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

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

The group of Richard Gregory was a first to discover the role of DIS3L2 exonuclease and RNA 3’-end 
oligouridylation in a cytoplasmic RNA metabolism in human cells (Chang et al., Nature 2013). DIS3L2 
malfunction had been earlier linked to a rare disease of the childhood, called Perlman syndrome, which 
affects mostly kidneys and increases the risk of developing Wilm’s tumor, however the molecular 
mechanism underlying pathogenesis of this disorder has remained unknown. Since their first publication 
on DIS3L2, R.I. Gregory and co-workers have documented the involvement of DIS3L2-mediated decay 
pathway (DMD) in the quality control of multiple non-coding RNA species, including pre-miRNA, RMRP 
RNA, 7SL component of signal recognition particle (SRP) (Pirouz et al., 2016). These findings have been 
recapitulated and extended to other ncRNA classes by many other research groups and have been 
shown to be conserved across eukaryotic lineage, with the exception of budding yeast, which do not 
have DIS3L2 homolog. Nonetheless, the key DIS3L2 RNA target in the context of Perlman syndrome has 
not been characterized to date. 

I would say that the present manuscript by Pirouz and colleagues is truly novel and ground-breaking in 
that it addresses for the first time the molecular basis of the Perlman syndrome. I have no doubts that it 
will be interesting to a wide community of researchers, because it touches many important aspects of 
cell physiology, including – apart from ncRNA metabolism – control of ER-associated translation, 
maintenance of calcium homeostasis as well as differentiation of stem cells. Therefore, I believe it fully 
meets the criteria of publication in Nature Communications. 

I am impressed by the exceptionally logical sequence of experiments, which make a coherent story, 
whereby DIS3L2 dysfunction leads to accumulation of aberrant 3’-oligouridylated 7SL RNA species, 
causing formation of non-functional SRP and this in turn results in the loss of ER-mediated translation 
and calcium leakage from the ER to the cytoplasm. This cascade of events entails deregulation of cell 
differentiation, which likely explains kidney overgrowth as one of the Perlman syndrome symptoms. The 
novelty of these results is indisputable and represents a significant steps towards our understanding 
why the disorder develops this specific way. 

All performed experiments, which required substantial expertise in up-to-date molecular and cell 
biology methods, are very well controlled and verified by mutually complementary techniques. With few 
little exceptions, which rather do not apply to key experiments from which the most interesting 
conclusions have been drawn, I assess the quality of the data presented in the manuscript as very high. 

Importantly, contrary to previous reports on DIS3L2 substrates, which utilized RNA-seq for DIS3L2 
mutant lines or depleted cells, or variations of CLIP high-throughput experiments, Pirouz et al. employed 
ribosome profiling on mESCs from DIS3L2 knock-out mice to assess how translation of particular mRNAs 
is affected by DIS3L2 dysfunction. 

<b>Remark 1: The authors emphasize that, in agreement with previous studies, global mRNA expression 
levels are not changed. However, there were some indications that transcripts coding for replication-
dependent histones might be direct targets of DIS3L2. Could they comment on how these mRNAs 
behaved in their analyses?</b> 

Ribo-seq approach revealed both translationally down- and upregulated transcripts. The authors next 
inspected the former class in detail, unveiling that RPFs distribution across coding sequences tends to 
decrease, albeit there is a specific accumulation of ribosome protected fragments in the vicinity of the 
CDS 5’-termini. 

<b>Remark 2: Could the authors elaborate a bit more on the possible mechanism of this phenomenon? 
Why is the translation impaired? Could be some ribosome stalling involved?</b> 

GO analysis identified many membrane- and ER-localized proteins encoded by translationally 
downregulated genes, suggesting that ER-associated translation might be impaired upon DIS3L2 
dysfunction. This was confirmed by metabolic labeling of newly synthesized proteins with radioactive 
methionine, which revealed decreased production of proteins secreted to the media (but not 
intracellular proteins). Furthermore, thapsigargin-mediated inhibition of ER-associated translation 
impaired de novo protein synthesis more robustly in DIS3L2 knock-out than in control cells. 

