GO:0035520 monoubiquitinated protein deubiquitination: Mechanism, Genes, Functions and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0035520 (monoubiquitinated protein deubiquitination) is the biological process that removes a single ubiquitin group from a monoubiquitinated protein.
• Deubiquitinases (DUBs) achieve substrate specificity through structural elements outside the catalytic domain, such as the N-terminus of USP1 that recognizes monoubiquitinated FANCD2.
• Monoubiquitination is a non-degradative signal, so its reversal by DUBs controls DNA repair, translation, chromatin state and cell survival rather than proteasomal turnover.
• Key DUBs in this process include USP1 (FANCD2), USP21 (histone H2AK119), OTUD6 (RPS7/eS7), BAP1 and ATAD5-associated factors.
• Deregulated monoubiquitinated protein deubiquitination contributes to cancer, leukaemogenesis and genome instability, making DUBs attractive drug targets.
• CRISPR knockout, point-mutation, knock-in and overexpression models are essential to dissect which DUB controls a specific monoubiquitinated substrate.
Description
Monoubiquitinated protein deubiquitination (GO:0035520) is the enzymatic removal of a single ubiquitin moiety from a protein that carries exactly one ubiquitin group. Unlike polyubiquitin chains that typically route proteins to the proteasome, monoubiquitination acts as a reversible signaling mark, and its erasure by deubiquitinases (DUBs) is a decisive regulatory event. The process is catalyzed by DUBs that hydrolyze the isopeptide bond between the ubiquitin C-terminus and a lysine residue on the substrate, and specificity is often conferred by domains outside the catalytic core. Researchers study GO:0035520 because it sits at the crossroads of DNA damage repair, chromatin regulation, translation control and cell-fate decisions. For example, USP1 specifically removes ubiquitin from monoubiquitinated FANCD2, a step required for the Fanconi anemia pathway to function correctly. Similarly, USP21 autoinhibition and its deubiquitination of histone H2AK119 illustrate how monoubiquitin removal shapes chromatin states. Because monoubiquitination is non-degradative, deubiquitination of monoubiquitinated proteins often acts as a molecular switch rather than a degradation signal. This makes GO:0035520 a high-value target for functional genomics, and CRISPR-based models are widely used to test causality between a DUB and a specific monoubiquitinated substrate.
monoubiquitinated protein deubiquitination At A Glance
| GO ID | GO:0035520 |
|---|---|
| GO term | monoubiquitinated protein deubiquitination |
| Ontology | biological_process |
| Synonym | monoubiquitinated protein deubiquitinylation; monoubiquitinated protein deubiquitylation |
| Major function | Removal of a single ubiquitin group from a monoubiquitinated protein by deubiquitinases |
| Substrate type | Proteins carrying exactly one ubiquitin moiety, including FANCD2, PCNA, histone H2AK119 and RPS7/eS7 |
| Key enzyme family | Deubiquitinases (DUBs) such as USP1, USP21, OTUD6 and BAP1 |
| Biological context | DNA repair, chromatin regulation, translation and stress responses |
What Is GO:0035520?
GO:0035520 is defined by QuickGO as the removal of the ubiquitin group from a monoubiquitinated protein. In other words, it is the reverse reaction of monoubiquitination: a deubiquitinase cleaves the bond that attaches one ubiquitin molecule to a substrate lysine, restoring the unmodified protein. The term covers DUB-catalyzed editing of monoubiquitin marks on histones, DNA repair factors, ribosomal proteins and other cellular targets.
Why Is monoubiquitinated protein deubiquitination Important in Cell Biology?
Monoubiquitinated protein deubiquitination is important because it reverses a non-degradative post-translational mark that controls protein localization, activity and interactions rather than protein stability. By erasing monoubiquitin, DUBs such as USP1, USP21 and OTUD6 directly influence DNA repair, chromatin state and translation, and their dysregulation is linked to cancer and genome instability. Consequently, GO:0035520 is both a fundamental cell-biology process and a source of therapeutic targets, as illustrated by ongoing efforts to inhibit USP1 in the clinic.
• Controls DNA repair: USP1 removes monoubiquitin from FANCD2, a step required for the Fanconi anemia pathway.
• Regulates chromatin: USP21 deubiquitinates histone H2AK119, affecting chromatin state and gene expression.
