Recombinant Xenopus laevis E3 ubiquitin-protein ligase RNF19B (rnf19b)

What is the structural and functional characterization of Xenopus laevis RNF19B?

RNF19B belongs to the E3 ubiquitin ligase family, which mediates the ubiquitination of multiple substrates through catalyzing the transfer of ubiquitin to target proteins. The protein contains characteristic RING finger domains responsible for its catalytic activity. In Xenopus, RNF19B plays critical roles in developmental processes and cellular functions through selective protein degradation pathways. Similar to its mammalian counterpart, Xenopus RNF19B is likely involved in immune response regulation and potentially in locomotor function regulation as seen in related amphibian species . Expression analysis in amphibian models has revealed that RNF19B shows tissue-specific patterns, with notable expression in immune cells and potentially in muscle tissues, reflecting its diverse physiological roles.

How does RNF19B expression differ across Xenopus laevis developmental stages?

RNF19B expression follows distinct temporal patterns throughout Xenopus development. While specific expression data for Xenopus laevis RNF19B across developmental stages is limited in the provided search results, research on related ubiquitin pathway genes suggests that expression typically begins in early embryogenesis and shows stage-specific regulation. Differential expression analysis would likely reveal peak expression during organogenesis, particularly during the development of tissues where RNF19B functions are critical, such as the immune system and potentially muscle tissue. Researchers studying developmental expression should employ techniques such as quantitative PCR, RNA sequencing at different developmental timepoints, and whole-mount in situ hybridization to visualize spatial expression patterns.

What are the key substrates of Xenopus laevis RNF19B?

While the complete substrate profile of Xenopus laevis RNF19B has not been fully characterized, comparative analysis with mammalian homologs provides insights into potential targets. Based on research with related E3 ligases, likely substrates include:

• Proteins involved in cell signaling pathways (possibly including components of TNF receptor signaling)

• Regulators of immune cell function, as RNF19B expression is essential for both natural killer (NK) cells and macrophages to facilitate cytotoxic function and signaling molecule release

• Proteins involved in muscle contraction and metabolism, particularly if RNF19B functions similarly to related proteins studied in Xenopus allofraseri that showed differential expression between locomotor phenotypes

Identification of novel substrates requires experimental approaches such as co-immunoprecipitation followed by mass spectrometry, proximity-dependent biotin identification (BioID), or ubiquitination assays with candidate proteins.

What are the optimal conditions for expressing recombinant Xenopus laevis RNF19B in bacterial systems?

For successful bacterial expression of functional Xenopus laevis RNF19B:

• Expression system selection: E. coli BL21(DE3) or Rosetta strains are preferred for eukaryotic protein expression.

• Vector optimization: Use vectors with T7 promoters and incorporate a TEV protease cleavage site between the tag and RNF19B sequence for tag removal.

• Expression conditions:

  • Induce expression at OD600 of 0.6-0.8

  • Use lower temperatures (16-18°C) for induction to enhance proper folding

  • Extend expression time to 16-20 hours

  • Consider adding 0.1-0.5 mM ZnCl2 to the medium to support proper folding of the RING finger domain

• Protein purification strategy:

  • Initial capture using affinity chromatography (His-tag or GST-tag)

  • Secondary purification using ion exchange chromatography

  • Final polishing with size exclusion chromatography

• Functional verification: Following purification, verify enzymatic activity using in vitro ubiquitination assays with known E1 and E2 enzymes.

The key challenge in recombinant expression of RNF19B is maintaining proper folding of the RING finger domains, which requires zinc coordination. Including reducing agents and zinc in buffers throughout purification helps maintain structural integrity.

How can researchers design effective CRISPR/Cas9 knockouts of RNF19B in Xenopus laevis?

