition, they show that they can rescue the defect by overexpressing full-length 7SL 
suggesting some sort of mass effect (i.e. reversible competition between uridylated and non-uridylated 
7SL) (fig. 3i). However, the overexpression increases total 7SL by less than 10% (supplementary fig. 5d). I 
cannot see how this very slight increase in normal 7SL can reverse the effect of what was a very small 
fraction of uridylated 7SL. As for figures 3d,e,g, they show a reasonably large fold increase of uridylated 
7SL in the various pull downs when comparing mutant to wt, but given that uridylated 7SL is extremely 
low to non-detectable in the wt, the fold increase is likely to reflect an extremely low absolute value, 
again making it hard to understand how it could be causing such severe downstream phenotypes. I 
appreciate that this may be a very difficult question to resolve, but it does represent a central theme of 
their paper. Maybe it just requires some finessing of their conclusions or maybe I am just missing 

something. If other reviewers disagree with my interpretation, I am happy to acquiesce. There are a lot 
of nice data in the paper. 

More minor issues: I remain unconvinced that the difference between ctrl and ko in figure 1d is 
significant and truly represents a block/delay in ribosome progression on transcripts in DIS3L knockouts. 
Also, now that they include a up group in their updated figure 1e, there appears to be an equal or near-
equal enrichment of ER-translated proteins in the down and up subsets (KO/Ctrl) when considering 
either the SignalP or Phobius ontology groups (new figure 1e). If DIS3L is supposedly inhibiting 
translation of ER -translated proteins, why are they also enriched in the up group. 

Reviewer #3 (Remarks to the Author): 

The authors have sufficiently addressed my main concerns in their revised manuscript. I support 
publication in Nature Communications, with a few minor points regarding the preciseness of the writing 
and descriptions of what occurs when treating cells with certain small molecules that can influence 
readers' interpretations of the results. 

Minor Points: 
1. In the explanation of Fig. 2: thapsigargin does not selectively inhibit translation of ER-targeted mRNAs. 
It inhibits the SERCA channel at the ER, which activates the integrated stress response (via the 
phosphorylation of eIF2-alpha), which inhibits protein synthesis globally (again, not specifically of ER-
targeted substrates) while upregulating stress response proteins. 
2. There is a similar issue with the explanation of the use of anisomycin in Fig. 4: anisomycin is primarily 
known to act as a translation elongation inhibitor. It directly binds ribosomes to inhibit peptide bond 
formation. The inhibition of calcium leak at ER translocons is a secondary effect from the inability in cells 
to clear ribosomes from translocons, but this is not clearly explained (and the citations to references 45 
and 54 do not sufficiently support a primary function of anisomycin in preventing calcium leakage) 
2. I am still struggling with the term "ER-mediated translation". It is not a term used in the long-standing 
fields of ER translocation or protein synthesis, and appears to only refer to translation that occurs at the 
ER membrane. However, the ER is not actively facilitating translation itself so the description seems 
somewhat misleading. Alternatives might be "ER-targeted" or "ER-localized" translation. 

R