Recombinant Xenopus laevis RING finger protein 126-A (rnf126-a)

What is RNF126-A and what is its primary function in Xenopus laevis?

RNF126-A is one of two paralogues (alongside RNF126-B) of the RING finger protein 126 in Xenopus laevis. It functions primarily as an E3 ubiquitin-protein ligase that mediates the ubiquitination of target proteins. Depending on the associated E2 ligase, RNF126-A can mediate both 'Lys-48'- and 'Lys-63'-linked polyubiquitination of substrates . The protein plays crucial roles in several cellular processes, including DNA repair mechanisms and protein quality control. In particular, RNF126 has been identified as a promoter of homologous recombination (HR) through regulation of BRCA1 expression and as a regulator of non-homologous end joining (NHEJ) by mediating the ubiquitylation of Ku80 .

What are the key structural domains of RNF126-A and how do they contribute to its function?

RNF126-A contains several important structural domains that contribute to its function:

• RING finger domain: This zinc-binding domain is essential for its E3 ubiquitin ligase activity, facilitating the transfer of ubiquitin from E2 enzymes to substrate proteins.

• Serine-rich array: RNF126 proteins contain a serine-rich array that is evolutionarily conserved and is similar to sequences found in transcriptional activators present at RNAPII-dependent promoters, suggesting a role in transcriptional regulation .

• E2F1-binding domain: Human RNF126 contains a critical 11-amino acid region (residues 185-195) that is essential for interaction with the transcription factor E2F1 . This suggests that the Xenopus orthologue may have similar binding capabilities.

The functional interplay between these domains enables RNF126-A to serve as both an E3 ubiquitin ligase and a potential transcriptional co-regulator, though the conservation of all these features in the Xenopus laevis protein requires further investigation.

How does RNF126-A compare with its orthologs in other species, particularly human RNF126?

While the search results don't provide comprehensive comparative data between Xenopus RNF126-A and human RNF126, several functional similarities can be inferred:

• Both Xenopus RNF126-A and human RNF126 function as E3 ubiquitin ligases .

• The serine-rich array found in human RNF126 is evolutionarily conserved, suggesting it may also be present in Xenopus RNF126-A .

• Human RNF126 has been shown to interact with transcription factors like E2F1 and to be involved in DNA repair mechanisms . Given the conservation of E3 ligase activity, similar interactions might exist in Xenopus.

A notable difference is that Xenopus laevis, being pseudotetraploid, has two paralogs (RNF126-A and RNF126-B), which may have undergone subfunctionalization or neofunctionalization compared to the single human ortholog .

What expression systems are optimal for producing recombinant Xenopus laevis RNF126-A?

Based on established protocols for similar proteins, several expression systems can be employed for producing recombinant Xenopus laevis RNF126-A:

• Bacterial expression (E. coli): Suitable for producing moderate amounts of protein for biochemical studies. GST-tagged or His-tagged RNF126-A constructs have been successfully used for similar RING finger proteins . The GST-RNF126 approach has been demonstrated to produce functional protein capable of direct binding to interaction partners in pull-down assays .

• Insect cell expression (Baculovirus): Provides better post-translational modifications and folding than bacterial systems, important for maintaining E3 ligase activity.

• Mammalian cell expression: HEK293 or HEK293T cells can be used to express FLAG-HA-tagged RNF126 (FH-RNF126) under inducible conditions, especially when prolonged overexpression might be cytotoxic .

For functional studies, the choice of expression system should be guided by the specific experimental requirements and the need for post-translational modifications.

What purification strategies yield the highest purity and activity of recombinant RNF126-A?

Based on protocols used for similar RING finger proteins:

Affinity Purification:

• For GST-tagged RNF126-A: Glutathione Sepharose affinity chromatography followed by PreScission protease cleavage if tag removal is desired .

• For His-tagged constructs: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins.

• For FLAG-HA-tagged proteins: Tandem affinity purification using anti-FLAG and anti-HA antibodies .

Further Purification Steps:

• Size exclusion chromatography to separate aggregates and ensure homogeneity

• Ion exchange chromatography to remove contaminating nucleic acids and proteins

Activity Preservation:

• Addition of zinc in buffers (typically 10-50 μM ZnCl₂) to maintain the integrity of the RING finger domain

• Inclusion of reducing agents (DTT or TCEP, 1-5 mM) to prevent oxidation of cysteine residues

• Storage in glycerol-containing buffers (10-20%) at -80°C

These purification strategies have been shown to yield functionally active recombinant RING finger proteins suitable for in vitro ubiquitination assays and interaction studies .

