MMD-associated RNF213 SNPs encode dominant-negative alleles that globally impair ubiquitylation

MMD-associated SNPs of RNF213 encode dominant-negative alleles that globally impair ubiquitylation by RNF213-UBE2D2.

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Full guidelines are available on our Instructions for Authors page, http://www.life-science-alliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** Bhardwaj et al studied the E3-ligase and ATPase activities of 293T or HeLa cells that overexpressed normal RNF213 protein and mutant RNF213 proteins with E2488Q, I2999A and R4810K. They found that RNF213 uses UBE2D2 to catalyze K6dependent poly-ubiquitination, and measured the E3-ligase activity by using this system. They found that the E3-ligase activities of mutant RNF213 proteins decreased, compared with normal RNF213. By contrast, the ATPase activity was not different between the normal and mutant RNF213 proteins. They concluded that RNF213 with R4810R mutation is a dominant negative mutant that decreases ubiquitylation globally.
Authors deal with critical issues how heterozygous RNF213 mutations increased the risk for MMD and the paper is well written. 1) There is a great concern regarding the assay system of E3-ligase activity of RNF213. Physiological E2 for RNF213 is not known and validation of use of UBE2D2 as E2 for the assay system remains unestablished. 2) All of the experiments were performed in the overexpressed RNF213 in cultured cells. Since overexpression experiments sometimes reflect unphysiological condition authors should provide the evidence of physiological conditions before they conclude that RNF213 with R4810R mutation works in a dominant-negative manner.
Reviewer #2 (Comments to the Authors (Required)): The manuscript by Bhardwaj et al. discusses ubiquitination activities of the E3 ligase RNF213 that is mutated in Moyamoya disease. With its size of >5200 Aa, this is a huge protein, and it is commendable that the authors attempt to tackle a protein of such size, and indeed the cell biological data with full-length proteins are of very good quality. Moreover, expression suggests that the protein can be generated in good quantity and quality, which should be exciting for any cryo-EM person on campus.
Mutations in the RING domain cause expected loss of ubiquitination activity. Interestingly, also Moyamoya SNPs disrupt E3 activity (while not affecting ATPase activity). What is striking yet not discussed, is why/how an MMD mutant, R4810K, can affect RING E3 activity of a domain some 800 Aa upstream. A second striking finding is that The RNF213 RING domain seemingly directs the known, non-specific E2 ubiquitination activity of a widely used E2 enzyme, UBE2D2, towards assembling predominantly Lys6-linkages.
Clearly, there are interesting intrinsic mechanistic features in this exciting E3 ligase, which scream for a more detailed molecular/structural characterisation. Such analysis would be beyond the scope of the manuscript, which I enjoyed reading for its clarity and compelling findings, and which I would support to publish in Life Science Alliance once below minor comments are addressed.
1) There are now many complementary methods to gain insights into ubiquitin chain types, and the authors use a somewhat outdated method, ubiquitin mutants. Especially concerning K6 chains, this has led to confusing data in the past (reason being, that K6 is also important for binding to and positioning Ub at the E2 enzyme). See work on MHC-I ubiquitination by Lehner and colleagues/Boname et al and also Goto et al. 2009/10. Better approaches that would be highly doable for the in-vitro Fig 1 and potentially for the cell-based figures, are massspectrometry, and use of the K6-affimer (though this may be off the market at the moment), to provide additional evidence for K6 chain assembly. 2) E3 ligases are typically not specific for individual members of the UBE2D family. Fig 1c is cause for some concern. The authors should perform control experiments to show that their E1 preparation can charge all E2 enzymes similarly, and work with charged E2 enzymes. An additional control would be to use a different, well expressed E3 ligase with known non-discriminatory E2 usage for E2Ds -there are many GST-RING-members members that should work here, and which should be commercially available.
3) Why does the intensity of the Ub signal change with the use of the K-only mutants? (1e). Again, this data suggests that the other mutants are very differently charged by the E1 enzyme. I don't understand the strong 135kDa signal labelled as E1~Ub. I agree that it is suggestive of a difference in activity, but it is not clear why it should light up. 4) While challenging on a 5200 aa protein, some attempt at sec structure prediction should be performed. Is the region beyond the RING domain ordered/domain character and are any repeat sequences recogniseable? What is known about other domains beyond those listed in Fig 1? What is the closest domain/protein similarity and species conservation for the region encompassing R4810K? this information would be valuable to add. 5) Methods lacks a section on molecular biology and sequencing, which given the size of the expressed protein, should be added. A Table with  This manuscript describes an E3 ubiquitin ligase in which mutations have been strongly linked to Moyamoya disease (MMD). Here, the authors demonstrate that RNF213 functions in concert with the E2 UBE2D2 in the conjugation of -K6-linked ubiquitin chains and demonstrate that high penetrance variants of RNF213 result in a reduction of global protein ubiquitination, yielding insight into MMD. Findings from this work attempt to settle controversy on RNF213 ATPase activity, multimerization and ubiquitin function as causative activities for MMD. Overall, these findings are interesting and I have few comments related to necessary controls and to help settle controversial functions of RNF213.