<b>Remark 3: The graphs and statistics are generally quite convincing, but I reckon it would be nice to 
see some SDS-PAGE gel with equal protein amounts loaded and its exposure against phosphoscreen to 
visualize differences in de novo secreted proteins’ synthesis efficiency between DIS3L2 KO and controls. 
Moreover, could some later time-point that 4h be included in the analysis – the differences would be 
probably more pronounced.</b> 

A more direct proof supporting the role of DIS3L2 in proper ER-mediated translation of secreted 
proteins was obtained in elegant experiments using secreted and non-secreted luciferase reporters in 
both mESC KO cells and in various human cell lines treated with siRNAs against DIS3L2. Importantly, 
rescue experiments with WT DIS3L2, but not its catalytically dead variant restored the wild-type levels of 
secreted luciferase in KO cells, showing that the enzymatic activity of the protein is indispensable for ER-
associated translation. 

<b>Remark 4: Why are the levels of exogenous, FLAG-tagged DIS3L2 proteins so much higher in control 
than in KO cells (Fig. 2D, upper panel)? Although it will probably not influence the conclusions, since the 
levels of WT and D389N mutant are comparable within either control or KO group, could the authors 
repeat the experiment so that the amounts of exogenously expressed DIS3L2 variants are more similar 
in control and KO cells?</b> 

<b>Remark 5: There is a clear technical problem with loading control (anti-tubulin antibodies) in the Fig. 
2E (upper panel), which should be eliminated in the revised version of the manuscript.</b> 

Next, Pirouz et al. investigated the cause of reduced ER-associated translation taking into account 
DIS3L2 targets in the cell. Their first guess was possible general impairment of the protein translation 
machinery due to ribosome malfunction or defective tRNAs. However, no change in mature rRNA levels 
or polysome profiles could be detected in DIS3L2 KO cells. Likewise, codon usage frequencies were 
unaffected, demonstrating that codon readout by (probably) defective tRNAs entering the ribosomes is 
rather unchanged. 

<b>Remark 6: To make the entire picture more complete, results of rRNA and tRNA analysis in the form 
of northern-blots which would demonstrate their misprocessing upon DIS3L2 loss should be shown in 
the Supplementary data, along with comparison of polysome profiles for KO and control cells.</b> 

The next series of experiments is dedicated to showing the involvement of DMD pathway in the 
surveillance of 7SL RNA, which appears to be the key DIS3L2 target in the context of correct ER-
associated translation. Several lines of evidence strongly support this conclusion. First, aberrantly 
truncated, oligouridylated 7SL RNA species bound to DIS3L2 mutant accumulated in DIS3L2 KO cells in 
RIP experiment. Co-IP analyses with antibodies against SRP component and ribosomal RPL23a in 
conjunction with polysome profile examination showed that these incorrect 7SL transcripts are present 
within SRP and associate with translationally active ribosomes. Defect of ER-associated translation 
resulting from abnormal SRP synthesis could be reversed by siRNA-mediated depletion of TUTases 4 and 
7, documenting also the role of these enzymes in uridylation-dependent 7SL RNA surveillance. 
Eventually, outcome of DIS3L2 defect was mimicked by overexpression of truncated 7SL RNA, which 
inhibited ER-associated translation of secreted luciferase reporter. 

While the data described so far have implications mainly for the field of RNA biology, their 
consequences are much broader. ER is not only the organelle important for protein folding and 
trafficking, but also a crucial reservoir of calcium. Accordingly, the authors demonstrate that DIS3L2 KO 
cells display increased efflux of calcium ions from the ER to the cytosol. This disturbed calcium 
homeostasis is synergistically increased by treatment of cells with reagents stimulating various calcium 
channels in ER membrane. 

Since calcium signaling affects cell differentiation and organ development, the observations made so far 
prompted the authors to check these phenomena in the context of Perlman syndrome pathogenesis. To 
this end, they utilized a cell line expressing renal-specific Osr1-GFP reporter, which was subjected to 
RNAi using shRNA targeting DIS3L2 or lacZ (negative control). 