• Modulates translation: OTUD6 deubiquitinates RPS7/eS7 on the free 40S ribosome to regulate global protein translation and stress responses.
• Impacts genome stability: ATAD5-BAZ1B interaction modulates PCNA ubiquitination during DNA repair.
• Links to cancer: BAP1, a tumor suppressor, regulates SLC7A11 and NADPH levels, connecting deubiquitination to disulfidptosis.
• Therapeutic target: USP1 inhibition has progressed from target discovery toward clinical translation.
• Relevant to leukaemogenesis: TET2 and chromatin state regulation intersect with ubiquitin-dependent processes in leukemia.
• Requires precise models: CRISPR knockout and knock-in are needed to assign a DUB to a specific monoubiquitinated substrate.
What Happens During monoubiquitinated protein deubiquitination?
Substrate recognition by the deubiquitinase
In simple terms: The enzyme first finds and grabs the protein that carries a single ubiquitin tag.
Deubiquitinases recognize monoubiquitinated substrates through a combination of the catalytic domain and auxiliary elements. For USP1, specificity for monoubiquitinated FANCD2 is driven by the N-terminus of the enzyme, demonstrating that regions outside the catalytic core can dictate substrate choice. More broadly, DUB specificity and regulation are achieved through structural modules that read the ubiquitin mark and the substrate surface simultaneously.
Cleavage of the isopeptide bond
In simple terms: The enzyme cuts the chemical link that holds ubiquitin onto the target protein.
Once bound, the DUB catalyzes hydrolysis of the isopeptide bond between the C-terminal glycine of ubiquitin and a lysine on the substrate, releasing free ubiquitin and the unmodified protein. This reaction is the defining biochemical event of GO:0035520 and is shared across cysteine-protease and metalloprotease DUB families.
Release and functional consequence
In simple terms: After the cut, the protein loses its ubiquitin signal and changes behavior.
Removal of the monoubiquitin mark alters the substrate's interactions and localization. For example, deubiquitination of FANCD2 by USP1 is required for proper Fanconi anemia pathway function, while USP21-mediated removal of ubiquitin from histone H2AK119 changes chromatin state. In translation, OTUD6 deubiquitination of RPS7/eS7 on the free 40S ribosome regulates global protein translation and stress responses.
Coupling to DNA repair and genome maintenance
In simple terms: The process helps cells fix DNA and keep their genome stable.
Monoubiquitinated protein deubiquitination is tightly coupled to DNA repair. ATAD5-BAZ1B interaction modulates PCNA ubiquitination during DNA repair, showing that the balance between ubiquitination and deubiquitination of PCNA is actively controlled. Similarly, USP1-dependent deubiquitination of FANCD2 is a checkpoint in the Fanconi anemia DNA repair pathway.
Regulation by autoinhibition and complex formation
In simple terms: The enzyme can switch itself off or need a partner to work.
DUB activity is often restrained by autoinhibitory domains or activated by binding partners. USP21 autoinhibition and its role in histone H2AK119 deubiquitination illustrate how conformational control shapes substrate targeting. General principles of DUB specificity and regulation, including complex formation and post-translational modification, are reviewed comprehensively.
Key Genes Involved in GO:0035520 monoubiquitinated protein deubiquitination
The following genes and proteins are experimentally implicated in monoubiquitinated protein deubiquitination (GO:0035520) or in the ubiquitination-deubiquitination balance of monoubiquitinated substrates.
