Trim39 regulates neuronal apoptosis by acting as a SUMO-targeted E3 ubiquitin-ligase for the transcription factor NFATc3

  1. Meenakshi Basu Shrivastava
  2. Barbara Mojsa
  3. Stéphan Mora
  4. Ian Robbins
  5. Guillaume Bossis
  6. Iréna Lassot
  7. Solange Desagher

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Abstract

NFATc3 is the predominant member of the NFAT family of transcription factor in neurons, where it plays a pro-apoptotic role. Mechanisms controlling NFAT protein stability are poorly understood. Here we identify Trim39 as an E3 ubiquitin-ligase of NFATc3. Indeed, Trim39 ubiquitinates NFATc3 in vitro and in cells, whereas silencing of endogenous Trim39 decreases NFATc3 ubiquitination. We also show that Trim17 inhibits Trim39-mediated ubiquitination of NFATc3 by reducing both the E3 ubiquitin-ligase activity of Trim39 and the NFATc3/Trim39 interaction. Moreover, mutation of SUMOylation sites in NFATc3 or SUMO-interacting motif in Trim39 reduces the NFATc3/Trim39 interaction and Trim39-induced ubiquitination of NFATc3. As a consequence, silencing of Trim39 increases the protein level and transcriptional activity of NFATc3, resulting in enhanced neuronal apoptosis. Likewise, a SUMOylation-deficient mutant of NFATc3 exhibits increased stability and pro-apoptotic activity. Taken together, these data indicate that Trim39 modulates neuronal apoptosis by acting as a SUMO-targeted E3 ubiquitin-ligase for NFATc3.

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    Reply to the reviewers

    We are grateful to Review Commons for the opportunity to get valuable comments on our manuscript “Trim39 regulates neuronal apoptosis by acting as a SUMO-targeted E3 ubiquitin-ligase for the transcription factor NFATc3”. We would like to acknowledge the very nice and constructive reviews that our manuscript received. We found all of the reviewer comments well founded and we are taking them into careful consideration in preparing the revised version. We are currently performing additional experiments to address the questions raised by the reviewers. We are not yet able to provide a revised version of the manuscript, but you will find below our response to the reviewers and our plan of revision. It is difficult to anticipate exactly how much time we will need to get the requested results and to prepare a complete revised version, as it will depend on whether we can work normally and whether we encounter technical problems. However, it should be possible within a few months.

    Reviewer #1

    **Summary:**

    Desagher and co-workers investigate the regulation of the NFAT family member NFATc3, a transcription factor in neurons with a pro-apoptotic role. They identify TRIM39 as a ubiquitin E3 ligase regulating NFATc3. They demonstrate that TRIM39 can bind and ubiquitinate NFATc3 in vitro and in cells. They identify a critical SUMO interaction motif in TRIM39, that is required for its interaction with NFATc3 and for its ability to ubiquitinate NFATc3. Moreover, mutating sumoylation sites in NFATc3 reduces the interaction with TRIM39 and reduces its ubiquitination. Silencing TRIM39 increases the protein levels of NFATc3 and its transcriptional activity, leading to apoptosis of neurons. TRIM17 modulates the TRIM39-NFATc3 axis. Combined, TRIM39 appears to be a SUMO-targeted ubiquitin ligase (STUbL) for NFATc3 in neurons.

    **Major points:**

    1.This manuscript containing two stories: the rather exciting story that TRIM39 is a STUbL for NFATc3 (as mentioned in the title) and the second less exciting story: TRIM17 modulates the regulation of NFATc3 by TRIM39. These stories are now mixed in a confusing manner, disrupting the flow of the first story. It would be better to focus the current manuscript on the first story and strengthen it further and develop the second story in a second manuscript.

    We understand that the reviewer is more interested in the part of our manuscript related to the characterization of Trim39 as a STUbL due to his/her field of expertise. However, the two other reviewers are also interested in the other parts of our work. Notably the third reviewer would like us to highlight the physiological importance of our findings. Indeed, the main goal of this article is to describe the mechanisms regulating the stability of the transcription factor NFATc3. Trim17 plays a role in this regulation by inhibiting Trim39. It is particularly important for understanding the impact of these mechanisms on neuronal apoptosis as Trim17 is induced in these conditions. As we want to reach a wide audience, we prefer not to focus our manuscript on the identification of a new STUbL. However, we agree with the reviewer that it would be very interesting to strengthen this part of our work and we are grateful for his/her suggestions.

    2.Whereas the cellular experiments to indicate that TRIM39 acts as a STUbL are properly carried out, the observed effects are not necessarily direct. Direct evidence that TRIM39 is indeed a STUbL for sumoylated NFATc3 needs to be obtained in vitro, using purified recombinant proteins. Does TRIM39 indeed preferentially ubiquitinate sumoylated NFATc3? Is ubiquitination reduced for non-sumoylated NFATc3? Is ubiquitination of sumoylated NFATc3 dependent on SIM3 of TRIM39? Do other SIMs in TRIM39 contribute?