<b>Remark 7: Most of the results presented until this point were based on DIS3L2 knock-out. I believe it 
needs to be explained why shRNA-mediated DIS3L2 depletion was used for the most impactful 
experiments presented in the manuscript. Wouldn’t it be better to express Osr1-GFP reporter in the 
DIS3L2 KO cell line instead? The authors should consider repeating at least some experiments on 
differentiation employing such experimental setup, which would be more complementary to other 
data.</b> 

While pluripotency of such mESCs was unaffected by DIS3L2 downregulation, spontaneous 
differentiation of these cells led to the formation of larger embryoid bodies (EBs) than in control cells. 
Poly(A)+ RNA-seq revealed significant changes in gene expression, particularly at later steps of EBs 
differentiation. Spectacularly, DEGs included genes coding for factors involved in calcium homeostasis, 
ECM organization as well as those associated with phenotypes characteristic for Perlman syndrome 
patients. Moreover, deregulation of renal-associated TFs accompanied ESCs differentiation. Accordingly, 
number of cells expressing Osr1-GFP reporter and renal-specific markers was increased during 
differentiation towards renal lineage, which is in line with Perlman syndrome manifestation in the form 
of kidney overgrowth and tendency of Wilms’ tumor development. 

Since this is the first such a comprehensive analysis of DIS3L2 role in the pathogenesis of Perlman 
syndrome and the experiments are very well planned and executed, the manuscript definitely deserves 
publication in Nature Communications provided that several small criticisms mentioned above will be 
addressed during revision phase. 

Minor comments: 
• throughout the manuscript, DIS3L2 protein name should be written with capital letters (as is in the list 
of key words at page 1); 
• There is still a mess with naming TUTases, however the most recent recommendations is to use names 
TENT3A and TENT3B for TUTase 4 and 7, respectively – this should be mentioned in the first paragraph 
of the Introduction at page 3 (line 6); 
• If authors decide to use the word “uridylyl transferase”, then they should also consequently change 
the term “uridylated” into “uridylylated’; otherwise. “uridylyl transferase” should be changed into 
“uridyl transferase”; 
• “poly-Uridine” should be changed into “poly-uridine” or “polyuridine” (page 3, line 11); 
• “abberant” should be changed into “aberrant” (page 4, line 10 and page 10, line 8); 
• “Inhibition of ER-mediated translation inhibition by thapsigargin” should be changed into “Inhibition of 
ER-mediated translation by thapsigargin” (page 5, line 8); 
• “in vitro” should be italicized (page 7, last line) – this also refers to other instances in the text; 
• “Dis3L2-defiecient” should be corrected into “DIS3L2-deficient” (page 9, line 5); 
• “mitochondrial” should be corrected into “mitochondria” (page 10, line 17); 
• Authors should decide which abbreviation – q.RT-PCR or qRT-PCR they want to use throughout the 
manuscript; 
• Fig. 5f Legend – “d10” should be changed into “d12”; 
• “bacteria” should be corrected into “plasmid” (page 27, line 2); 
• “normalization” should be corrected into “normalized” (page 27, line 8); 
• “edegR” should be corrected into “edgeR” (page 29, line 18); 
• “disitonin” should be corrected into “digitonin” (page 31, line 2). 

Reviewer #2 (Remarks to the Author): 

In “The Perlman Syndrome Dis3l2 exonuclease safeguard ER-associated translation, Ca2+ homeostasis, 
and stem cell differentiation,” Richard Gregory and colleagues aim to determine the functionally most 
important target(s) of Dis3l2, a protein known to degrade multiple classes of non-coding RNAs when 
their 3’ ends are oligouridylated. They present data that suggest that the specific failure to degrade the 
critical RNA component of the signal recognition particle (SRP), 7SL, leads to decreased translation of 
proteins at the ER, which in turns leads to defects in Ca homeostasis and ES differentiation. The basic 
idea is the retention of truncated and uridylated 7SL gums up the ability to translate transcripts that 
encode a signal peptide. The paper presents a number of experiments to support the model. The 
connections between each step though (accumulation of 7SL, reduced translation, calcium homeostasis, 
and stem cell differentiation) represent rather large leaps. For example, it is unclear how uridylated 7SL 
would gum up the system, why only a subset of ER translated proteins are affected, or how defects in ER 
proteins would lead to specific differentiation defects. Presumably these questions are being saved for 
another day. The findings should be of interest to readers of NC, especially those interested in RNA 
surveillance. However, a number of details surrounding their experiments need to be addressed first 
before being suitable for publication. 

Figure 1. 1a: According to methods, cutoff is set at 1.5x, with no statistical measure. Authors should 
include a statistical cutoff and clearly state cutoffs in figure and/or legend. 1b: Again, need to present 
some statistical argument that the difference is meaningful. 1c: Would like to see same for up genes. 1d: 
again, would like to see broken down into down, unchanged, and up. Importantly, there needs to be a 
discussion of why only a small fraction of ER translated proteins are affected. 