Major Comments: -The high molecular weight products in Figure 1c, 1d, and 1e must show that these modifications are indeed assembled on GST-RING RNF213 and not due to autoubiquination of other enzymes present in the in vitro reaction such as E1 and E2s themselves. The authors must further show that the catalytic activity of GST-RING RNF213 is required for ubiquitination via UBE2D2. It is unclear how the ubiquitin chain mutants were generated in this assay as it is not clearly stated in the supplemental methods section. Further, the quantities/concentrations of all enzymes used in these reactions must be reported in the methods section.
-The authors' claim based on the data in figures 1d and 1e that other chain linkages can be formed on RNF213 based on the finding that adding back K6-linked chains only partially recovers poly-Ub of RNF213 to WT. It would be important for the authors to further explore this possibility through measurements of chain formation or adding back lysines (especially -K63) within ubiquitin in an attempt to recover levels to WT.
-Given the prior publication (Takeda_et_al, 2020) demonstrating Ubc13/Uev1a to have strong activity for RNF213 RING fragment and no activity with UbcH5b (UBE2D2), it will be critical for the authors to compare their findings in a side-by-side experiment using these two E2s and explain why the authors in this paper were not able to observe autoubiquitination activity in their assays.
-Given that full-length RNF213 can be expressed in cells, it is critical to establish that the full-length version of RNF213 can indeed assemble -K6 ubiquitin linkages.
-I disagree with the authors' conclusion that RNF213 ubiquitination is substantially reduced in UBE2D2 knockdown cells. The molecular weight of the HA-Ub reactive band is lower than the FLAG-RNF213 bands shown in the image (Figure 1h). How do the authors reconcile this discrepancy? And, if the assembly of RNF213 via UBE2D2 takes place via -K6 polyubiquitin linkages, why does this not appear as a higher molecular weight smear on the membrane, similar to Figure 3c? -In addition to the coomassie gel stain blot, please provide a FLAG immunoblot of the immune purified proteins along with a vector only control with FLAG antibody lane.
-The ubiquitination experiment shown in Figure 3c should also be done in cells as opposed to a post-hoc ubiquitin assay.
-It is unclear how the experiments were conducted in Figure 3e. The amount of total protein ubiquitination is not a reflection of the E3 ligase activity of RNF213 as one needs to show the ubiquitin is directly conjugated to RNF213. Thus, at minimum, the authors need to purify these proteins similar to what was done in Figure 3c or interpret their results based on global changes in ubiquitin patterning.
-Are the increases in Ub conjugates observed with RNF213 overexpression -K6 linked?
-The quantification of experiments from Figures 4a and b appear that the ATPase mutant (E2488Q) does not increase ubiquitination like the WT, suggesting that this activity is not serving in a dominant negative manner but is important to enhance protein ubiquitination. Moreover, it is unclear why the authors are not using the double mutant (E2488Q/E2845Q) that shows a lack of ATPase activity from Figure 2c.
Minor comments: -In the introduction, state that protein ubiquitination selectively modifies lysine residues on a substrate.
-The authors discuss the common RNF213 R4810 mutation in the results section but never introduce this mutation in the prior text.
-There are other studies that assessed E3 ligase activity with one of the most common RNF213 variants. It is important to cite these key papers in the Introduction.
-Please provide original citations for the RNF213 reported patient mutations used in this study.
-There are a few grammatical errors in the text that need to be corrected.

Detailed Response to Reviewers' Comments
Reviewer #1 (Comments to the Authors (Required)): <Summary> Bhardwaj et al studied the E3-ligase and ATPase activities of 293T or HeLa cells that overexpressed normal RNF213 protein and mutant RNF213 proteins with E2488Q, I2999A and R4810K. They found that RNF213 uses UBE2D2 to catalyze K6-dependent poly-ubiquitination, and measured the E3-ligase activity by using this system. They found that the E3-ligase activities of mutant RNF213 proteins decreased, compared with normal RNF213. By contrast, the ATPase activity was not different between the normal and mutant RNF213 proteins. They concluded that RNF213 with R4810R mutation is a dominant negative mutant that decreases ubiquitylation globally. <Comments> Authors deal with critical issues how heterozygous RNF213 mutations increased the risk for MMD and the paper is well written. 1) There is a great concern regarding the assay system of E3-ligase activity of RNF213. Physiological E2 for RNF213 is not known and validation of use of UBE2D2 as E2 for the assay system remains unestablished.
