DTX3L ubiquitin ligase ubiquitinates single-stranded nucleic acids

  1. Cancer Research UK Scotland Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, United Kingdom
  2. School of Cancer Sciences, University of Glasgow, Glasgow, G61 1QH, United Kingdom
  3. Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom
  4. School of Molecular Biosciences, University of Glasgow, Glasgow, G12 8QQ, United Kingdom

Peer review process

Not revised: This Reviewed Preprint includes the authors’ original preprint (without revision), an eLife assessment, public reviews, and a provisional response from the authors.

Read more about eLife’s peer review process.

Editors

  • Reviewing Editor
    Amy Andreotti
    Iowa State University, Ames, United States of America
  • Senior Editor
    Amy Andreotti
    Iowa State University, Ames, United States of America

Reviewer #1 (Public Review):

In the article by Dearlove et al., the authors present evidence in strong support of nucleotide ubiquitylation by DTX3L, suggesting it is a promiscuous E3 ligase with capacity to ubiquitylate ADP ribose and nucleotides. The authors include data to identify the likely site of attachment and the requirements for nucleotide modification.

While this discovery potentially reveals a whole new mechanism by which nucleotide function can be regulated in cells, there are some weaknesses that should be considered. Is there any evidence of nucleotide ubiquitylation occurring cells? It seems possible, but evidence in support of this would strengthen the manuscript. The NMR data could also be strengthened as the binding interface is not reported or mapped onto the structure/model, this seems of considerable interest given that highly related proteins do have the same activity.

The paper is for the most part well well-written and is potentially highly significant, but it could be strengthened as follows:

(1) The authors start out by showing DTX3L binding to nucleotides and ubiquitylation of ssRNA/DNA. While ubiquitylation is subsequently dissected and ascribed to the RD domains, the binding data is not followed up. Does the RD protein alone bind to the nucleotides? Further analysis of nucleotide binding is also relevant to the Discussion where the role of the KH domains is considered, but the binding properties of these alone have not been analysed.
(2) With regard to the E3 ligase activity, can the authors account for the apparent decreased ubiquitylation activity of the 232-C protein in Figure 1/S1 compared to FL and RD?
(3) Was it possible to positively identify the link between Ub and ssDNA/RNA using mass spectrometry? This would overcome issues associated with labels blocking binding rather than modification.
(4) Furthermore, can a targeted MS approach be used to show that nucleotides are ubiquitylated in cells?
(5) Do the authors have the assignments (even partial?) for DTX3L RD? In Figure 4 it would be helpful to identify the peaks that correspond to the residues at the proposed binding site. Also do the shifts map to a defined surface or do they suggest an extended site, particularly for the ssDNA.
(6) Does sequence analysis help explain the specificity of activity for the family of proteins?
(7) While including a summary mechanism (Figure 5I) is helpful, the schematic included does not necessarily make it easier for the reader to appreciate the key findings of the manuscript or to account for the specificity of activity observed. While this figure could be modified, it might also be helpful to highlight the range of substrates that DTX3L can modify - nucleotide, ADPr, ADPr on nucleotides etc.

Reviewer #2 (Public Review):

Summary:

The manuscript by Dearlove et al. entitled "DTX3L ubiquitin ligase ubiquitinates single-stranded nucleic acids" reports a novel activity of a DELTEX E3 ligase family member, DTX3L, which can conjugate ubiquitin to the 3' hydroxyl of single-stranded oligonucleotides via an ester linkage. The findings that unmodified oligonucleotides can act as substrates for direct ubiquitylation and the identification of DTX3 as the enzyme capable of performing such oligonucleotide modification are novel, intriguing, and impactful because they represent a significant expansion of our view of the ubiquitin biology. The authors perform a detailed and diligent biochemical characterization of this novel activity, and key claims made in the article are well supported by experimental data. However, the studies leave room for some healthy skepticism about the physiological significance of the unique activity of DTX3 and DTX3L described by the authors because DTX3/DTX3L can also robustly attach ubiquitin to the ADP ribose moiety of NAD or ADP-ribosylated substrates. The study could be strengthened by a more direct and quantitative comparison between ubiquitylation of unmodified oligonucleotides by DTX3/DTX3L with the ubiquitylation of ADP-ribose, the activity that DTX3 and DTX3L share with the other members of the DELTEX family.