    We agree with the reviewer that additional in vitro experiments using purified recombinant proteins would strengthen the characterization of Trim39 as a STUbL. In order to answer the specific questions of the reviewer, we propose to perform in vitro ubiquitination using different forms of GST-Trim39 (WT/mSIM3/mSIM1&2) following in vitro SUMOylation (or not) of NFATc3 produced by TnT (wheat germ) and purified by immunoprecipitation. Preliminary results using WT Trim39 show that indeed the in vitro ubiquitination of NFATc3 is improved by prior in vitro SUMOylation. We have to confirm these results and to test the SIM mutants of Trim39 in the same conditions.

    3.Rule out potential roles for other STUbLs by including control knockdowns of RNF4 and RNF111 and verify the sumoylation of NFATc3 and ubiquitination of wildtype and sumoylation-mutant NFATc3.

    Our data show that silencing of Trim39 deeply decreases the ubiquitination level of NFATc3 in Neuro2A cells, indicating that Trim39 plays a major role in this process. We agree that this does not exclude the possible involvement of other STUbLs in NFATc3 ubiquitination in this model but their potential contribution would be limited. This point will be better addressed in the discussion.

    4.Figure 6B: use SUMO inhibitor ML-792 to demonstrate that ubiquitination of wildtype NFATc3 by TRIM39 is dependent on sumoylation.

    We thank the reviewer for suggesting this experiments that can easily improve the strength of our demonstration. Our preliminary results indeed indicate that in vivo ubiquitination of NFATc3 by Trim39 is strongly decreased following treatment with the SUMO inhibitor ML-792. We have to confirm these results.

    **Minor points:**

    5.Figure 1A and B: demonstrate by immunoprecipitation and Western that the endogenous counterparts indeed interact.

    We are currently setting the conditions to immunoprecipitate endogenous NFATc3 and Trim39 in order to demonstrate that they indeed interact.

    6.Figure 1C and 1E: Quantify the PLA results properly and perform statistics.

    We will perform these quantification and statistical analysis as requested.

    7.Figure 2B: Correct unequal loading of samples.

    We agree with the reviewer (as with reviewer #2) that the blots showing the total lysates of this experiment are confusing. As mentioned in the legend, some material has been lost during the TCA precipitation resulting in unequal loading. However, these experiments have been performed a very long time ago and we do not have the protein extracts anymore. We are currently trying to produce efficient shRNA-expressing lentiviruses to reproduce this experiment and provide a better figure.

    8.Figure 6B: proper statistics are needed here from at least three independent experiments.

    The reviewer is right. Statistics are needed to reinforce the significance of these results. We have quantified three independent experiments and made graphs and statistics that will be presented in the revised version of the manuscript. They better support our conclusion.

    Reviewer #1 (Significance (Required)):

    *Humans have over 600 different ubiquitin E3s. Currently, RNF4 and RNF111 are the only known human SUMO-Targeted Ubiquitin Ligases (STUbLs). Here, the authors present evidence that the ubiquitin E3 ligase TRIM39 is a STUbL for sumoylated NFATc3. Identification of a new STUbL **is an exciting finding for the ubiquitin and SUMO field and for the field of ubiquitin-like signal transduction in general, *but needs to be strengthened as outlined above. My field of expertise is SUMO and ubiquitin signal transduction.

    Reviewer #2

    *In this manuscript, the authors analyze the effect of TRIM39, a ubiquitin E3 ligase, on NFATc3, a transcription factor that regulates apoptosis in the nervous system. The authors show that TRIM39 can promote the ubiquitination of NFATc3 and regulate its half-life. Furthermore, ubiquitination depends on the SUMOylation state of NFATc3, which suggests that TRIM39 could be a new example of SUMOylation-dependent ubiquitin ligase or STUbL. *In addition, the authors show that TRIM17 interferes with TRIM39 ubiquitination, representing a new regulatory level for NFATc3 degradation. This has consequences on the regulation of apoptosis in cells derived from the nervous system.

    The authors show well-controlled, sound results for the most part. The manuscript is well written, and argumentation is convincing. Given the fact that only 2 STUbLs were previously characterized in mammals, the results are relevant and represent an advance in the field. Overall, this is a nice piece of work. Here are some comments.

    **Major comments**

    *-In Fig. 2B, the levels of material loaded are uneven, which difficult the interpretation. *

    We agree with the reviewer (as with reviewer #1) that the blots showing the total lysates of this experiment are confusing. As mentioned in the legend, some material has been lost during the TCA precipitation, resulting in unequal loading. In the other experiments, we have the same problem or the background is too high. We are currently trying to produce efficient shRNA-expressing lentiviruses to reproduce this experiment and provide a better figure.

    However, it seems that the control shRNA also has an effect on NFATc3 ubiquitination, which should not be the case.

    It is true that, in the present figure, the ubiquitination signal is decreased in cells transduced with the control shRNA. However, this is likely due to reduced expression of transfected NFATc3 following lentiviral infection, as it can be seen on the western blot of total lysates.

    Also, by reducing ubiquitination by TRIM39, shouldn't you expect an increase in the levels of NFATc3, if this ubiquitination was driving degradation? The authors do not specify whether those cells were treated or not with proteasomal inhibitor.

    We agree that an increase in the protein level of NFATc3 is expected following silencing of Trim39. However, in the assay presented in Figure 2B, NFATc3 is transfected and the part of overexpressed NFATc3 that is ubiquitinated by endogenous Trim39 is certainly low. Therefore, silencing of Trim39 cannot have a visible impact on the total protein level of NFATc3.