We thank the Reviewer for this perceptive comment. While we were attempting to address his/her and the other Reviewers' concerns, it was reported that RNF213 has a novel E3 domain ("RZ"), distinct from the RING (Otten et al., 2021). Given these data, our initial findings using the isolated RING are at best incomplete and at worst potentially misleading. Consequently, we repeated all of the initial studies using purified full length (FL) RNF213. We also performed an extensive analysis of potential E2s.
Specifically, our new in vitro ubiquitylation and siRNA knockdown data suggest that UBE2D2 is an E2 for RNF213 in vitro and in vivo (Revised Figure 1d-g). Consistent with our results, an unbiased screen to find E2s for RNF213 showed that UBE2D2 can promote RNF213 autoubiquitylation, although it was not the only E2 with this activity (Ahel et al., 2020). Another unbiased screen indicated that RNF213 can interact with UBE2D2 as well as UBE2L3 (Pao et al., 2018).
That said, the aforementioned papers as well as our own new data (New Figure 1) indicate that other E2s (e.g., UBE2L3) also can act with RNF213, at least in vitro. Notably, however, we find that these E2s catalyze the addition of distinct UB linkages. We discuss this important issue in the revision.
2) All of the experiments were performed in the overexpressed RNF213 in cultured cells. Since overexpression experiments sometimes reflect unphysiological condition authors should provide the evidence of physiological conditions before they conclude that RNF213 with R4810R mutation works in a dominant-negative manner.
We agree conceptually with the Reviewer's concern regarding potential artifacts of overexpression. However, please note that WT RNF213 and various RNF213 mutants were reexpressed in RNF213 KO cells under doxycycline control, and the dox-induced RNF213 level is comparable to that of endogenous expression of RNF213 in these cells (e.g., Fig. 1C in the revision). Hence, we are not examining the effects of over-expression but rather, nearnormal levels of these proteins.
The manuscript by Bhardwaj et al. discusses ubiquitination activities of the E3 ligase RNF213 that is mutated in Moyamoya disease. With its size of >5200 Aa, this is a huge protein, and it is commendable that the authors attempt to tackle a protein of such size, and indeed the cell biological data with fulllength proteins are of very good quality. Moreover, expression suggests that the protein can be generated in good quantity and quality, which should be exciting for any cryo-EM person on campus.
Mutations in the RING domain cause expected loss of ubiquitination activity. Interestingly, also Moyamoya SNPs disrupt E3 activity (while not affecting ATPase activity). What is striking yet not discussed, is why/how an MMD mutant, R4810K, can affect RING E3 activity of a domain some 800 Aa upstream. A second striking finding is that The RNF213 RING domain seemingly directs the known, nonspecific E2 ubiquitination activity of a widely used E2 enzyme, UBE2D2, towards assembling predominantly Lys6-linkages.
Clearly, there are interesting intrinsic mechanistic features in this exciting E3 ligase, which scream for a more detailed molecular/structural characterisation. Such analysis would be beyond the scope of the manuscript, which I enjoyed reading for its clarity and compelling findings, and which I would support to publish in Life Science Alliance once below minor comments are addressed.
1) There are now many complementary methods to gain insights into ubiquitin chain types, and the authors use a somewhat outdated method, ubiquitin mutants. Especially concerning K6 chains, this has led to confusing data in the past (reason being, that K6 is also important for binding to and positioning Ub at the E2 enzyme). See work on MHC-I ubiquitination by Lehner and colleagues/Boname et al and also Goto et al. 2009/10. Better approaches that would be highly doable for the in-vitro Fig 1 and potentially for the cell-based figures, are mass-spectrometry, and use of the K6-affimer (though this may be off the market at the moment), to provide additional evidence for K6 chain assembly.
We appreciate the Reviewer's criticisms, and we spent extensive time trying to perform the suggested experiments (indeed, much of the long time spent on this revision was aimed at addressing this comment). Also, as noted in our response to Reviewer 1, the experiments in the original submission analyzed the activity of the isolated RING domain and UBED2. In the revision, for the reasons discussed in our response to his/her comments, we repeated all of these experiments using FL RNF213 and UBE2D2 and UBE2L3.