| Gene | Major Role | Research Relevance |
|---|---|---|
| USP1 | Deubiquitinates monoubiquitinated FANCD2; N-terminus drives specificity | Fanconi anemia pathway, DNA repair, USP1 inhibitor development |
| USP21 | Deubiquitinates histone H2AK119; regulated by autoinhibition | Chromatin state, histone modification, cancer biology |
| OTUD6 | Deubiquitinates RPS7/eS7 on the free 40S ribosome | Global translation, stress response, ribosome biology |
| BAP1 | Tumor suppressor linked to SLC7A11 and NADPH regulation | Disulfidptosis, cancer metabolism, deubiquitinase biology |
| FANCD2 | Monoubiquitinated substrate of USP1 | Fanconi anemia, DNA interstrand crosslink repair |
| PCNA | Monoubiquitinated during DNA repair; modulated by ATAD5-BAZ1B | Translesion synthesis, genome stability |
| ATAD5 | Interacts with BAZ1B to modulate PCNA ubiquitination | DNA repair, replication-coupled ubiquitination |
| BAZ1B | Partner of ATAD5 in PCNA ubiquitination control | Chromatin remodeling, DNA repair |
| RPS7/eS7 | Ribosomal protein deubiquitinated by OTUD6 | Translation regulation, stress response |
| H2AK119 | Histone mark deubiquitinated by USP21 | Chromatin regulation, gene silencing |
| TET2 | Chromatin state regulator linked to leukaemogenesis | Epigenetics, leukemia, ubiquitin-chromatin crosstalk |
| SLC7A11 | Regulated downstream of BAP1; affects NADPH and disulfidptosis | Cancer metabolism, ferroptosis/disulfidptosis |
| USP1-UAF1 complex | Regulatory complex for USP1 activity | Target validation for USP1 inhibitors |
| DUB catalytic domain | Catalyzes isopeptide bond hydrolysis | Mechanistic enzymology, inhibitor design |
| Ubiquitin | The monoubiquitin group removed in GO:0035520 | Substrate tagging, signaling |
| 40S ribosome | Platform for OTUD6-mediated RPS7/eS7 deubiquitination | Translation initiation and stress |
How Is monoubiquitinated protein deubiquitination Regulated?
Monoubiquitinated protein deubiquitination is regulated at multiple levels. DUBs can be controlled by autoinhibitory domains, as shown for USP21, whose autoinhibition governs histone H2AK119 deubiquitination. Substrate specificity can be encoded by regions outside the catalytic domain, exemplified by the USP1 N-terminus that drives recognition of monoubiquitinated FANCD2. In addition, DUBs often function within multi-protein complexes, and their activity is tuned by interacting partners and post-translational modifications. The balance between ubiquitination and deubiquitination of PCNA during DNA repair is modulated by the ATAD5-BAZ1B interaction, illustrating how protein-protein interactions set the threshold for monoubiquitin removal. Finally, chromatin-associated processes such as TET2-dependent regulation of chromatin state can intersect with ubiquitin signaling in leukaemogenesis.
monoubiquitinated protein deubiquitination and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| USP1 | Fanconi anemia pathway, cancer, genome instability | USP1 knockout and point-mutation cell lines; FANCD2 monoubiquitination assays |
| BAP1 | Cancer metabolism, disulfidptosis suppression | BAP1 knockout with SLC7A11/NADPH readouts |
| USP21 | Chromatin dysregulation, cancer | USP21 autoinhibition mutants; H2AK119 deubiquitination assays |
| OTUD6 | Translation stress, ribosome dysfunction | OTUD6 knockout with polysome profiling and stress assays |
| ATAD5/BAZ1B | DNA repair defects, genome instability | ATAD5 or BAZ1B knockout; PCNA ubiquitination immunoblots |
Cancer and genome instability
Deregulated monoubiquitinated protein deubiquitination contributes to cancer through effects on DNA repair and genome stability. USP1 removes monoubiquitin from FANCD2, and USP1 inhibition has advanced from target discovery toward clinical translation, highlighting its therapeutic potential. ATAD5-BAZ1B control of PCNA ubiquitination further links this process to DNA repair fidelity and genome maintenance. BAP1, a tumor suppressor deubiquitinase, regulates SLC7A11 and NADPH levels and suppresses disulfidptosis, connecting GO:0035520 to cancer metabolism and cell death pathways.
Leukaemogenesis and chromatin regulation
Chromatin-associated deubiquitination is relevant to leukemia. USP21 autoinhibition and histone H2AK119 deubiquitination influence chromatin state, and TET2-mediated chromatin regulation is implicated in leukaemogenesis. These findings suggest that monoubiquitin removal from histones and chromatin regulators participates in leukemic transformation.
Translation stress and ribosome-associated disease
OTUD6 deubiquitination of RPS7/eS7 on the free 40S ribosome regulates global protein translation and stress responses. Because ribosomal protein imbalances are linked to ribosomopathies and stress-related phenotypes, this branch of GO:0035520 may be relevant to diseases of translation dysfunction.