    Indeed, cells were treated with proteasome inhibitor. It is mentioned in the legend of Figure 2A. To avoid repeating it in the legend of Figure 2B, we just wrote that, after 24h transfection, cells were treated as in A, with includes MG-132 treatment for 6h.

    Same applies in Figure 4B, where no reduction in NFATc3 are seen after including TRIM39 in the reaction (beyond the fact that it looks reduced because the presence of ubiquitinated forms).

    In Figure 4B, the reaction of ubiquitination is performed in an acellular medium with purified recombinant proteins. Although NFATc3 is produced by in vitro transcription/translation in wheat germ extract, it is purified by immunoprecipitation before in vitro ubiquitination. Therefore, the reaction does not contain any proteasome and NFATc3 should not be degraded following its ubiquitination by TRIM39.

    -After the experiments in vitro shown in Fig. 2C, the authors conclude that the NFATc3 is a direct substrate of TRIM39. I think the authors used the right approach by using bacterially produced GST-TRIM39 for the ubiquitination reaction. However NFATc3 is produced by an in vitro transcription-translation system, which could in principle provide other contaminant proteins to the reaction. Did the authors try to use bacterially produced NFATc3? This might be difficult in the case of big proteins, in which case the authors could add some caution note in the text. Same applies in Figure 4B.

    The reviewer is right. It would have been preferable to use NFATc3 produced in bacteria. Indeed, we started with this approach. However, it was very difficult to get NFATc3 expressed in bacteria, and when we succeeded, most of the protein was degraded. We tried different protease inhibitor cocktails and we used a strain of bacteria (BL21-CodonPlus(DE3)-RP) that is mutated on the genes coding for the proteases Lon and OmpT and is further engineered to express tRNAs that are often limiting when expressing mammalian proteins. Unfortunately, this did not improve our production enough.

    We agree that, in principle, in vitro transcription-translation (TnT) systems can include contaminant proteins. However, we used wheat germ extract to produce NFATc3 by TnT. Moreover, we immunopurified NFATc3 from the TnT reaction prior to the ubiquitination reaction. The probability that proteins modifying NFATc3 are expressed in plants and are co-purified with NFATc3 is low. Nevertheless, we will discuss this point in the result section of the revised version of the manuscript, when describing results of Figure 2B and 4B.

    *-In Fig. 6B, higher levels of ubiquitination in the different SUMOylation mutants are shown. Is this effect consistent? How this can be explained? *

    We are grateful to the reviewer for pointing out this inconsistency in our manuscript. It will be corrected. Indeed, the values indicated in red in Figure 6B are confusing and are certainly not consistent. We calculated them by normalizing the intensity of the ubiquitination signal by the intensity of NFATc3 in total lysates, which seems to have introduced a bias. Variations in NFATc3 levels are probably responsible for the artificially higher levels of ubiquitination for different SUMOylation mutants after normalization. When quantifying three independent experiments, as requested by reviewer #1, we realized that results are much more consistent without normalization. Therefore, in the revised version of manuscript, we will add a graph showing the average and standard deviation of three independent experiments quantified without normalization. We will also replace the experiment currently presented in Figure 6B by another one in which the levels of NFATc3 show lower variations in the total lysates.

    In addition, variations in the levels of NFATc3 are shown in the total lysate, despite the use of proteasomal inhibitors. How the author explain this effect?

    These variations in NFATc3 levels in the total lysates may be due to differential protein precipitation by TCA. That is why, in more recent experiments, we collected a portion of the homogenous cell suspension before lysis in the guanidinium buffer, to assess the expression level of transfected proteins (as presented in Figures 4A and 7E).

    It is true that treatment with proteasome inhibitor should attenuate differences in protein level due to different ubiquitination levels. However, cells are transfected for 24h and then treated with MG-132 for 6h before lysis. Proteasome inhibition cannot compensate for what occurred in the cells during the 24h transfection. It is added essentially to accumulate poly-ubiquitinated forms of NFATc3.

    Somehow, this is contradictory with the general message of SUMOylation-dependent ubiquitination.

    The reduced levels of SUMOylation mutants in total lysates may appear to be contradictory with SUMOylation-dependent ubiquitination. However, as mentioned above, this could be due to differential protein precipitation by TCA or to different transfection efficiencies. In contrast, the half-life measurement of WT and EallA mutant, that does not rely on initial expression levels, clearly shows a stabilization of the SUMOylation mutant. Moreover, the average of the three ubiquitination experiments is really convincing. Therefore, we believe that the data that will be presented in the revised manuscript will strongly support our hypothesis.

    -In Fig. 7E, not clear to me what the big bands above 130 KDa is after the nickel beads. Do they correspond to monoUb NFATc3 or to the unmodified protein that is sticky to the beads? Do the authors have side-by-side gels of the initial lysate next to the nickel beads eluates to show the increase in molecular weight?

    The big bands above 130 kD among nickel bead-purified proteins in Figure 7A are unlikely to be unmodified NFATc3 sticking to the beads. Indeed, in the control condition, in which NFATc3 is overexpressed in the absence of His-ubiquitin, these bands are not visible. Therefore, they might be mono-ubiquitinated forms of NFATc3, or degradation products of poly-ubiquitinated NFATc3. We will correct the figure to clarify this point. Unfortunately, we do not have a gel with nickel bead eluates and total lysates side by side for this experiment.