Before submission of the initial manuscript, we had already attempted to use K6 affimers to assess RNF213 ubiquitylation. However, we found that this reagent (at least as provided by the company at the time) was unable to visualize either RNF213 or any other proteins (perhaps this is why this reagent is no longer on the market!). We also tried to identify RNF213 Ub sites by MS. Although we did not see K6-Ub in tryptic digests of immunopurified RNF213, we also did (could) not get full coverage of this large protein. To simplify the analysis, we also tried Lbpro (a ubiquitin clipase), but we found this enzyme be very inefficient.
Given these difficulties, we had no choice but to repeat the initial experiments using UB mutants. The results revealed clear, E2-dependent differences in the type of UB modification: UBE2D2 promoted primarily K6 ubiquitylation, whereas UBE2L3 catalyzed K48 and K11 ubiquitylation (and, to a much lower extent, K6 ubiquitylation). These results are found in Figures 1f and 1g of the revision. Although this approach might be "older", we believe that the results obtained are both reliable and highly interesting.
2) E3 ligases are typically not specific for individual members of the UBE2D family. Fig 1c is cause for some concern. The authors should perform control experiments to show that their E1 preparation can charge all E2 enzymes similarly, and work with charged E2 enzymes. An additional control would be to use a different, well expressed E3 ligase with known non-discriminatory E2 usage for E2Ds -there are many GST-RING-members members that should work here, and which should be commercially available. This issue is no longer relevant as, for the reasons detailed above and in response to Reviewer 1, we have removed all experiments using the GST-RING domain and only study purified FL RNF213.
3) Why does the intensity of the Ub signal change with the use of the K-only mutants? (1e). Again, this data suggests that the other mutants are very differently charged by the E1 enzyme. I don't understand the strong 135kDa signal labelled as E1~Ub. I agree that it is suggestive of a difference in activity, but it is not clear why it should light up.
We appreciate this point as well, but it too is no longer relevant, as we have repeated these experiments with FL RNF213. 4) While challenging on a 5200 aa protein, some attempt at sec structure prediction should be performed. Is the region beyond the RING domain ordered/domain character and are any repeat sequences recogniseable? What is known about other domains beyond those listed in Fig 1? What is the closest domain/protein similarity and species conservation for the region encompassing R4810K? this information would be valuable to add.
After the initial manuscript was submitted, the RNF213 structure was deciphered using cryoEM (Ahel et al., 2020) 5) Methods lacks a section on molecular biology and sequencing, which given the size of the expressed protein, should be added. A Table with sequencing primers to dissect the 15kb insert should be included. We apologize for these errors, which have been corrected in the revised manuscript.

Reviewer #3 (Comments to the Authors (Required)):
This manuscript describes an E3 ubiquitin ligase in which mutations have been strongly linked to Moyamoya disease (MMD). Here, the authors demonstrate that RNF213 functions in concert with the E2 UBE2D2 in the conjugation of -K6-linked ubiquitin chains and demonstrate that high penetrance variants of RNF213 result in a reduction of global protein ubiquitination, yielding insight into MMD. Findings from this work attempt to settle controversy on RNF213 ATPase activity, multimerization and ubiquitin function as causative activities for MMD. Overall, these findings are interesting and I have few comments related to necessary controls and to help settle controversial functions of RNF213.
Major Comments: -The high molecular weight products in Figure 1c, 1d, and 1e must show that these modifications are indeed assembled on GST-RING RNF213 and not due to autoubiquination of other enzymes present in the in vitro reaction such as E1 and E2s themselves. The authors must further show that the catalytic activity of GST-RING RNF213 is required for ubiquitination via UBE2D2. It is unclear how the ubiquitin chain mutants were generated in this assay as it is not clearly stated in the supplemental methods section. Further, the quantities/concentrations of all enzymes used in these reactions must be reported in the methods section.

RNF213 has a novel E3 domain (RZ) (Otten et al., 2021), calling into question our initial experiments with GST-RNF213-RING. For this reason, we repeated all of the relevant experiments with full length RNF213, rendering the first part of this comment no longer relevant.
We apologize for not being clear in our Methods section. The requested information is present in the revision.
-The authors' claim based on the data in figures 1d and 1e that other chain linkages can be formed on RNF213 based on the finding that adding back K6-linked chains only partially recovers poly-Ub of RNF213 to WT. It would be important for the authors to further explore this possibility through measurements of chain formation or adding back lysines (especially -K63) within ubiquitin in an attempt to recover levels to WT.