Figure 2. 2b/c: not sure what the addition of thapsigargin shows. The effect is small, non-specific to KO 
in case of the Gluc, and any effect is likely very indirect. I recommend removing. Also, in C, it looks like 
downregulation is as at translation level rather than translocation across ER (which is the presumed 
function of SRP). Would be helpful to have some discussion of why a defect in SRP, which directs 
transcript to ER, is inhibiting translation. This is similar to comment above, i.e. how does 
uridylated/truncated 7SL gum up translation of signal peptide containing proteins. 

Figure 3. 3a,d,e: From these data, it is unclear what fraction of total 7SL transcripts are 
uridylated/truncated in each experiment. This number is important. If it is really small (say less than 5%), 
it is unclear how it could be gumming up the entire system. 3c: Needs much better explanation. Not sure 
it adds much. 3f: not sure what the point of this panel is. Is the idea that the uridylated 7SL is enriched in 
the translated fraction? If so, need to show data for monosome fraction. 3h: Given that the overall 
increase in 7SL expression is minimal (fig. S2C), it is difficult to understand the basis for the results. Is 
there something special about expressing mouse 7SL in human cells that even a small overall (human 
and mouse) increase can rescue? 

Figure 4. minor issue: very difficult to read fig. 4b. 

Figure 5. 5c: Why are the DEGs almost entirely upregulated genes, especially d12? 5d: In contrast to text 
I would conclude from this clustering that it is embryonic day not genotype that is predominantly 

separating samples. A principal component analysis might help to see if there is a principal component 
that nicely separates genotype and determine what genes are most heavily weighted in that PC. 5g: not 
sure how to interpret this analysis or what value it adds. 

Figure 6. 6a,b: for this data, would like to see fold change in addition to z score. That is what is the actual 
change in expression. If small changes with minimal variance are underlying the z score, that would be 
less meaningful. 6d-f: I appreciate what authors are trying to do, but these results are a very far stretch 
from describing the disease phenotype associated with the kidney. The interpretation is overstated and 
oversimplified given the minimal data. 

Reviewer #3 (Remarks to the Author): 

Pirouz et al. analyze the consequences of removing Dis3l2 function in mammalian cells. Based on 
ribosome profiling, they report impaired translation of secretory and membrane proteins and claim a 
mechanistic link to impaired quality control of the 7SL RNA component of the signal recognition particle 
(SRP) that targets most proteins to the endoplasmic reticulum (ER) in mammalian cells. The authors try 
to link these findings to ER calcium leakage and dysregulated ESC differentiation. The experiments are 
well-executed and the independent observations of (i) different translation efficiencies, (ii) aberrant SRP 
7SL RNA, (iii) calcium dysregulation, and (iv) differentiation phenotypes are clear. These results should 
be useful towards understanding the role of Dis3l2-mediated RNA decay in physiology. However, the 
suggested causality between Dis3l2 function, protein synthesis at the ER, and the proposed downstream 
“SRPopathy” phenotypes are weak and overinterpreted in this manuscript as written. At minimum, the 
text implying these links should be significantly toned down before publication. 

Major comments: 
1. Although the data showing aberrant SRP 7SL RNA in Dis3l2 KO cells (Fig. 3) are clear, the evidence 
linking Dis3l2 function and impaired “ER-mediated translation” is not convincing and requires more 
controls and/or reinterpretation: 
a. In Fig. 1, if predicted ER-destined proteins make up at most 20% of translationally downregulated 
genes in KO cells (Fig. 1d), can a difference in ribosome occupancy by metagene analysis of the entire set 
of downregulated genes (Fig. 1b) be linked specifically to ER-associated translation? 
Do downregulated non-ER transcripts show the same RPF pattern in the KO cells? How do 
downregulated non-ER transcripts compared to unaffected non-ER transcripts? More results are needed 
to demonstrate that Dis3L2 KO is not inducing a more general translational repression mechanism. 
b. Is there a Dis3l2-independent difference of ribosomal occupancy on ER-destined transcripts vs. 
others? What happens if the same analysis shown in Supplemental Fig. 1c is done for control cells? 
c. Fig. 1d shows some preference of predicted ER proteins in the translationally downregulated genes, 
but these are still only ~1/8 of all predicted ER-targeted proteins. What makes these clients particularly 
sensitive to Dis3l2 KO? Are SRP clients de-enriched in the translationally up-regulated genes in the KO 
cells? 