How can the E3 ubiquitin ligase activity of RNF126-A be assayed in vitro?

The E3 ubiquitin ligase activity of RNF126-A can be assessed using established in vitro ubiquitination assays. Based on protocols used for human RNF126:

Standard In Vitro Ubiquitination Assay Components:

• Purified recombinant RNF126-A (50-200 nM)

• Ubiquitin-activating enzyme (E1; Uba1, 50-100 nM)

• Ubiquitin-conjugating enzyme (E2; UBE2D3 has been used with human RNF126, 200-500 nM)

• Substrate protein (e.g., Ku80, 100-500 nM)

• Ubiquitin (10-50 μM)

• ATP regeneration system (ATP, creatine phosphate, creatine kinase)

• Buffer components (Tris-HCl pH 7.5, MgCl₂, DTT)

Detection Methods:

• Western blotting using substrate-specific antibodies to detect mobility shifts

• Anti-ubiquitin antibodies to detect ubiquitinated species

• MS/MS analysis for identification of specific ubiquitination sites

An example from human RNF126 research shows that it possesses E3 activity for Ku80 and Ku70 but not for Hsc70 in the presence of the appropriate E1 and E2 enzymes , demonstrating substrate specificity that can be evaluated in similar assays for the Xenopus ortholog.

What are the known substrates of RNF126-A in Xenopus laevis and how were they identified?

• Ku80/Ku70: Human RNF126 directly ubiquitylates Ku80, promoting its dissociation from DNA damage sites to complete DNA repair . This interaction is enhanced following DNA damage.

• TRC35: RNF126 has been implicated in TRC35 ubiquitylation, with RNF126 knockdown reducing TRC35 ubiquitylation .

Methods for Substrate Identification:

• Immunoprecipitation followed by mass spectrometry

• Yeast two-hybrid screening

• Protein arrays probed with recombinant RNF126-A

• Comparative ubiquitinome analysis using stable isotope labeling with amino acids in cell culture (SILAC)

For Xenopus-specific studies, researchers could employ Xenopus egg extract systems, which have been used previously to study ubiquitylation dynamics of proteins like Ku80 .

What types of ubiquitin linkages does RNF126-A preferentially catalyze?

According to the available information, RNF126 can mediate both 'Lys-48'- and 'Lys-63'-linked polyubiquitination depending on the associated E2 ubiquitin-conjugating enzyme . These different linkage types have distinct functional outcomes:

• 'Lys-48'-linked chains: Typically target proteins for proteasomal degradation

• 'Lys-63'-linked chains: Often involved in signaling, DNA damage response, and protein trafficking

The ability to mediate different ubiquitin linkage types suggests that RNF126-A may have diverse roles in cellular processes depending on context and binding partners. The specific E2 enzymes that partner with RNF126-A in Xenopus to determine these linkage preferences have not been fully characterized in the provided search results.

How does RNF126-A contribute to DNA repair in Xenopus compared to mammals?

While the search results don't provide Xenopus-specific data on RNF126-A's role in DNA repair, insights from human RNF126 suggest potential mechanisms:

Homologous Recombination (HR):
Human RNF126 promotes HR by enhancing the transcription of BRCA1 through direct binding to E2F1 . This interaction increases E2F1 binding to the BRCA1 promoter, thereby upregulating BRCA1 expression. Given the conservation of DNA repair mechanisms, RNF126-A may play a similar role in Xenopus.

Non-Homologous End Joining (NHEJ):
In human cells, RNF126 regulates NHEJ by ubiquitylating Ku80, which promotes the release of the Ku70/80 heterodimer from DNA damage sites, allowing completion of DNA repair . RNF126 accumulates at sites of DNA damage and colocalizes with Ku70 and γH2AX (a marker for double-strand breaks) .

Experimental Systems to Study RNF126-A in Xenopus DNA Repair:

• Xenopus egg extracts, which have been used to study DNA repair processes including Ku80 ubiquitylation and degradation

• Microirradiation studies in Xenopus cell lines to observe recruitment of RNF126-A to sites of DNA damage

• Depletion experiments using morpholinos or CRISPR/Cas9 to assess the impact on DNA repair efficiency

Understanding these roles in Xenopus could provide important evolutionary insights into the conservation of DNA repair mechanisms across vertebrates.

What is the molecular mechanism by which RNF126 regulates Ku70/80 in the context of DNA repair?