We thank the Reviewer for this perceptive comment. As noted above, we have repeated the experiments with FL RNF213 and relevant E2s. Under these conditions, the relevant addback mutants alone or in combination have similar activity to the WT (Fig. 1g,h of the revision).
-Given the prior publication (Takeda et al., 2020) demonstrating Ubc13/Uev1a to have strong activity for RNF213 RING fragment and no activity with UbcH5b (UBE2D2), it will be critical for the authors to compare their findings in a side-by-side experiment using these two E2s and explain why the authors in this paper were not able to observe autoubiquitination activity in their assays.
For the reasons discussed above, we no longer present any data on the GST-RING (because it might be confusing), but restrict our analysis to FL RNF213. We trust that the Reviewer will agree that our revised approach is more relevant than either the cited prior publication or our own initial data.
-Given that full-length RNF213 can be expressed in cells, it is critical to establish that the full-length version of RNF213 can indeed assemble -K6 ubiquitin linkages.
We thank the Reviewer for this comment, which motivated the aforementioned experiments on FL RNF213. As described above, our data suggest that UBE2D2-RNF213, and to a much lower extent, UBE2L3-RNF213, promote K6 ubiquitin linkage (Fig. 1f-g).
-I disagree with the authors' conclusion that RNF213 ubiquitination is substantially reduced in UBE2D2 knockdown cells. The molecular weight of the HA-Ub reactive band is lower than the FLAG-RNF213 bands shown in the image (Figure 1h). How do the authors reconcile this discrepancy? And, if the assembly of RNF213 via UBE2D2 takes place via -K6 polyubiquitin linkages, why does this not appear as a higher molecular weight smear on the membrane, similar to Figure 3c? We apologize for the confusion, which was the result of inaccurate labeling of the original figure. We have repeated this result and provide the data in Figure 1d. In addition, as we used ECF (LICOR) with different colors for RNF213 and HA-UB, we provide the merged color blot, which shows the bands are the same. Also, please note that the "smeary" UB bands are from in vitro ubiquitylation experiments.
-In addition to the coomassie gel stain blot, please provide a FLAG immunoblot of the immune purified proteins along with a vector only control with FLAG antibody lane. Fig. 2b of the revision.

We have included the requested immunoblot in
-The ubiquitination experiment shown in Figure 3c should also be done in cells as opposed to a post-hoc ubiquitin assay.
We respectfully suggest that our new data with FL RNF213 clearly show that it has E2dependent K6 Ub (as well as K11 and K48 Ub) activity.
-It is unclear how the experiments were conducted in Figure 3e. The amount of total protein ubiquitination is not a reflection of the E3 ligase activity of RNF213 as one needs to show the ubiquitin is directly conjugated to RNF213. Thus, at minimum, the authors need to purify these proteins similar to what was done in Figure 3c or interpret their results based on global changes in ubiquitin patterning.
We agree with the Reviewer's point regarding our interpretation of the results. Accordingly, we have changed the Results section to focus on global changes in ubiquitin patterning.
-Are the increases in Ub conjugates observed with RNF213 overexpression -K6 linked?
As stated in the manuscript (p. 12 in the revision), we reported previously (Banh et al, NCB 2016) that in cells with increased RNF213 activity, total K6-Ub was increased. Minor comments: -In the introduction, state that protein ubiquitination selectively modifies lysine residues on a substrate.
We have modified the Introduction to state that "Mono-ubiquitylation joins a single UB molecule to a lysine residue of the substrate, although multiple sites on the same substrate can be mono-ubiquitylated ("multi-mono-ubiquitylation"). Poly-ubiquitylation occurs when UB residues are added sequentially to a specific lysine residue in a previously conjugated UB, forming a UB chain." -The authors discuss the common RNF213 R4810 mutation in the results section but never introduce this mutation in the prior text.

Respectfully, this allele was mentioned on page 3 of the original version,
where it also appears in the revision.
-There are other studies that assessed E3 ligase activity with one of the most common RNF213 variants.
It is important to cite these key papers in the Introduction.
We are not clear on which papers the Reviewer is referring to, but would be happy to add these references if he/she provides.
-Please provide original citations for the RNF213 reported patient mutations used in this study.
Again respectfully, we did state in the initial (and do in the revised) Results that "Different -There are a few grammatical errors in the text that need to be corrected.
We have tried to ensure that all grammar is correct. Thank you for submitting your revised manuscript entitled "MMD-associated RNF213 SNPs Encode Dominant Negative Alleles That Globally Impair Ubiquitylation". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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