From monoubiquitinated protein deubiquitination-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Is a DUB required for removal of monoubiquitin from a specific substrate? | CRISPR knockout of the DUB gene followed by substrate ubiquitination assays |
| Does a point mutation in the catalytic domain abolish deubiquitinase activity? | CRISPR point-mutation knock-in of catalytic cysteine or histidine residues |
| Does a disease-associated variant alter substrate specificity? | Knock-in of the variant allele and comparison with wild type |
| Where does the DUB act on the substrate in cells? | Tagged knock-in of the DUB or substrate with epitope/fluorescent tags |
| Does overexpression of the DUB phenocopy loss of the monoubiquitin mark? | Doxycycline-inducible overexpression cell lines |
| Which pathways depend on the DUB? | Knockout plus transcriptomics, proteomics or CRISPR library screening |
How to Study the monoubiquitinated protein deubiquitination Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Immunoblotting with ubiquitin antibodies | Levels of monoubiquitinated substrate | Monitoring FANCD2 or PCNA monoubiquitination after DUB perturbation |
| In vitro deubiquitination assay | Catalytic removal of ubiquitin from a substrate | Testing USP1 specificity for monoubiquitinated FANCD2 |
| Mass spectrometry ubiquitin remnant profiling | Site-specific ubiquitination changes | Global substrate discovery for DUBs |
| RNA-seq | Transcriptional consequences of deubiquitination | Chromatin and leukemia-related gene expression |
| Chromatin immunoprecipitation | Histone modification and factor occupancy | H2AK119 deubiquitination by USP21 |
| Polysome profiling | Translation efficiency and ribosome association | OTUD6 effects on global translation |
| CRISPR library screening | Fitness and pathway dependencies | Identifying DUB dependencies in cancer cells |
| Proximity labeling or imaging | Subcellular localization of DUB-substrate interactions | Tagged knock-in studies of DUB dynamics |
Ubiquitination and deubiquitination assays
Direct measurement of monoubiquitinated protein deubiquitination typically uses immunoblotting with ubiquitin or substrate-specific antibodies, often after denaturing immunoprecipitation to preserve ubiquitin conjugates. In vitro assays with recombinant DUBs and monoubiquitinated substrates can define catalytic requirements and specificity, as shown for USP1 and FANCD2.
Proteomics and ubiquitin remnant profiling
Mass spectrometry-based ubiquitin remnant profiling can quantify changes in monoubiquitination sites after DUB perturbation. Such approaches help assign specific substrates to DUBs and reveal global shifts in the ubiquitin landscape.
Transcriptomics and chromatin assays
RNA-seq and chromatin immunoprecipitation can reveal downstream consequences of deubiquitination, particularly for histone substrates such as H2AK119 targeted by USP21. TET2-related chromatin state changes in leukemia further illustrate how transcriptomic and epigenomic readouts complement ubiquitin assays.
Translation profiling
Polysome profiling and ribosome footprinting can assess the impact of OTUD6-mediated RPS7/eS7 deubiquitination on global translation and stress responses. These methods connect GO:0035520 to translational control.
How CRISPR Can Be Used to Study GO:0035520 monoubiquitinated protein deubiquitination
Knockout
CRISPR knockout of DUB genes such as USP1, USP21, OTUD6, BAP1 or ATAD5 is used to test whether the enzyme is required for removal of monoubiquitin from a specific substrate. Knockout cells can be challenged with DNA damage or stress to reveal pathway-specific phenotypes.
Point Mutation
Point-mutation knock-in of catalytic residues or regulatory phosphorylation sites can separate enzymatic activity from scaffolding functions. For example, mutations that disrupt USP21 autoinhibition can be introduced to study H2AK119 deubiquitination.
Knock-in
Tagged knock-in of DUBs or their substrates enables localization and interaction studies in a physiological context. Knock-in of disease-associated variants can also test whether a specific allele alters monoubiquitinated protein deubiquitination.
Overexpression
Inducible overexpression of a DUB can amplify deubiquitination of monoubiquitinated substrates and reveal downstream phenotypes. Overexpression models are particularly useful when knockout causes lethality or redundancy among DUB family members.
How EDITGENE Supports monoubiquitinated protein deubiquitination Research
Researchers studying monoubiquitinated protein deubiquitination-related genes often need to determine whether a candidate gene is causally involved in removing a monoubiquitin mark from a specific substrate, or whether it acts indirectly through a network. Establishing causality requires precise genetic models that can delete, mutate, tag or overexpress the gene of interest in a relevant cell background, followed by quantitative readouts of substrate ubiquitination and downstream phenotypes.