    -Quantifications in some pictures (i.e. Figures 5A, 5B, 6B, 7) is shown in red above or below the bands. Not clear whether the quantifications shown correspond to that single experiment or is the average of several experiments. In the first case, the number would not be very valuable. Authors could add quantification graphs with standard deviations or error bars to the experiments if they want to make the point of changes (significant or not) in the levels. Alternatively, indicate in the Figure legends whether the numbers correspond to the average of several experiments.

    These quantifications correspond to the representative experiments shown in the different figures. We will clarify this point in the Figure legends of the revised manuscript. We added these quantifications to normalize the amount of co-precipitated proteins by the amount of the precipitated partner (Fig 5A, 5B, 7B, 7C, 7D) which is not always precipitated with the same efficiency in the different conditions. We think that it should help the reader to assess the degree of interaction. We also added quantifications to Figure 7E to normalize the ubiquitination signal by the amount of NFATc3 expressed in the total lysate. However, we did not want to overload the figures by adding too many graphs.

    For Figure 6B, where TCA precipitation of total lysates created an inconsistency, we will provide a graph with the average and standard deviation of three independent experiments, as requested by reviewer #1.

    -In Fig. 8, the quantification of apoptotic nuclei has been done just based on the morphology after DAPI staining. Could you use an apoptosis marker (i.e. cleaved caspase Abs) to label the apoptotic cells?

    We have been using primary cultures of cerebellar granule neurons (CGN) as an in vitro model of neuronal apoptosis for many years. Nuclear condensation, visualized after DAPI staining, is very characteristic in these neurons and allows a reliable assessment of neuronal apoptosis. In a previous study (Desagher et al. JBC 2005), we have shown that the kinetics of apoptosis in CGN is the same whether we measure cytochrome c release, active caspase 3 or nuclear condensation (Fig 1b). We therefore believe that the counting of apoptotic nuclei is sufficient to support our conclusions, notably for transfection experiments in Figure 8A which would require a lot of work to be repeated with active caspase 3 staining. However, if we can produce efficient shRNA-expressing lentiviruses, we will reproduce the experiment presented in Figure 8B and we will perform a western blot using anti-active caspase 3 to confirm our conclusion.

    **Minor comments**

    -In Figs. 1 and 5, the red channel should be put in black and white, as it is much easier to see the signal. Not relevant to have DAPI alone in B&W (it does not hurt either), as it is well visible in the merge picture. Also, quantification of the PLA positive dots should be shown in Fig. 1.

    We thank the reviewer for these suggestions. We will modify the figures and we will quantify the PLA dots in Figure 1 as requested.

    -In Fig. 3C, is the difference in TRIM17 expression between empty plasmid and NFATc3 plasmid significant? If so, indicate it in the graph. The same in panels D, E, indicate all significant differences. Same in other Figures.

    No, the difference in Trim17 expression is not statistically significant between NFATc3 and empty plasmid although it clearly increases. However, we agree with the reviewer that more significant differences could be shown in the figures, particularly in Figure 3. Nonetheless, we will try not to overload the figures and will restrict ourselves to comparisons that make sense.

    -It would be nice to show a scheme on the location of SIMs in TRIM39 in relation to the other feature of the protein.

    We are grateful to the reviewer for this suggestion. We will be happy to add a scheme of Trim39 showing its different domains and the location of its SIMs in the revised Figure 7.

    -In Fig. 2 legend, "Note that in the presence of ubiquitin the unmodified form of WT GST-Trim39 is lower due to high Trim39 ubiquitination." Please change to "...in the presence of ubiquitin the levels of the unmodified form..."

    -In Fig. 7 legend, the phrases "The intensity of the bands ... " are not clear. Please rephrase.

    *-In Fig. 8 legend, "*** * PWe thank the reviewer for pointing out typographical errors and awkward sentences in our manuscript. Changes will be made as requested.

    Reviewer #2 (Significance (Required)):

    In this manuscript, the authors analyze the effect of TRIM39, a ubiquitin E3 ligase, on NFATc3, a transcription factor that regulates apoptosis in the nervous system. The authors show that TRIM39 can promote the ubiquitination of NFATc3 and regulate its half-life. Furthermore, ubiquitination depends on the SUMOylation state of NFATc3, which suggests that TRIM39 could be a new example of SUMOylation-dependent ubiquitin ligase or STUbL. In addition, the authors show that TRIM17 interferes with TRIM39 ubiquitination, representing a new regulatory level for NFATc3 degradation. This has consequences on the regulation of apoptosis in cells derived from the nervous system.

    The authors show well-controlled, sound results for the most part. The manuscript is well written, and argumentation is convincing. Given the fact that only 2 STUbLs were previously characterized in mammals, the results are relevant and represent an advance in the field. Overall, this is a nice piece of work.

