Peer review of: OTUD5 cooperates with TRIM25 in transcriptional regulation and tumor progression via deubiquity (2020)

Reviewers' comments: 
 
Reviewer #1 (Remarks to the Author): 
 
Li et al. present a study that reveals a link between OTUD5 (deubiquitinase) and E3 ubiquitin 
ligase TRIM25 in the context of oncogenic cellular processes. They show that depletion of OTUD5 
results in enhanced proliferation and that this effect is dependent on the interaction with TRIM25. 
They suggest that OTUD5 specifically deubiquitinates autoubiquitinated TRIM25. Moreover, they 
link OTUD5 with the expression of tumour suppressor protein PML (TRIM19). Finally, they provide 
evidence that reduced levels of OTUD5 are associated with an aggressive phenotype and poor 
clinical outcome of human cancers. 
 
Overall, it is a thorough study with multiple angles and comprehensive results. That said, I have 
several issues that shed light on the key conclusions of the manuscript. 
 
1. Why there is no signal of OUTD5 and TRIM25 in the inputs in Co-IP experiments presented in 
Figure 2B and C? 
 
2. How was TRIM25-ubs purified? If the purification was done in the same way described for 
OUTD5 we cannot be sure that these are truly purified proteins. Even though, high stringency 
buffer was use there is a possibility that some tight interactions are persistent. This could be dealt 
with by following a proper protein purification protocol, which authors do not. The SDS page gels 
of 'purified proteins' should be at least presented. 
 
3. Point 2 brings me to Figure 3. There is no evidence to support that the ubiquitin smear we see 
on the blots is indeed autoubiquitinated TRIM25. The fact that it most likely is not is reinforced by 
results presented in Figure 5F where TRIM25 without its RING domain is still heavily ubiquitinated. 
Thus, the smear could be coming from other proteins, tightly bound by TRIM25. 
 
The only way to discern if the ubiquitin smear is detecting autubiquitinated TRIM25 is to perform 
similar experiments with the TRIM25 K117 mutant. K117 is the key residue responsible for 
autoubiquitination. 
 
This also brings me to the discussion section (lanes 402 - 404. 'It is possible that ubiquitination on 
the RING domain of TRIM25 makes structural transform or behaves as a signal to help the 
enrichment of TRIM25 on the target genes.' In the light of what we know about TRIM25 
autoubiqutination this speculation (without proper control) is ungrounded. 
 
If the authors can show that the TRIM25 K117R is phenocopying OUTD5 this will be a substantial 
discovery as thus far the role TRIM25 autoubiqutination remains unknown. 
 
4. The TRIM25 K117R mutant should be used in other assays, including CHIP presented in Figure 
5G. Currently, the only conclusion that we can draw from this experiment is that catalytically dead 
TRIM25 is not enriched at the PML TSS. The only way to prove that this is due to the OUTD5-
dependent deubiquitination of TRIM25 would be with the above mentioned mutant. 
 
5. The RNAseq as well as Mass Spectrometry should be publicly available. This is very important as 
the authors base the whole manuscript on a relatively weak mass spectrometry result (2 unique 
peptides of TRIM25 in the OUTD5 IP). 
 
Minor comments 
 
1. Wine 69 should read '...innate immunity' 
 
2. Are the transcripts upregulated upon TRIM25 knockdown detected by RT-PCR (Fig.4G) also 
upregulated in RNAseq experiment (Fig. 4C). If so they should be indicated in the same way as 
PML. 
 
 
 
Reviewer #2 (Remarks to the Author): 
 
In the manuscript entitled “OTUD5 cooperates with TRIM25 in transcriptional regulation and tumor 
progression via the deubiquitination activity” by F. Li et al., the authors investigated the role of the 
OTUD deubiquitinases in cancer, in particular growth suppressive effects of OTUD5 on 
hepatocarcinoma cell lines. 
Mechanistically, the authors propose that OTUD5 controls the deubiquitination of TRIM25, 
inhibiting its repressive effects on PML expression. The latter would account for the increase in 
tumor cell growth induced by OTUD5 knock down (KD). 
TCGA database analyses shows that low expression of OTUD5 is associated with a decrease in 
overall survival of patients with six different types of carcinoma (not hepatocarcinomas). Although, 
in principle, this is a important observation, the role of OTUD5 in tumor development should have 
been assessed in more physio-pathological relevant models than merely cell lines. 
The authors clearly established that OTUD5 interacts and debubiquitinylates TRIM25, and that 
OTUD5 KD increases tumor size in hepatocarninoma cell line xenografts. However, the role of 
TRIM25 deubiquitination in the latter is only correlative. While anti-tumoral properties of PML are 
well known in many cancers, its involvement in tumor growth as a target of OTUD5-mediated 
TRIM25 deubiquitination was not clearly established in this study. 
 