When designing CRISPR/Cas9 strategies for RNF19B knockout in Xenopus laevis, consider these methodological approaches:

• Target site selection:

  • Design sgRNAs targeting early exons (preferably exons 1-3) to ensure complete loss-of-function

  • Target conserved domains like the RING finger domains

  • Account for Xenopus laevis' allotetraploid genome by identifying targets conserved across homeologs

• sgRNA design parameters:

  • Select targets with minimal off-target effects

  • Ensure GC content between 40-60%

  • Avoid poly-T sequences (4+ consecutive Ts)

  • Verify specificity using Xenopus-specific CRISPR design tools

• Delivery method:

  • Microinjection into fertilized eggs at one-cell stage (typical volume: 2-4 nl)

  • Cas9 can be delivered as protein (recommended) or mRNA

  • Typical concentrations: 500-1000 pg Cas9 protein with 300-400 pg sgRNA

• Validation approaches:

  • T7 endonuclease assay or high-resolution melt analysis for initial screening

  • Sanger sequencing to confirm mutations

  • Western blot to verify protein knockout

  • Phenotypic analysis to correlate with expected outcomes based on RNF19B function

• Control strategies:

  • Include Cas9-only and non-targeting sgRNA control groups

  • Design rescue experiments with RNF19B mRNA containing synonymous mutations at the sgRNA target site

This approach accounts for the specific challenges of Xenopus laevis as a model organism while targeting the RNF19B gene effectively.

What methods are recommended for studying RNF19B-mediated ubiquitination in Xenopus laevis tissues?

To study RNF19B-mediated ubiquitination in Xenopus tissues:

• Tissue preparation and ubiquitination detection:

  • Harvest tissues of interest (likely immune tissues based on known RNF19B functions)

  • Homogenize in buffer containing deubiquitinase inhibitors (N-ethylmaleimide, PR-619)

  • Perform immunoprecipitation of potential substrates followed by ubiquitin western blotting

  • Use ubiquitin linkage-specific antibodies (K48, K63, linear) to determine ubiquitin chain topology

• In vitro reconstitution assays:

  • Express and purify recombinant Xenopus E1, E2s, and RNF19B

  • Combine with recombinant substrate, ATP, and ubiquitin

  • Analyze reaction products by SDS-PAGE and western blotting

  • Include controls lacking individual components to verify specificity

• Cell-based approaches:

  • Establish Xenopus cell lines (e.g., XTC-2) with modulated RNF19B expression

  • Treat with proteasome inhibitors (MG132) to stabilize ubiquitinated proteins

  • Use cycloheximide chase assays to measure substrate stability

  • Employ proximity ligation assays to visualize RNF19B-substrate interactions in situ

• Ubiquitin proteomics:

  • Compare ubiquitinomes of wild-type and RNF19B-depleted tissues using tandem ubiquitin binding entities (TUBEs) enrichment

  • Perform mass spectrometry to identify differentially ubiquitinated proteins

  • Validate candidates with targeted approaches

These methods allow for comprehensive analysis of RNF19B's ubiquitination activity and substrate specificity in the Xenopus system.

How can Xenopus laevis RNF19B expression patterns inform our understanding of locomotor performance trade-offs?

Research on related species suggests intriguing connections between ubiquitin pathway genes and locomotor performance. In Xenopus allofraseri, transcriptomic analysis revealed differential gene expression between burst-performant and endurance-performant individuals . While RNF19B was not specifically identified in that study, other genes involved in muscle function regulation showed performance-specific expression patterns.

To investigate whether RNF19B plays a role in locomotor performance trade-offs in Xenopus laevis:

• Characterize expression patterns in different muscle fiber types using immunohistochemistry and qRT-PCR

• Correlate expression levels with measurable locomotor parameters (burst speed, endurance time)

• Perform knockdown/overexpression studies to determine if altering RNF19B levels affects locomotor performance

• Identify potential muscle-specific substrates that might mediate effects on contractile properties

The likely mechanistic pathways would involve:

• Calcium signaling and muscle contraction regulation

• Endoplasmic reticulum stress responses

• Metabolism of energy substrates in muscle tissue

Such studies would bridge the gap between molecular function and whole-organism performance, potentially revealing novel roles for ubiquitin ligases in physical performance trade-offs.