    Audience: researchers interested on proteostasis in general and on nervous system regulation

    My expertise: postranslational modifications

    Reviewer #3

    **Summary:**

    In this study, Shrivastava et al. elucidated the previously unknown function of TRIM39 in regulating protein stability of NFATc3, the predominant member of the NFAT family of transcription factor in neurons, where it plays a pro-apoptotic role. NFATs have been shown to be regulated by multiple mechanisms, including at the level of protein stability. In this study, the authors identify TRIM39 as the E3 ligase for NFATc3. Interestingly, TRIM39 recognizes the SUMOylated form of NFATc3 and the interaction facilitates its ubiquitylation and subsequent proteasomal degradation. They further showed that binding of TRIM39 to NFATc3 can also be regulated by TRIM17. Like TRIM39, TRIM17 is a ring-finger containing protein previously shown by this group that it binds NFATc3 but the interaction resulted in an up- rather than down-regulation of NFATc3. In this study, they offer insight to the paradox that overexpression of TRIM17 binding to TRIM39 is to inhibit TRIM39-mediated ubiquitylation of NFATc3. Furthermore, they showed activation of NFATc3 transcriptionally activates TRIM17 expression, thus forming a feedback loop between NFATc3 and TRIM17. Hence, an TRIM17-TRIM39-NFATc3 signaling axis for modulating the protein stability for promoting the activity of NFATc3 in regulating apoptosis in the cerebellar granule neurons induced by KCl deprivation is proposed

    *The key conclusions are convincing. The data in general are of good quality and with many of the key interactions vigorously documented *by conducting reciprocal interaction analysis. For knockdown expeRIMents, two shRNA independent sequences were used. However, some issues remain to be addressed:

    **Major comments:**

    1.Figure 1D - the authors should demonstrate that the depletion of TRIM39 expression by shRNA in Neuro2A by Western blotting

    We agree with the reviewer that it would be better to provide this control. Unfortunately, we have never been able to observe a convincing decrease in the protein level of Trim39, following knockdown, by Western blotting in Neuro2A cells. This is surprising because the decrease is clearly visible by immunofluorescence in Neuro2A cells, and by western blotting in neurons (see Figure 8C). It is possible that Neuro2A cells, but not neurons, express a protein that is non-specifically recognized by our best anti-Trim39 antibody in western blots and that migrates at the same size as Trim39, thus preventing the investigator to detect the depletion of Trim39. We will test additional anti-Trim39 antibodies to address this question.

    2.Figure 3 - the author should show overexpression of TRIM39 resulted in reduction of basal level of endogenous NFATc3 due to its effect on protein stability by using CHX or other pulse chase method.

    This is an important point and we have performed many experiments using cycloheximide to measure the half-life of NFATc3 in the presence or the absence of overexpressed Trim39. The results were neither consistent nor reproducible. This is certainly due to the fact that the half-life of endogenous NFATc3 is longer than that of overexpressed Trim39 and that cycloheximide inhibits the expression of both proteins. Therefore, we will perform pulse-chase experiments after metabolic labelling of cells with [35S]-Met. We are currently setting up the conditions to immunoprecipitate endogenous NFATc3 to be able to perform these experiments.

    3.Figure 3 - Does overexpression or knockdown of TRIM39 has an effect on affecting levels of NFATc3 mRNAs?

    The reviewer is right. It is important to control that overexpression and knockdown of Trim39 do not modify the mRNA level of NFATc3. Therefore, we are currently measuring NFATc3 mRNA levels in all the experiments used to make the graphs of Figure 3. These results will be added to the revised version of the manuscript as supplemental data. First results show no significant change of NFATc3 mRNA levels in these experiments.

    4.Figure 6A - the authors should confirm the multiple bands that are slower migrating are SUMO form of NFATc9 by demonstrating the presence of SUMO in these forms of NFATc3, or alternatively, perform His-SUMO pull-down and probe for NFATc3.

    The reactions shown in Figure 6B have been performed in vitro, with purified recombinant proteins and with NFATc3 produced by in vitro transcription/translation. The wheat germ extract used to produce NFATc3 is unlikely to provide the material needed for post-translation modification of a mammalian protein. However, we agree that it would be better to confirm that slower migrating bands are indeed SUMOylated forms of NFATc3. We may hybridize the membranes with an anti-SUMO antibody but it would give a smear as the enzymes added to the reaction mix are themselves SUMOylated. Therefore, we will show an experiment in which the reaction mix has been incubated with and without SUMO. The results show no slower migrating bands in the absence of SUMO although all conditions were otherwise identical. This will be added to the revised Figure 6.

    5.Figure 7C - the quantification for mSIM1 does not seem to agree with the band intensity.

    Yes, we agree with the reviewer that the quantification (122%) does not seem to reflect the amount of SUMO-chains bound to GST-Trim39 mSIM1. This is due to the normalization of the SUMO signals by the intensity of GST-Trim39 bands. Indeed, it is difficult to control exactly how much recombinant protein is used. GST-Trim39 mSIM1 was slightly less abundant than the other GST-Trim39 proteins in this experiment, explaining why less SUMO-chains were eluted in this condition. The normalization is mentioned in the legend of Figure 7C.

    6.TRIM17 reduces TRIM39/NFATc3 interaction and inhibits TRIM39 E3 activity, which results in stabilization of NFATc3. NFATc3 in turn transcriptionally induces TRIM17 expression, thus forming a feedback loop between NFATc3 and TRIM17. It will be good if the authors can discuss the possibility of the existence of this feedback mechanism in physiological context? Is the protein level of NFATc3 level, which should be low abundance at the resting state, elevated by KCI deprivation? If so, can the authors discuss the possible signalling event(s) that that lead to activation of NFATc3 upon KCI deprivation? For instance, does KCL deprivation cause de-SUMOylation of NFATc3?