Strengths:

The manuscript reports a novel and exciting observation that ubiquitin can be directly attached to the 3' hydroxyl of unmodified, single-stranded oligonucleotides by DTX3L. The study builds on the extensive expertise and the impactful previous studies by the Huang laboratory of the DELTEX family of E3 ubiquitin ligases. The authors perform a detailed and diligent biochemical characterization of this novel activity, and all claims made in the article are well supported by experimental data. The manuscript is clearly written and easy to read, which further elevates the overall quality of submitted work. The findings are impactful and will help illuminate multiple avenues for future follow-up investigations that may help establish how this novel biochemical activity observed in vitro may contribute to the biological function of DTX3L. The authors demonstrate that the activity is unique to the DTX3/DTX3L members of the DELTEX family and show that the enzyme requires at least two single-stranded nucleotides at the 3' end of the oligonucleotide substrate and that the adenine nucleotide is preferred in the 3' position. Most notably, the authors describe a chimeric construct containing RING domain of DTX3L fused to the DTC domain DTX2, which displays robust NAD ubiquitylation, but lacks the ability to ubiquitylate unmodified oligonucleotides. This construct will be invaluable in the future cell-based studies of DTX3L biology that may help establish the physiological relevance of 3' ubiquitylation of nucleic acids.

Weaknesses:

The main weakness of the study is in the lack of direct evidence that the ubiquitylation of unmodified oligonucleotides reported by the authors plays any role in the biological function of DTX3L. The study leaves plenty of room for natural skepticism regarding the physiological relevance of the reported activity, because, akin to other DELTEX family members, DTX3 and DTX3L can also catalyze attachment of ubiquitin to NAD, ADP ribose and ADP-ribosylated substrates. Unfortunately, the study does not offer any quantitative comparison of the two distinct activities of the enzyme, which leaves plenty of room for doubt. One is left wondering, whether ubiquitylation of unmodified oligonucleotides is just a minor and artifactual side activity owing to the high concentration of the oligonucleotide substrates and E2~Ub conjugates present in the in-vitro conditions and the somewhat lower specificity of the DTX3 and DTX3L DTC domains (compared to DTX2 and other DELTEX family members) for ADP ribose over other adenine-containing substrates such as unmodified oligonucleotides, ADP/ATP/dADP/dATP, etc. The intriguing coincidence that DTX3L, which is the only DTX protein capable of ubiquitylating unmodified oligonucleotides, is also the only family member that contains nucleic acid interacting domains in the N-terminus, is suggestive but not compelling. A recently published DTX3L study by a competing laboratory (PMID: 38000390), which is not cited in the manuscript, suggests that ADP-ribose-modified nucleic acids could be the physiologically relevant substrates of DTX3L. That competing hypothesis appears more convincing than ubiquitylation of unmodified oligonucleotides because experiments in that study demonstrate that ubiquitylation of ADP-ribosylated oligos is quite robust in comparison to ubiquitylation of unmodified oligos, which is undetectable. It is possible that the unmodified oligonucleotides in the competing study did not have adenine in the 3' position, which may explain the apparent discrepancy between the two studies. In summary, a quantitative comparison of ubiquitylation of ADP ribose vs. unmodified oligonucleotides could strengthen the study.

Author response:

Public Reviews:

Reviewer #1 (Public Review):

In the article by Dearlove et al., the authors present evidence in strong support of nucleotide ubiquitylation by DTX3L, suggesting it is a promiscuous E3 ligase with capacity to ubiquitylate ADP ribose and nucleotides. The authors include data to identify the likely site of attachment and the requirements for nucleotide modification.

While this discovery potentially reveals a whole new mechanism by which nucleotide function can be regulated in cells, there are some weaknesses that should be considered. Is there any evidence of nucleotide ubiquitylation occurring cells? It seems possible, but evidence in support of this would strengthen the manuscript. The NMR data could also be strengthened as the binding interface is not reported or mapped onto the structure/model, this seems of considerable interest given that highly related proteins do have the same activity.

The paper is for the most part well well-written and is potentially highly significant, but it could be strengthened as follows:

(1) The authors start out by showing DTX3L binding to nucleotides and ubiquitylation of ssRNA/DNA. While ubiquitylation is subsequently dissected and ascribed to the RD domains, the binding data is not followed up. Does the RD protein alone bind to the nucleotides? Further analysis of nucleotide binding is also relevant to the Discussion where the role of the KH domains is considered, but the binding properties of these alone have not been analysed.