d. I am not convinced that the metabolic labeling experiment in Fig. 2a is a fair assessment of secreted 
vs. non-secreted protein synthesis. It seems that the lysate and supernatant fractions were collected in 
separate experiments. In addition, while the protein content of the lysates was normalized before 
scintillation counting, that of the supernatant was not addressed, allowing for far more variability that 
may arise not only from translation and secretion efficiency, but also different rates of cell growth and 
amino acid metabolism. It’s also not clear if the radioactivity measured in the supernatant is truly 
incorporated into proteins vs. free radiolabeled methionine. A more equal comparison would be to start 
with the same number of WT and KO cells, collect the supernatant and lysate in the same experiment, 
and remove free amino acids from each sample. 
e. Many experiments examining ER-mediated translation are done with a secreted luciferase reporter. 
To rule out SRP-independent effects, controls should be included showing the level of mRNA expression 
of this reporter in the conditions analyzed in Fig. 2c-2e, especially since the secreted and cytosolic 
reporters are in different plasmid backbones, encode different enzymes, and are expressed by transient 
transfection, which is additional subject to variable transfection efficiencies and cell-to-cell 
heterogeneity. 
f. Stronger support for a link between Dis3l2 function and SRP client translation could be achieved by 
biochemically analyzing endogenous Dis3l2-dependent and independent clients by metabolic labeling 
and immunoprecipitations in control and KO cells with and without Dis3l2 re-expression. Alternatively, 
the authors could use matched reporter constructs that differ in the presence of an ER-targeting signal 
sequence, or compared to an SRP-independent ER targeting signal, such as a C-terminal tail-anchored 
transmembrane anchor. 
g. Can the authors comment on the mechanism by which impaired SRP function in Dis3l2 KO cells leads 
to reduced translation, rather than protein mislocalization as observed in Costa et al. (2018), ref. 39? 
2. The authors suggest a causal link between translational downregulation of ER-destined proteins and 
calcium leakage. While the data independently showing translational downregulation of some genes and 
calcium dysregulation in Dis3l2 cells are clear, there is no evidence supporting the claim that the first 
causes the second. As an example, thapsigargin treatment causes cytosolic calcium accumulation, which 
also leads to impaired protein secretion in Dis3l2 KO cells (Fig. 2b and 2c). This already suggests that the 
two observations of impaired protein secretion/translation and calcium dysregulation cannot be cleanly 
uncoupled in these experiments. 
3. Similarly, while the experiments looking at cellular differentiation are beautiful, they remain 
observational. Descriptions suggesting causal links should be avoided in the text. Alternatively, the 
hypothesis should be directly tested. For example, rescuing the differentiation phenotype by re-
expressing wildtype SRP 7SL RNA would give more credence to an “SRPopathy” model. As it stands, 
there is no data in the paper to rule out other plausible hypotheses. Perhaps Dis3l2 KO have 
misreguated miRNA targets that lead to the observed phenotypes. It also remains unclear why only a 
minority of predicted SRP clients are affected in an “SRPopathy” (see point 1b). 

Minor points: 
1. The authors demonstrate their expertise in RNA biology and metabolism in the first paragraph of the 
introduction. In contrast, the second paragraph describing SRP function was vague, lacking specific 
information that could influence interpretations in the manuscript. The general mechanism of SRP-

mediated targeting of ribosomes synthesizing secretory and membrane proteins to the ER, as well as the 
scope and characteristics of SRP clients, are well studied and included in most college-level biology 
textbooks, such as Molecular Biology of the Cell by Alberts et al. It would be helpful if the relevant parts 
of this established work are introduced to help place the authors’ findings in context. 
2. The term/concept of “ER-mediated translation” is not clear to me. It implies that ribosomes at the ER 
translate more efficiently. Is this what the authors mean (and if so, what is the evidence for this)? Or 
does it refer only to protein synthesis at the ER? 
3. Please be wary of the term “protein translation”. mRNAs are translated, proteins are not. Alternatives 
are “translation” or “protein synthesis”. 
4. “aberrant” is misspelled as “abberant” throughout the text.