    We thank the reviewer for these suggestions. Our preliminary results suggest that the protein level of NFATc3 is increased in neurons following KCl deprivation. We are currently performing additional experiments to confirm this result. If proved, this increase may be due to the transcriptional induction of Trim17 that should result in the stabilization of NFATc3 through the inhibition of Trim39. It may also be due to a possible deSUMOylation of NFATc3 following apoptosis induction, as suggested by the reviewer. To address the latter point, we are currently setting up PLA using anti-NFATc3 and anti-SUMO antibodies to assess the SUMOylation level of endogenous NFATc3 in neurons. If they are of good quality, we will add these data to Figure 8 and we will discuss the possible existence of feedback loops in neuronal apoptosis, as suggested by the reviewer.

    **Minor comments:**

    1.Line 294 - it should be "SUMOylation" instead of "SUMO".

    We thank the reviewer for pointing out this typographical error that will be corrected.

    2.Figure 8 - to include TRIM39/NFATc3 double knockdown to show the effect on increased neuronal apoptosis in the cells with TRIM39 knocked down was due to elevation of NFATc3 rather than other target(s) of TRIM39.

    We agree that it would be interesting to test whether the increase on neuronal apoptosis following Trim39 silencing is mainly due to its effect on NFATc3. We will therefore perform double silencing of Trim39 and NFATc3 in neurons in order to address this point.

    3.The discussion may be shortened and revised to highlight the physiological importance of the findings linked to cerebellar granule neurons survival.

    As suggested by the reviewer, we will modify the discussion to better highlight the physiological implications of our data, particularly by discussing the results of the additional experiments we will conduct in neurons.

    Reviewer #3 (Significance (Required)):

    *Prior to this study, the mechanism by which protein stability of NFATc3, the pre-dominant member of the NFAT family of transcription factor in neurons, is regulated remains poorly understood. Shrivastava et al. have unravelled the interplay between ubiquitylation and SUMOylation involving TRIM39 and TRIM17 to have an important role in regulating protein stability of NFATc3. **The work is interesting and bears significance *towards understanding how apoptosis could be finely controlled in cerebellar granule neurons. Furthermore, the study has also expanded the understanding of the role and regulation of the TRIM family of proteins. The senior author is an expert in this field and over the years, her group has contributed many key discoveries on the function of TRIM family of E3 ubiquitin ligases and their critical ubiquitylation substrates in neuronal survival and its relevance to neuronal biology and diseases.

    The referee's field of expertise in in the field of mitochondrial apoptosis signalling. The referee extensively involved in studying how protein stability of regulators in apoptosis signalling are regulated by the ubiquitin-proteasome system (UPS) and how does the regulation play a role in physiology and diseases.

    Key words: apoptosis, ubiquitylation, cell signaling, liver diseases

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    In this study, Shrivastava et al. elucidated the previously unknown function of TRIM39 in regulating protein stability of NFATc3, the predominant member of the NFAT family of transcription factor in neurons, where it plays a pro-apoptotic role. NFATs have been shown to be regulated by multiple mechanisms, including at the level of protein stability. In this study, the authors identify TRIM39 as the E3 ligase for NFATc3. Interestingly, TRIM39 recognizes the SUMOylated form of NFATc3 and the interaction facilitates its ubiquitylation and subsequent proteasomal degradation. They further showed that binding of TRIM39 to NFATc3 can also be regulated by TRIM17. Like TRIM39, TRIM17 is a ring-finger containing protein previously shown by this group that it binds NFATc3 but the interaction resulted in an up- rather than down-regulation of NFATc3. In this study, they offer insight to the paradox that overexpression of TRIM17 binding to TRIM39 is to inhibit TRIM39-mediated ubiquitylation of NFATc3. Furthermore, they showed activation of NFATc3 transcriptionally activates TRIM17 expression, thus forming a feedback loop between NFATc3 and TRIM17. Hence, an TRIM17-TRIM39-NFATc3 signaling axis for modulating the protein stability for promoting the activity of NFATc3 in regulating apoptosis in the cerebellar granule neurons induced by KCl deprivation is proposed.

    The key conclusions are convincing. The data in general are of good quality and with many of the key interactions vigorously documented by conducting reciprocal interaction analysis. For knockdown expeRIMents, two shRNA independent sequences were used. However, some issues remain to be addressed:

    Major comments:

    1.Figure 1D - the authors should demonstrate that the depletion of TRIM39 expression by shRNA in Neuro2A by Western blotting

    2.Figure 3 - the author should show overexpression of TRIM39 resulted in reduction of basal level of endogenous NFATc3 due to its effect on protein stability by using CHX or other pulse chase method.

    3.Figure 3 - Does overexpression or knockdown of TRIM39 has an effect on affecting levels of NFATc3 mRNAs?

    4.Figure 6A - the authors should confirm the multiple bands that are slower migrating are SUMO form of NFATc9 by demonstrating the presence of SUMO in these forms of NFATc3, or alternatively, perform His-SUMO pull-down and probe for NFATc3.

    5.Figure 7C - the quantification for mSIM1 does not seem to agree with the band intensity.

