Recombinant Xenopus laevis E3 ubiquitin-protein ligase RNF149 (rnf149)

What is RNF149 and what are its key structural features?

RNF149 (RING finger protein 149) is a 400-amino acid protein containing a RING finger domain, which is a specialized type of zinc finger of 40–60 residues that binds two atoms of zinc . This domain is critical for its E3 ubiquitin ligase activity. The protein contains a transmembrane domain and a cytoplasmic RING domain that catalyzes the transfer of ubiquitin to target substrates.

When working with Xenopus laevis RNF149, researchers should note that the conserved RING domain is essential for its enzymatic function. Sequence alignment analysis between mammalian and Xenopus RNF149 reveals conservation of critical cysteine and histidine residues that coordinate zinc atoms in the RING domain, confirming its evolutionary importance.

What biological processes does RNF149 regulate?

Based on research findings, RNF149 regulates several critical cellular processes through targeted protein degradation:

• Cell growth regulation: RNF149 attenuates cell growth induced by wild-type BRAF through its degradation .

• Inflammatory responses: In macrophages, RNF149 restricts inflammation by promoting the degradation of IFNGR1 (interferon gamma receptor 1) .

• Tissue repair: RNF149 plays a role in infarct healing and cardiac function by modulating macrophage-driven inflammation .

For Xenopus research, these functions suggest potential roles in developmental processes, immune response regulation, and tissue homeostasis that could be explored in embryonic and adult tissues.

How is RNF149 expression regulated in different tissues and conditions?

RNF149 expression demonstrates context-dependent regulation:

• In cardiac tissue following myocardial infarction (MI), RNF149 is highly expressed in infiltrating macrophages but not in neutrophils .

• RNF149 expression is upregulated specifically in MHC-II highCCR2+ macrophages following MI, compared to other macrophage subpopulations .

• Studies indicate that STAT1 (signal transducer and activator of transcription 1) activation induces Rnf149 transcription, establishing a feedback control mechanism .

In Xenopus models, researchers should investigate tissue-specific expression patterns during development and in response to environmental stressors or tissue damage to determine conserved regulatory mechanisms.

What is the mechanism of substrate recognition by RNF149?

RNF149 exhibits selective substrate recognition:

• RNF149 binds directly to the C-terminal kinase-containing domain of wild-type BRAF, but does not bind to mutant BRAF (V600E) .

• The substrate specificity appears to depend on protein conformation, as RNF149 distinguishes between wild-type and mutant forms of the same protein .

• For IFNGR1 recognition, RNF149 targets specific lysine residues for ubiquitination, leading to proteasomal degradation .

When studying Xenopus RNF149, researchers should perform domain mapping experiments to identify regions responsible for substrate interaction. Co-immunoprecipitation assays with domain deletion mutants can reveal the binding interfaces between RNF149 and its substrates in Xenopus cellular contexts.

What are the consequences of RNF149 dysfunction in disease models?

Research demonstrates significant consequences when RNF149 function is disrupted:

• RNF149 mutations have been reported in human breast, ovarian, and colorectal cancers, suggesting a potential tumor suppressor role .

• Knockout of RNF149 in myocardial infarction models leads to worsened cardiac dysfunction, increased infiltration of proinflammatory monocytes/macrophages, and impaired infarct healing .

• Loss of RNF149 function results in excessive type-II interferon signaling due to stabilization of IFNGR1, leading to exaggerated inflammatory responses .

For Xenopus models, researchers could explore the effects of RNF149 knockdown on developmental processes, tissue regeneration, and inflammation using morpholinos or CRISPR-Cas9 gene editing techniques.

What are the optimal methods for expressing and purifying recombinant Xenopus laevis RNF149?