We thank the reviewer for the suggestion. We have tested DTX3L RD for ssDNA binding using NMR (see Figure 4A and Figure S2), which showed that DTX3L RD binds ssDNA. We also tested the DTX3L KH domains for RNA/ssDNA binding using an FP experiment. However, the FP experiment did not show significant changes upon titrating RNA/ssDNA. It seems that the KH domains alone are not sufficient to bind RNA/ssDNA and both KH and RD domains are required for binding. Understanding how DTX3L binds RNA/ssDNA is an ongoing research in the lab. We will revise the Discussion on the KH domains.

(2) With regard to the E3 ligase activity, can the authors account for the apparent decreased ubiquitylation activity of the 232-C protein in Figure 1/S1 compared to FL and RD?

We will address this question in the revision.

(3) Was it possible to positively identify the link between Ub and ssDNA/RNA using mass spectrometry? This would overcome issues associated with labels blocking binding rather than modification.

We have tried to use mass spectrometry to detect the linkage between Ub and ssDNA/RNA, but was unable to do so. We suspect that the oxyester linkage might be labile, posing a challenge for mass spectrometry techniques. Similarly, a recent preprint from Ahel lab, which utilises LC-MS, detects the Ub-NMP product rather than the linkage (https://www.biorxiv.org/content/10.1101/2024.04.19.590267v1.full.pdf).

(4) Furthermore, can a targeted MS approach be used to show that nucleotides are ubiquitylated in cells?

This will require future development and improvement of the MS approach, specifically the isolation of labile oxyester-linked products from cells and the optimisation of the MS detection method.

(5) Do the authors have the assignments (even partial?) for DTX3L RD? In Figure 4 it would be helpful to identify the peaks that correspond to the residues at the proposed binding site. Also do the shifts map to a defined surface or do they suggest an extended site, particularly for the ssDNA.

We only collected HSQC spectra which was insufficient for assignments. We have performed a competition experiment using ADPr and labelled ssDNA, showing that ADPr competes against the ubiquitination of ssDNA (Figure 4D). We will provide an additional experiment showing that ssDNA with a blocked 3’-OH can compete against ubiquitination of ADPr. These data, together with our NMR analysis, will further strengthen the evidence that ssDNA and ADPr compete the same binding pocket in DTX3L RD. Understanding how DTX3L RD binds ssDNA/RNA is an ongoing research in the lab.

(6) Does sequence analysis help explain the specificity of activity for the family of proteins?

We will performed sequence alignment of DTX proteins RD domains and discuss this point in the revision.

(7) While including a summary mechanism (Figure 5I) is helpful, the schematic included does not necessarily make it easier for the reader to appreciate the key findings of the manuscript or to account for the specificity of activity observed. While this figure could be modified, it might also be helpful to highlight the range of substrates that DTX3L can modify - nucleotide, ADPr, ADPr on nucleotides etc.

We will modify this Figure as suggested.

Reviewer #2 (Public Review):

Summary:

The manuscript by Dearlove et al. entitled "DTX3L ubiquitin ligase ubiquitinates single-stranded nucleic acids" reports a novel activity of a DELTEX E3 ligase family member, DTX3L, which can conjugate ubiquitin to the 3' hydroxyl of single-stranded oligonucleotides via an ester linkage. The findings that unmodified oligonucleotides can act as substrates for direct ubiquitylation and the identification of DTX3 as the enzyme capable of performing such oligonucleotide modification are novel, intriguing, and impactful because they represent a significant expansion of our view of the ubiquitin biology. The authors perform a detailed and diligent biochemical characterization of this novel activity, and key claims made in the article are well supported by experimental data. However, the studies leave room for some healthy skepticism about the physiological significance of the unique activity of DTX3 and DTX3L described by the authors because DTX3/DTX3L can also robustly attach ubiquitin to the ADP ribose moiety of NAD or ADP-ribosylated substrates. The study could be strengthened by a more direct and quantitative comparison between ubiquitylation of unmodified oligonucleotides by DTX3/DTX3L with the ubiquitylation of ADP-ribose, the activity that DTX3 and DTX3L share with the other members of the DELTEX family.