    6.TRIM17 reduces TRIM39/NFATc3 interaction and inhibits TRIM39 E3 activity, which results in stabilization of NFATc3. NFATc3 in turn transcriptionally induces TRIM17 expression, thus forming a feedback loop between NFATc3 and TRIM17. It will be good if the authors can discuss the possibility of the existence of this feedback mechanism in physiological context? Is the protein level of NFATc3 level, which should be low abundance at the resting state, elevated by KCI deprivation? If so, can the authors discuss the possible signalling event(s) that that lead to activation of NFATc3 upon KCI deprivation? For instance, does KCL deprivation cause de-SUMOylation of NFATc3?

    Minor comments:

    1.Line 294 - it should be "SUMOylation" instead of "SUMO".

    2.Figure 8 - to include TRIM39/NFATc3 double knockdown to show the effect on increased neuronal apoptosis in the cells with TRIM39 knocked down was due to elevation of NFATc3 rather than other target(s) of TRIM39.

    3.The discussion may be shortened and revised to highlight the physiological importance of the findings linked to cerebellar granule neurons survival.

    Significance

    Prior to this study, the mechanism by which protein stability of NFATc3, the pre-dominant member of the NFAT family of transcription factor in neurons, is regulated remains poorly understood. Shrivastava et al. have unravelled the interplay between ubiquitylation and SUMOylation involving TRIM39 and TRIM17 to have an important role in regulating protein stability of NFATc3. The work is interesting and bears significance towards understanding how apoptosis could be finely controlled in cerebellar granule neurons. Furthermore, the study has also expanded the understanding of the role and regulation of the TRIM family of proteins. The senior author is an expert in this field and over the years, her group has contributed many key discoveries on the function of TRIM family of E3 ubiquitin ligases and their critical ubiquitylation substrates in neuronal survival and its relevance to neuronal biology and diseases.

    The referee's field of expertise in in the field of mitochondrial apoptosis signalling. The referee extensively involved in studying how protein stability of regulators in apoptosis signalling are regulated by the ubiquitin-proteasome system (UPS) and how does the regulation play a role in physiology and diseases.

    Key words: apoptosis, ubiquitylation, cell signaling, liver diseases

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, the authors analyze the effect of TRIM39, a ubiquitin E3 ligase, on NFATc3, a transcription factor that regulates apoptosis in the nervous system. The authors show that TRIM39 can promote the ubiquitination of NFATc3 and regulate its half-life. Furthermore, ubiquitination depends on the SUMOylation state of NFATc3, which suggests that TRIM39 could be a new example of SUMOylation-dependent ubiquitin ligase or STUbL. In addition, the authors show that TRIM17 interferes with TRIM39 ubiquitination, representing a new regulatory level for NFATc3 degradation. This has consequences on the regulation of apoptosis in cells derived from the nervous system. The authors show well-controlled, sound results for the most part. The manuscript is well written, and argumentation is convincing. Given the fact that only 2 STUbLs were previously characterized in mammals, the results are relevant and represent an advance in the field. Overall, this is a nice piece of work. Here are some comments.

    Major comments

    -In Fig. 2B, the levels of material loaded are uneven, which difficult the interpretation. However, it seems that the control shRNA also has an effect on NFATc3 ubiquitination, which should not be the case. Also, by reducing ubiquitination by TRIM39, shouldn't you expect an increase in the levels of NFATc3, if this ubiquitination was driving degradation? The authors do not specify whether those cells were treated or not with proteasomal inhibitor. Same applies in Figure 4B, where no reduction in NFATc3 are seen after including TRIM39 in the reaction (beyond the fact that it looks reduced because the presence of ubiquitinated forms).

    -After the experiments in vitro shown in Fig. 2C, the authors conclude that the NFATc3 is a direct substrate of TRIM39. I think the authors used the right approach by using bacterially produced GST-TRIM39 for the ubiquitination reaction. However NFATc3 is produced by an in vitro transcription-translation system, which could in principle provide other contaminant proteins to the reaction. Did the authors try to use bacterially produced NFATc3? This might be difficult in the case of big proteins, in which case the authors could add some caution note in the text. Same applies in Figure 4B.

    -In Fig. 6B, higher levels of ubiquitination in the different SUMOylation mutants are shown. Is this effect consistent? How this can be explained? In addition, variations in the levels of NFATc3 are shown in the total lysate, despite the use of proteasomal inhibitors. How the author explain this effect? Somehow, this is contradictory with the general message of SUMOylation-dependent ubiquitination.

    -In Fig. 7E, not clear to me what the big bands above 130 KDa is after the nickel beads. Do they correspond to monoUb NFATc3 or to the unmodified protein that is sticky to the beads? Do the authors have side-by-side gels of the initial lysate next to the nickel beads eluates to show the increase in molecular weight?

    -Quantifications in some pictures (i.e. Figures 5A, 5B, 6B, 7) is shown in red above or below the bands. Not clear whether the quantifications shown correspond to that single experiment or is the average of several experiments. In the first case, the number would not be very valuable. Authors could add quantification graphs with standard deviations or error bars to the experiments if they want to make the point of changes (significant or not) in the levels. Alternatively, indicate in the Figure legends whether the numbers correspond to the average of several experiments.

    -In Fig. 8, the quantification of apoptotic nuclei has been done just based on the morphology after DAPI staining. Could you use an apoptosis marker (i.e. cleaved caspase Abs) to label the apoptotic cells?