For successful expression and purification of recombinant Xenopus laevis RNF149:

• Expression system selection:

  • Bacterial systems (E. coli BL21(DE3)) may be suitable for the RING domain alone

  • Eukaryotic systems such as insect cells (Sf9) or mammalian cells (HEK293T) are recommended for full-length protein to ensure proper folding and post-translational modifications

• Purification strategy:

  • Incorporate affinity tags (His6, GST, or DDK/FLAG) at either the N- or C-terminus

  • Use tandem affinity purification when high purity is required

  • Include zinc in purification buffers (10-50 μM ZnCl2) to maintain RING domain structure

• Protein stability considerations:

  • Add protease inhibitors to prevent degradation

  • Include reducing agents (DTT or β-mercaptoethanol) to maintain cysteine residues

  • Consider fusion proteins or solubility enhancers (SUMO, MBP) if encountering solubility issues

Based on published protocols for E3 ligases, optimal expression conditions would include induction at lower temperatures (16-20°C) to enhance proper folding.

In vitro ubiquitination assays:

• Components required:

  • Purified recombinant RNF149

  • E1 ubiquitin-activating enzyme

  • E2 ubiquitin-conjugating enzyme (test a panel to identify optimal E2 partner)

  • Ubiquitin (consider using tagged or labeled ubiquitin)

  • ATP and buffer system

  • Purified substrate protein

• Detection methods:

  • Western blotting with substrate-specific antibodies

  • Autoradiography if using radiolabeled ubiquitin

  • Mass spectrometry to identify ubiquitination sites

In vivo ubiquitination assays:

• Experimental approach in Xenopus:

  • Co-express RNF149 with tagged ubiquitin in embryos or cell lines

  • Use proteasome inhibitors (MG132) to prevent degradation of ubiquitinated proteins

  • Immunoprecipitate the substrate protein and detect ubiquitination by Western blotting

• Techniques to validate specificity:

  • Use RNF149 variants with mutations in the RING domain as negative controls

  • Perform siRNA-mediated knockdown of endogenous RNF149

  • Employ CRISPR-Cas9 to generate RNF149-null cells

Research with mammalian RNF149 has demonstrated its capability to induce ubiquitination of wild-type BRAF but not mutant BRAF , providing a methodological framework that can be adapted for Xenopus studies.

What techniques are most effective for studying RNF149 protein-protein interactions?

Several complementary approaches are recommended:

• Co-immunoprecipitation (Co-IP):

  • Use antibodies against endogenous proteins or epitope tags

  • Include appropriate controls (IgG, tag-only constructs)

  • Consider crosslinking approaches for transient interactions

• Proximity-based labeling:

  • BioID or TurboID fusion with RNF149 to identify proximity partners

  • APEX2 fusion for rapid labeling of proximal proteins

  • These methods are particularly valuable for identifying weaker or transient interactions

• Yeast two-hybrid screening:

  • Use the full-length RNF149 or specific domains as bait

  • Screen against Xenopus cDNA libraries to identify novel interaction partners

• Advanced microscopy techniques:

  • Fluorescence resonance energy transfer (FRET)

  • Bimolecular fluorescence complementation (BiFC)

  • These approaches allow visualization of interactions in living cells

Previous studies employed tandem affinity purification followed by mass spectrometry to identify RNF149 as a wild-type BRAF-interacting protein . This approach can be adapted for Xenopus systems to discover novel interaction partners.

How should researchers interpret contradictory findings between studies on RNF149?

When encountering contradictory findings regarding RNF149 function or interactions:

• Consider context-dependent effects:

  • Cell type specificity: RNF149 functions differently in various cell types, as demonstrated by its macrophage-specific activity in cardiac tissue

  • Species differences: Evaluate whether contradictions arise from species-specific functions

  • Experimental conditions: Analyze differences in experimental approaches (in vitro vs. in vivo)

• Substrate specificity analysis:

  • RNF149 shows remarkable specificity for wild-type BRAF over mutant BRAF

  • Contradictions might arise from differences in substrate conformations or post-translational modifications

• Resolution strategies:

  • Perform side-by-side comparisons using identical experimental conditions

  • Use multiple complementary approaches to validate findings

  • Collaborate with groups reporting contradictory results

When studying Xenopus RNF149, consider developmental stage-specific effects and tissue context as potential sources of seemingly contradictory results.