The mechanism by which RNF126 regulates Ku70/80 during DNA repair has been studied in human cells and provides a model that may apply to Xenopus RNF126-A:

• Recruitment to DNA Damage Sites: Following DNA damage, RNF126 is recruited to double-strand break (DSB) sites where it colocalizes with Ku70 and the DSB marker γH2AX .

• Direct Binding to Ku Proteins: RNF126 directly binds to both Ku80 and Ku70. This interaction is enhanced by gamma radiation that induces DNA damage . The binding involves:

  • Either the NH₂-terminal vWAD domain or the Ku core domain (CD) of Ku proteins

  • The NH₂-terminal portion of RNF126

• Ubiquitylation of Ku80: RNF126 catalyzes the ubiquitylation of Ku80 as a late response to DSB generation (not detected at 30 minutes post-irradiation) . Proteomics analysis identified 19 lysine residues in Ku80 as ubiquitylation sites .

• Dissociation from DNA: The ubiquitylation triggers the release of Ku70/80 from damaged DNA, allowing the completion of DNA repair .

• Proteasomal Degradation: Following ubiquitylation, Ku80 undergoes proteasomal degradation, with this effect being more prominent in the chromatin fraction than in the soluble fraction .

Knockdown of RNF126 inhibits the dissociation of Ku70/80 from chromatin, impairs DNA damage response and DSB repair, and increases susceptibility to DSB-induced cell death , demonstrating the critical role of this mechanism in DNA repair.

How can CRISPR/Cas9 technology be optimized for studying RNF126-A function in Xenopus?

CRISPR/Cas9 technology offers powerful approaches for studying RNF126-A function in Xenopus laevis:

Design Considerations for Xenopus laevis:

• Target Selection: Due to the pseudotetraploid nature of X. laevis, both homeologs (RNF126-A and RNF126-B) may need to be targeted. Design guide RNAs (gRNAs) targeting conserved exons in both homeologs or design homeolog-specific gRNAs for differential analysis.

• gRNA Design:

  • Use Xenopus-specific CRISPR design tools that account for the organism's genomic peculiarities

  • Target early exons to ensure functional disruption

  • Verify low off-target potential using Xenopus genome databases

• Delivery Methods:

  • Microinjection of Cas9 protein with gRNAs into fertilized eggs (protein-gRNA ribonucleoprotein complexes)

  • Cas9 mRNA with gRNAs for longer expression during development

  • Tissue-specific CRISPR using tissue-specific promoters driving Cas9 expression

Verification Strategies:

• T7 Endonuclease I assay or direct sequencing to confirm mutations

• Western blotting to verify protein knockdown

• Quantitative PCR to measure changes in transcript levels

Functional Analysis Approaches:

• Phenotypic analysis during development

• DNA damage response assays in CRISPR-modified embryos or tissues

• Rescue experiments by co-injecting wild-type mRNA

While the search results don't specifically mention CRISPR studies in Xenopus for RNF126-A, these approaches have been successfully applied to other genes in Xenopus and could be adapted for studying this E3 ubiquitin ligase.

What methodologies can be used to identify novel interaction partners of RNF126-A?

Several complementary approaches can be employed to identify novel interaction partners of RNF126-A:

Affinity Purification-Mass Spectrometry (AP-MS):

• Express tagged RNF126-A (FLAG-HA, GST, or His) in Xenopus cells or embryos

• Perform immunoprecipitation under various conditions (normal vs. DNA damage)

• Analyze co-precipitated proteins by LC-MS/MS

• Validate interactions by co-immunoprecipitation and functional assays

Proximity-Based Labeling:

• BioID: Fusion of RNF126-A with a promiscuous biotin ligase (BirA*) to biotinylate proximal proteins

• APEX: Fusion with an engineered ascorbate peroxidase for proximity labeling

• TurboID: A faster version of BioID for more efficient labeling

Yeast Two-Hybrid Screening:
Create a bait construct with RNF126-A (or specific domains) to screen against a Xenopus cDNA library

Protein Arrays:

• Probe commercial protein arrays with recombinant RNF126-A

• Develop custom Xenopus protein arrays for more species-specific interactions

In Silico Approaches:

• Structural modeling to predict potential interaction interfaces

• Network analysis based on orthologous interactions in other species

These methods have been successfully used to identify interaction partners of human RNF126, including E2F1 and Ku70/80 , and can be adapted for the Xenopus ortholog.

How does RNF126-A expression and function change during Xenopus development?