Strengths:

The manuscript reports a novel and exciting observation that ubiquitin can be directly attached to the 3' hydroxyl of unmodified, single-stranded oligonucleotides by DTX3L. The study builds on the extensive expertise and the impactful previous studies by the Huang laboratory of the DELTEX family of E3 ubiquitin ligases. The authors perform a detailed and diligent biochemical characterization of this novel activity, and all claims made in the article are well supported by experimental data. The manuscript is clearly written and easy to read, which further elevates the overall quality of submitted work. The findings are impactful and will help illuminate multiple avenues for future follow-up investigations that may help establish how this novel biochemical activity observed in vitro may contribute to the biological function of DTX3L. The authors demonstrate that the activity is unique to the DTX3/DTX3L members of the DELTEX family and show that the enzyme requires at least two single-stranded nucleotides at the 3' end of the oligonucleotide substrate and that the adenine nucleotide is preferred in the 3' position. Most notably, the authors describe a chimeric construct containing RING domain of DTX3L fused to the DTC domain DTX2, which displays robust NAD ubiquitylation, but lacks the ability to ubiquitylate unmodified oligonucleotides. This construct will be invaluable in the future cell-based studies of DTX3L biology that may help establish the physiological relevance of 3' ubiquitylation of nucleic acids.

Weaknesses:

The main weakness of the study is in the lack of direct evidence that the ubiquitylation of unmodified oligonucleotides reported by the authors plays any role in the biological function of DTX3L. The study leaves plenty of room for natural skepticism regarding the physiological relevance of the reported activity, because, akin to other DELTEX family members, DTX3 and DTX3L can also catalyze attachment of ubiquitin to NAD, ADP ribose and ADP-ribosylated substrates. Unfortunately, the study does not offer any quantitative comparison of the two distinct activities of the enzyme, which leaves plenty of room for doubt. One is left wondering, whether ubiquitylation of unmodified oligonucleotides is just a minor and artifactual side activity owing to the high concentration of the oligonucleotide substrates and E2~Ub conjugates present in the in-vitro conditions and the somewhat lower specificity of the DTX3 and DTX3L DTC domains (compared to DTX2 and other DELTEX family members) for ADP ribose over other adenine-containing substrates such as unmodified oligonucleotides, ADP/ATP/dADP/dATP, etc. The intriguing coincidence that DTX3L, which is the only DTX protein capable of ubiquitylating unmodified oligonucleotides, is also the only family member that contains nucleic acid interacting domains in the N-terminus, is suggestive but not compelling. A recently published DTX3L study by a competing laboratory (PMID: 38000390), which is not cited in the manuscript, suggests that ADP-ribose-modified nucleic acids could be the physiologically relevant substrates of DTX3L. That competing hypothesis appears more convincing than ubiquitylation of unmodified oligonucleotides because experiments in that study demonstrate that ubiquitylation of ADP-ribosylated oligos is quite robust in comparison to ubiquitylation of unmodified oligos, which is undetectable. It is possible that the unmodified oligonucleotides in the competing study did not have adenine in the 3' position, which may explain the apparent discrepancy between the two studies. In summary, a quantitative comparison of ubiquitylation of ADP ribose vs. unmodified oligonucleotides could strengthen the study.

We thank the reviewer for the constructive feedback. We agree that evidence for the biological function is lacking. While we have tried to detect Ub-ssDNA/RNA from cells, we found that Isolating and detecting labile oxyester-linked Ub-ssDNA/RNA products remain challenging due to (1) low levels of Ub-ssDNA/RNA products, (2) the presence of DUBs and nucleases that rapidly remove the products during the experiments, and (3) our lack of a suitable MS approach to detect the product. For these reasons, we feel that discovering the biological function will require future effort and expertise and is beyond the scope of our current manuscript.

In the manuscript (PMID: 38000390), the authors used PARP10 to catalyse ADP-ribosylation onto 5’-phosphorylated ssDNA/RNA. They used the following sequences which lacks 3’-adenosine, which could explain the lack of ubiquitination.

E15_5′P_RNA [Phos]GUGGCGCGGAGACUU

E15_5′P_DNA [Phos]GTGGCGCGGAGACTT

We will perform the experiment using this sequence to verify this. We have cited this manuscript but for some reasons, Pubmed has updated its published date from mid 2023 to Jan 2024. We will update the Endnote in the revised manuscript.

We agree that it is crucial to compare ubiquitination of oligonucleotides and ADPr by DTX3L to find its preferred substrate. We have challenged oligonucleotide ubiquitination by adding excess ADPr and found that ADPr efficiently competes with oligonucleotide (Figure 4D). We will perform more thorough competition experiments by titrating with increasing molar excess of either ADPr or ssDNA to examine the effect on the ubiquitination of ssDNA and ADPr, respectively.

  1. Howard Hughes Medical Institute
  2. Wellcome Trust
  3. Max-Planck-Gesellschaft
  4. Knut and Alice Wallenberg Foundation