What statistical approaches are most appropriate for analyzing RNF149 degradation kinetics?

To rigorously analyze RNF149-mediated protein degradation:

• For cycloheximide chase experiments:

  • Apply non-linear regression analysis to determine protein half-life

  • Use two-way ANOVA to compare degradation rates between conditions

  • Consider Bayesian hierarchical modeling for complex experimental designs

• For quantifying western blot data:

  • Normalize to appropriate loading controls

  • Apply linear mixed-effects models to account for blot-to-blot variation

  • Use repeated measures analysis when appropriate

• Sample size determination:

  • Power analysis based on expected effect sizes from preliminary data

  • Minimum n=3 independent biological replicates

  • Consider increased replication for experiments with high variability

A proposed analysis framework for RNF149-mediated degradation studies:

How can researchers distinguish between direct and indirect effects of RNF149 on cellular pathways?

Distinguishing direct from indirect effects requires multiple lines of evidence:

• Biochemical approaches:

  • In vitro ubiquitination assays with purified components

  • Domain mapping to identify interaction surfaces

  • Mutational analysis of putative ubiquitination sites on substrates

• Temporal analysis:

  • Time-course experiments to establish sequence of events

  • Rapid induction systems (e.g., auxin-inducible degron-tagged RNF149)

  • Kinetic modeling of degradation pathways

• Specific controls and rescue experiments:

  • Use catalytically inactive RNF149 (RING domain mutations)

  • Employ ubiquitin mutants (K48R, K63R) to dissect ubiquitin chain types

  • Perform substrate rescue with ubiquitination-resistant mutants

Research on RNF149's effect on BRAF demonstrated that RNF149 directly interacts with wild-type BRAF's C-terminal kinase domain and induces its ubiquitination, providing a methodological template for investigating direct effects .

What are the emerging research areas for RNF149 in comparative biology?

Future research on Xenopus laevis RNF149 could explore:

• Evolutionary conservation of substrate specificity:

  • Comparative analysis of RNF149 target recognition across species

  • Identification of conserved degrons in substrate proteins

  • Structural studies of RNF149-substrate complexes

• Developmental roles:

  • RNF149 function during embryonic development in Xenopus

  • Tissue-specific expression patterns and temporal regulation

  • Impact on key developmental signaling pathways (Wnt, BMP, FGF)

• Regenerative processes:

  • Role in Xenopus limb and tail regeneration

  • Regulation of inflammatory responses during tissue repair

  • Interaction with stem cell maintenance pathways

Given RNF149's role in restricting inflammation through IFNGR1 degradation in mammals , similar immune regulatory functions could be explored in the context of Xenopus immune system development and tissue regeneration.

What technologies are emerging that could advance RNF149 research?

Several cutting-edge technologies hold promise for advancing Xenopus RNF149 research:

• Genome editing technologies:

  • CRISPR-Cas9 for generating RNF149 knockout Xenopus models

  • Base editing for introducing specific mutations

  • Prime editing for precise genetic modifications

• Proteomics approaches:

  • Proximity-dependent biotin identification (BioID) for mapping interaction networks

  • Ubiquitinome analysis using diGly remnant antibodies

  • Crosslinking mass spectrometry for structural insights

• Single-cell technologies:

  • scRNA-seq to map RNF149 expression across cell types

  • Spatial transcriptomics to visualize expression patterns

  • Single-cell proteomics to quantify protein levels and modifications

• Structural biology:

  • Cryo-EM for visualization of RNF149-substrate complexes

  • Hydrogen-deuterium exchange mass spectrometry for conformational analysis

  • AlphaFold and related AI tools for structure prediction and docking