Contact EDITGENE today to design your custom CRISPR model for monoubiquitinated protein deubiquitination research.
Frequently Asked Questions About monoubiquitinated protein deubiquitination
What is monoubiquitinated protein deubiquitination?
It is the biological process defined by GO:0035520 in which a deubiquitinase removes a single ubiquitin group from a monoubiquitinated protein.
What genes are involved in monoubiquitinated protein deubiquitination?
Key genes include USP1, USP21, OTUD6, BAP1, ATAD5 and BAZ1B, along with substrates such as FANCD2, PCNA, H2AK119 and RPS7/eS7.
Why is monoubiquitinated protein deubiquitination important?
It reverses a non-degradative ubiquitin signal that controls DNA repair, chromatin state and translation, and its dysregulation is linked to cancer and genome instability.
How does USP1 deubiquitinate FANCD2?
The N-terminus of USP1 drives specificity for monoubiquitinated FANCD2, allowing the enzyme to remove the ubiquitin mark required for Fanconi anemia pathway function.
What is the role of USP21 in chromatin?
USP21 is autoinhibited and deubiquitinates histone H2AK119, thereby influencing chromatin state.
How does OTUD6 regulate translation?
OTUD6 deubiquitinates RPS7/eS7 on the free 40S ribosome, regulating global protein translation and stress responses.
Is monoubiquitinated protein deubiquitination a drug target?
Yes, USP1 inhibition has progressed from target discovery toward clinical translation, indicating therapeutic interest in this process.
What methods study monoubiquitinated protein deubiquitination?
Common methods include ubiquitin immunoblotting, in vitro deubiquitination assays, mass spectrometry, RNA-seq, chromatin immunoprecipitation and polysome profiling.
How do CRISPR models help study GO:0035520?
CRISPR knockout, point-mutation, knock-in and overexpression models establish causality between a DUB and a specific monoubiquitinated substrate.
Which diseases are linked to defects in this process?
Cancers, genome instability syndromes, leukemia and translation-stress-related conditions have been linked to altered monoubiquitinated protein deubiquitination.
Conclusion
GO:0035520 (monoubiquitinated protein deubiquitination) is a central regulatory process that erases a non-degradative ubiquitin mark from proteins such as FANCD2, PCNA, histone H2AK119 and RPS7/eS7. Its importance spans DNA repair, chromatin regulation, translation and cancer biology, with DUBs like USP1, USP21, OTUD6 and BAP1 as key players and emerging therapeutic targets. Because substrate specificity is often encoded outside the catalytic domain and regulated by autoinhibition or partner proteins, precise genetic models are essential. CRISPR knockout, point-mutation, knock-in and overexpression platforms, combined with ubiquitin profiling and functional genomics, provide the tools needed to move from correlation to causality in this field.
References
- 1. Mevissen TET et al.. 2017. Mechanisms of Deubiquitinase Specificity and Regulation.. Annu Rev Biochem 86:159-192 PMID: 28498721
- 2. Zou Z et al.. 2024. RNA m(5)C oxidation by TET2 regulates chromatin state and leukaemogenesis.. Nature 634(8035):986-994 PMID: 39358506
- 3. Torrado C et al.. 2025. USP1 inhibition: A journey from target discovery to clinical translation.. Pharmacol Ther 271:108865 PMID: 40274197
- 4. Villa S et al.. 2024. OTUD6 deubiquitination of RPS7/eS7 on the free 40 S ribosome regulates global protein translation and stress.. Nat Commun 15(1):6873 PMID: 39127721
- 5. Arkinson C et al.. 2018. Specificity for deubiquitination of monoubiquitinated FANCD2 is driven by the N-terminus of USP1.. Life Sci Alliance 1(5):e201800162 PMID: 30456385
- 6. Kim Y et al.. 2024. ATAD5-BAZ1B interaction modulates PCNA ubiquitination during DNA repair.. Nat Commun 15(1):10496 PMID: 39627214
- 7. Wang J et al.. 2024. Tumor suppressor BAP1 suppresses disulfidptosis through the regulation of SLC7A11 and NADPH levels.. Oncogenesis 13(1):31 PMID: 39266549
- 8. Rahman S et al.. 2025. Mechanism of USP21 autoinhibition and histone H2AK119 deubiquitination.. Sci Adv 11(41):eady2604 PMID: 41071870