Expression Analysis:

• Temporal Expression: RT-qPCR and RNA-seq analysis of RNF126-A expression across developmental stages from fertilization through metamorphosis

• Spatial Expression: Whole-mount in situ hybridization to determine tissue-specific expression patterns

• Protein Localization: Immunohistochemistry with RNF126-A-specific antibodies to track protein localization during development

Functional Analysis:

• Knockdown Studies: Morpholino oligonucleotides or CRISPR/Cas9 to reduce RNF126-A expression during specific developmental windows

• Overexpression Studies: Microinjection of RNF126-A mRNA to assess gain-of-function phenotypes

• Domain-Specific Studies: Expression of truncated or mutated RNF126-A to determine domain-specific functions during development

Potential Developmental Roles:
Given RNF126's known functions in DNA repair mechanisms , it may be particularly important during periods of rapid cell division and genomic stress, such as early cleavage stages, gastrulation, and neurulation. Additionally, its role in regulating protein quality control could be critical during tissue remodeling during metamorphosis.

A thorough developmental analysis would provide insights into when and where RNF126-A functions during Xenopus development, complementing the biochemical and cellular studies of its molecular functions.

How can research on Xenopus RNF126-A inform therapeutic approaches for human diseases?

Research on Xenopus RNF126-A can inform therapeutic approaches for human diseases in several ways:

Cancer Therapeutics:
Human RNF126 has been implicated in cancer biology through its regulation of DNA repair mechanisms. Studies show that RNF126 depletion leads to increased sensitivity to ionizing radiation (IR) and poly (ADP-ribose) polymerase (PARP) inhibition . Xenopus RNF126-A research could:

• Validate conservation of these mechanisms across vertebrates

• Provide a developmental context for understanding oncogenesis

• Offer a platform for screening small molecule inhibitors of RNF126 before moving to mammalian systems

DNA Repair Disorders:
Given RNF126's role in both homologous recombination and non-homologous end joining , understanding its function through Xenopus studies could provide insights into:

• Genetic disorders characterized by defective DNA repair

• Age-related diseases associated with accumulated DNA damage

• Radiation sensitivity syndromes

Drug Discovery Approaches:

• Target Validation: Confirm whether structural features critical for RNF126 function in humans are conserved in Xenopus

• High-Throughput Screening: Develop Xenopus egg extract-based assays for screening compounds that modulate RNF126 activity

• Structure-Activity Relationships: Use recombinant Xenopus RNF126-A to determine binding sites for potential therapeutics

As noted in the research literature, RNF126 "not only offers novel insights into our current understanding of the biological functions of RNF126 but also provides a potential therapeutic target for cancer treatment" , highlighting the translational relevance of fundamental research on this protein across model organisms.

What experimental approaches can be used to study the effects of RNF126-A mutations on its function?

Several experimental approaches can be employed to study the effects of RNF126-A mutations on its function:

Structure-Function Analysis:

• Site-Directed Mutagenesis: Generate point mutations, deletions, or truncations in recombinant RNF126-A. Critical regions to target include:

  • The RING finger domain (for E3 ligase activity)

  • The 11-amino acid region (residues 185-195 in human RNF126) critical for E2F1 binding

  • Serine-rich arrays implicated in transcriptional regulation

• Functional Assays for Mutants:

  • In vitro ubiquitination assays to assess E3 ligase activity

  • GST pull-down assays to evaluate binding to interaction partners like E2F1

  • Luciferase reporter assays to measure effects on transcriptional activation (e.g., BRCA1 promoter activity)

  • Chromatin immunoprecipitation (ChIP) to assess recruitment to target gene promoters

Cellular and Developmental Assays:

• Rescue Experiments: Test the ability of mutant RNF126-A constructs to rescue phenotypes in RNF126-A-depleted Xenopus embryos or cells

• Dominant-Negative Effects: Assess whether mutants like RNF126-Δf (lacking the E2F1 interaction domain) exhibit dominant-negative effects on wild-type function

• DNA Repair Assays: Measure the impact of mutations on DNA repair efficiency following induced damage

Structural Studies:

• X-ray crystallography or cryo-EM of wild-type and mutant RNF126-A to understand conformational changes

• Hydrogen-deuterium exchange mass spectrometry to assess dynamic structural changes upon mutation

These approaches would provide comprehensive insights into structure-function relationships in RNF126-A and potentially identify critical regions for therapeutic targeting.

Comparison of Ubiquitination Targets of RNF126