    Minor comments

    -In Figs. 1 and 5, the red channel should be put in black and white, as it is much easier to see the signal. Not relevant to have DAPI alone in B&W (it does not hurt either), as it is well visible in the merge picture. Also, quantification of the PLA positive dots should be shown in Fig. 1.

    -In Fig. 3C, is the difference in TRIM17 expression between empty plasmid and NFATc3 plasmid significant? If so, indicate it in the graph. The same in panels D, E, indicate all significant differences. Same in other Figures.

    -It would be nice to show a scheme on the location of SIMs in TRIM39 in relation to the other feature of the protein.

    -In Fig. 2 legend, "Note that in the presence of ubiquitin the unmodified form of WT GST-Trim39 is lower due to high Trim39 ubiquitination." Please change to "...in the presence of ubiquitin the levels of the unmodified form..."

    -In Fig. 7 legend, the phrases "The intensity of the bands ... " are not clear. Please rephrase.

    -In Fig. 8 legend, "*** * P<0.001". Change to "*** P<0.001".

    Significance

    In this manuscript, the authors analyze the effect of TRIM39, a ubiquitin E3 ligase, on NFATc3, a transcription factor that regulates apoptosis in the nervous system. The authors show that TRIM39 can promote the ubiquitination of NFATc3 and regulate its half-life. Furthermore, ubiquitination depends on the SUMOylation state of NFATc3, which suggests that TRIM39 could be a new example of SUMOylation-dependent ubiquitin ligase or STUbL. In addition, the authors show that TRIM17 interferes with TRIM39 ubiquitination, representing a new regulatory level for NFATc3 degradation. This has consequences on the regulation of apoptosis in cells derived from the nervous system.

    The authors show well-controlled, sound results for the most part. The manuscript is well written, and argumentation is convincing. Given the fact that only 2 STUbLs were previously characterized in mammals, the results are relevant and represent an advance in the field. Overall, this is a nice piece of work.

    Audience: researchers interested on proteostasis in general and on nervous system regulation

    My expertise: postranslational modifications

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Desagher and co-workers investigate the regulation of the NFAT family member NFATc3, a transcription factor in neurons with a pro-apoptotic role. They identify TRIM39 as a ubiquitin E3 ligase regulating NFATc3. They demonstrate that TRIM39 can bind and ubiquitinate NFATc3 in vitro and in cells. They identify a critical SUMO interaction motif in TRIM39, that is required for its interaction with NFATc3 and for its ability to ubiquitinate NFATc3. Moreover, mutating sumoylation sites in NFATc3 reduces the interaction with TRIM39 and reduces its ubiquitination. Silencing TRIM39 increases the protein levels of NFATc3 and its transcriptional activity, leading to apoptosis of neurons. TRIM17 modulates the TRIM39-NFATc3 axis. Combined, TRIM39 appears to be a SUMO-targeted ubiquitin ligase (STUbL) for NFATc3 in neurons.

    Major points:

    1.This manuscript containing two stories: the rather exciting story that TRIM39 is a STUbL for NFATc3 (as mentioned in the title) and the second less exciting story: TRIM17 modulates the regulation of NFATc3 by TRIM39. These stories are now mixed in a confusing manner, disrupting the flow of the first story. It would be better to focus the current manuscript on the first story and strengthen it further and develop the second story in a second manuscript.

    2.Whereas the cellular experiments to indicate that TRIM39 acts as a STUbL are properly carried out, the observed effects are not necessarily direct. Direct evidence that TRIM39 is indeed a STUbL for sumoylated NFATc3 needs to be obtained in vitro, using purified recombinant proteins. Does TRIM39 indeed preferentially ubiquitinate sumoylated NFATc3? Is ubiquitination reduced for non-sumoylated NFATc3? Is ubiquitination of sumoylated NFATc3 dependent on SIM3 of TRIM39? Do other SIMs in TRIM39 contribute?

    3.Rule out potential roles for other STUbLs by including control knockdowns of RNF4 and RNF111 and verify the sumoylation of NFATc3 and ubiquitination of wildtype and sumoylation-mutant NFATc3.

    4.Figure 6B: use SUMO inhibitor ML-792 to demonstrate that ubiquitination of wildtype NFATc3 by TRIM39 is dependent on sumoylation.

    Minor points:

    5.Figure 1A and B: demonstrate by immunoprecipitation and Western that the endogenous counterparts indeed interact.

    6.Figure 1C and 1E: Quantify the PLA results properly and perform statistics.

    7.Figure 2B: Correct unequal loading of samples.

    8.Figure 6B: proper statistics are needed here from at least three independent experiments.

    Significance

    Humans have over 600 different ubiquitin E3s. Currently, RNF4 and RNF111 are the only known human SUMO-Targeted Ubiquitin Ligases (STUbLs). Here, the authors present evidence that the ubiquitin E3 ligase TRIM39 is a STUbL for sumoylated NFATc3. Identification of a new STUbL is an exciting finding for the ubiquitin and SUMO field and for the field of ubiquitin-like signal transduction in general, but needs to be strengthened as outlined above. My field of expertise is SUMO and ubiquitin signal transduction.

  5. Version published to 10.1101/2020.09.29.317958 on bioRxiv