 
Main points 
 
My main concern is linked to the cellular models. The authors based their study on 2 
hepatocarcinoma cell lines, mainly used in culture. Cross talks between OTUD5 and TRIM25/PML 
should have been investigated (by RNA seq, ChiP, TRIM25 ubiquitination etc.) directly from mice, 
at least from the xenografted cells, more relevant for tumorogenesis. Double KD for OTUD5 and 
TRIM25 or PML in xenografts are also required to validate the mechanism of action proposed by 
the authors for OTUD5 function in tumor progression. 
 
Since OTUD5 was shown to target various substrates, RNAseq analysis of OTUD5 KD cells should 
be performed and compared to TRIM25 KD to determine overlapping and non-overlapping 
pathways/ genes more broadly. 
GSEA/GO analyses would provide information on the pathways targeted by TRIM25KD in these cell 
lines compared to other models, as those found by TRIM25 ChIPseq and RNAseq upon TRIM25 KD 
in breast cancer in vivo models. 
 
The pattern of PML protein migrations presented here, in these cell lines, is quite unusual. PML is 
expressed as various isoforms and the most abundant one is PML-I with a size >120 kDa. Here, all 
WB analyses shows a band close to 75kDa. How do the authors explain this difference? Do other 
anti-PML antibodies show the same pattern, especially antibodies specific for different PML 
isoforms? 
 
In figure 4D, the authors focused on the expression of genes potentially regulated by PML (such as 
CDKN1A or BAX), showing by qRT-PCR significant induction upon TRIM25 KD. Are these transcripts 
also found upregulated in the RNAseq analysis? 
To really assess any PML involvement in this TRIM25 targets, the authors should KD PML together 
with that of TRIM25 and assess expression of those genes, or more broadly on TRIM25 tagets. 
 
It is not clear why the authors used TSA to activate apoptosis. P53 is mutated in these two cell 
lines. A more classical way would have been to use TNF or irradiation known to activate a PML-
dependent apoptosis. More importantly, a KD of a pro-apoptotic gene is expected to increase cell 
viability, this does not mean that it is specifically involved in TRIM25 KD effects. The authors would 
have to test the effect of PML KD alone on cell survival and compare to the double KD. Propidium 
Iodide staining is indicative for cell survival rather than apoptosis. 
 
Although PML increase upon TRIM25 siRNA in cell lines is clear, the mechanism proposed by the 
author is not. Using ChIP-PCR (upon HA-TRIM25 or HA-IRF8 overexpression), the authors 
proposed that TRIM25 binds PML TSS and competes with IRF8-binding on interferon responsive 
elements within PML promoter. However, the effect of TRIM25 shRNA on HA-IRF8 enrichment at 
PML promoter was far too low to explain PML upregulation. In addition, although OTUD5 KD 
increased TRIM25 binding onto PML promoter, it only slightly decreased in PML mRNA level (Fig 6C 
and D). 
Since PML is heavily regulated by SUMO and ubiquitin conjugation, the authors should investigate 
whether the TRIM25 ubiquitin-ligase and the OTUD5 DUB could affect PML stability by directly 
regulating its ubiquitinylation. 
 
More globally, this is not clear whether OTUD5 only affects TRIM25 ubiquitinylation or its ubiquitin 
ligase activity and which of the two is involved in TRIM25 cell growth effects. Indeed, the authors 
only assessed trim25 auto-ubiquitination as reflecting it ubiquitin ligase activity, while other 
TRIM25 protein targets may be also debuquitinated within OTUD5 complex. The authors could test 
at least global ubiquitination in cells expressing HA-TRIM25 +/- OTUD5. 
TRIM25 mutants for its ubiquitinylated lysines should be compared to delta-RING mutant, both in 
colony formation or PML regulation. The authors could check which type of ubiquitin chains is 
affected by the DUB, in particular in vivo, using K63 or K48 chain specific antibodies in their IP/ 
WB analyses. 
 
Similarly, the role of OTUD5 DUB activity was investigated neither on PML nor on tumor growth in 
xenografts using OTUD5 mutant for its catalytic cysteine or UIM. Thus, the title overstates the role 
of DUB activity in tumor progression. 
 
Finally, it is not clear whether the authors propose a general mechanism of OTUD5/TRIM25 
function in cancer or a specific one to hepatocarcinoma. Assessing OTUD5 in the metastatic 
function of TRIM25, in breast cancer models, is particularly important to conclude on any role of 
OTUD5 in “tumor progression”, as stated in the title. 
 
 
Minor points 
 
While PML mRNA expression was 3 fold-induced by acute down-regulation of TRIM25 using siRNA 
(Fig 4c and D), TRIM25 KD had very low effects shRNA in fig 5H, where shRNA were apparently 
used. The authors did mention the shRNA before, why do the authors switch to sh for some 
experiments? 
 
Quantifications on the total PML protein amount is required to validate the effects of the double 
TRIM25/OTUD5 KD onto PML expression and conclude that OTUD5-induced down regulation of PML 
is TRIM25 dependent. 
 
Since PML is an IFN-target gene and OTUD5 was shown to down regulate IFN type I response, up 
regulation of IFN pathway in OTUD5 KD might have opposite effect on PML expression that would 
even limit PML down regulation upon OTUD5 KD. How the authors take that into account? Is there 
any IFN pathways signature activated by OTUD5 KD in these cell lines? 
 
 
Reviewer #3 (Remarks to the Author): 
 
OTUD5 cooperates with TRIM25 in transcriptional regulation and tumor progression via the 
deubiquitination activity 
 
This interesting new manuscript describes a ubiquitin based signaling axis that controls 
proliferation, gene expression and which likely plays a role in cancer. It centers on a 
deubiquitinase OTUD5, which the authors suggest regulates the ubiquitination of a transcriptional 
repressor TRIM25. This in turn control the expression of PML, among other genes, to control 
tumorigenesis. The manuscript was well organized, the data was clear, logical and well put 
together, and the findings were new and interesting. I am therefore enthusiastic about the 
eventual impact of the manuscript and supportive of its publication. Below are a few points and 
suggested experiments that I would hope the reviewers can first address prior to full acceptance of 
the paper. 
 
Major points 
1. In figure 3, the authors make the case that TRIM25 ubiquitination is controlled by OTUD5. 
However, in all of the panels in this experiment, the authors are not directly assessing the covalent 
modification of TRIM25 with ubiquitin, and instead, rely on TRIM25 pulldowns followed by ubiquitin 
immunoblots. Thus, it is unclear if the smeared band in the pulldown in in fact TRIM25, or some 
other co-precipitating protein. I understand that these are routinely done under near-denaturing 
conditions, but this cannot rule of this concern. Since this is a top-line conclusion of the 
manuscript, it must be addressed. I do not think all of the experiment needs to be redone, but just 
one or two, to show convincingly that it is in fact TRIM25 that is ubiquitin modified. I can imagine 
this being done in two ways. First, they could blot the TRIM25 pulldowns for TRIM25 (or its tag) to 
show that it in fact smears into high-molecular weight bands. Alternatively, the authors can 
pulldown ubiquitin and show that TRIM25 comes with it. 
2. The authors suggest that the data in Fig 2 shows that the increased growth of OTUD5 depleted 
cells is TRIM25 dependent. However, there data are inconclusive in this respect. The depletion of 
TRIM25 reduces proliferation is cells with OTUD5, and the relative decrease in proliferation 
between control and OTUD5-depeleted cells that are depleted for TRIM25 appears almost the 
same. I have no issue with the result, but believe it is being over-interpreted in this case. 
3. The authors refer to OTUD5 as a tumor suppressor in several places. The bar for calling it that 
is, I think, quite high. Since there is no good evidence that TRIM25 is lost or mutated in tumors, 
and by itself, would cause tumors, I think it would be more accurate to say that it has tumor 
suppressive properties. Not everything that suppresses proliferation is a tumor suppressor. 
4. Since the ubiquitination of trim25 appears to not be degradative it would be interesting to know 
what the chain linkage is, or to at least show that it is not K48 linked. This could be done using a 
K48R mutated version of ubiquitin. 
5. The mechanism for how trim25 is regulated is unclear. Although not necessary to figure out for 
publication, I would suggest simply testing if it might affect TRIM25 nuclear localization or 
chromatin binding. These experiments could be done easily, using existing reagents, with simple 
fractionation techniques. 
6. The authors say that they tested to see if trim25 regulates PML and that it doesn’t, but the data 
is not shown. This is actually a rather important finding and the negative data should be included. 
7. In a couple of places, the authors performed chip experiments and used IgG IPs in 
untransfected cells as a control for an HA IP in a transfected cell population. These should actually 
be done using an HA IP in control transfected cells. See figure 5 in particular. 
8. Figure 3B is not well controlled since phosphatase inhibitors are only used to control some of 
these conditions. Because these will block all dephosphorylation it makes the interpretation very 
difficult. 
 
 
Minor points 
1. Would be interested to test OTUD5 overexpression in some of these assays. 

2. The inputs in Fig 2A don’t make any sense. There shouldn’t be three inputs for this experiment. 
The authors need to clarify and explain better how this is done. 
3. Fig 2G- need to show TRIM25 in the input of the IP to interpret the result that the UIM mediates 
binding partially. 
4. The authors should be more clear that the phosphorylation site has already been shown to 
modulate OTUD5 activity. 
5. Authors should indicate that OTUD5 is also called DUBA. 
6. Need show evidence that the TRIM25 shRNA works 
7. Typo in fig 5H (mmRNA levels) 
8. In figure 5 there is some confusion of the use of flag or ha tags between the text and figure 
9. Remove 2F and 5E- they are not needed