What is the relationship between RNF19B and immune function in Xenopus laevis?

Based on mammalian studies, RNF19B plays essential roles in immune cell function, particularly in NK cells and macrophages . Investigating this relationship in Xenopus laevis provides an opportunity to understand the evolutionary conservation of these functions.

To explore RNF19B's immune functions in Xenopus:

• Characterize expression in immune tissues and isolated immune cell populations

• Analyze RNF19B regulation during immune challenges (infection, inflammation)

• Perform functional studies using CRISPR/Cas9 knockout or morpholino knockdown

• Assess impacts on:

  • Immune cell development and differentiation

  • Cytotoxic activity of NK-like cells

  • Macrophage polarization (similar to findings in human systems)

  • Cytokine production and signaling

Research in mammalian systems demonstrates that RNF19B positively correlates with several immune checkpoint-related genes, particularly PD-1 and CTLA4 . A comparative analysis in Xenopus could reveal conserved regulatory mechanisms controlling immune checkpoints across vertebrate evolution.

How does alternative splicing affect the function of Xenopus laevis RNF19B?

Alternative splicing represents an important regulatory mechanism for many genes, and the search results indicate that transcript isoforms can impact protein function in related systems . For RNF19B in Xenopus laevis, investigating alternative splicing:

• Identify splicing variants using RNA-seq and targeted RT-PCR across tissues and developmental stages

• Characterize isoform-specific domains and their potential functional implications

• Express recombinant isoforms to determine differences in:

  • Substrate specificity

  • Subcellular localization

  • Catalytic activity

  • Interaction partners

Potential functional differences might include:

• Altered E2 enzyme preference

• Different ubiquitin chain topology specification (K48 vs. K63 vs. linear)

• Tissue-specific activities reflecting the specialized needs of different cell types

This research direction would significantly expand our understanding of how alternative splicing contributes to the functional diversity of ubiquitin ligases in vertebrate development.

How conserved is RNF19B structure and function between Xenopus laevis and other vertebrate models?

Comparative analysis of RNF19B across vertebrate species provides insights into evolutionary conservation and divergence of ubiquitin ligase functions. A systematic comparison would include:

Research approaches should include:

• Sequence alignment and phylogenetic analysis

• Domain structure comparison using predictive algorithms

• Heterologous expression studies to test functional complementation

• Cross-species substrate validation

Understanding the evolutionary trajectory of RNF19B function will illuminate how ubiquitin ligase specificity evolves and adapts to species-specific physiological demands.

What can Xenopus laevis RNF19B teach us about the evolution of ubiquitin signaling pathways?

Xenopus laevis occupies an important evolutionary position between fish and mammals, making it valuable for understanding the evolution of complex signaling systems. Research on RNF19B in this context would:

• Trace the appearance and diversification of RNF19B across vertebrate evolution

• Identify conserved and divergent substrates suggesting core vs. species-specific functions

• Compare regulatory mechanisms controlling RNF19B expression and activity

• Analyze key features like:

  • RING domain evolution and zinc coordination structures

  • Substrate recognition motifs

  • Regulatory domains influencing activity or localization

The pathways in which RNF19B functions, particularly immune regulation and potentially muscle function, represent systems that have undergone significant adaptation throughout vertebrate evolution. By studying how RNF19B's role has changed across species, researchers can gain insights into how ubiquitin signaling networks adapt to meet evolving physiological demands.

How does RNF19B interact with other components of the ubiquitin-proteasome system in Xenopus laevis?

RNF19B functions within a complex network of ubiquitin-proteasome system (UPS) components. In Xenopus laevis, these interactions likely include:

• E2 conjugating enzyme partnerships:

  • Identify specific E2 enzymes that partner with RNF19B

  • Determine whether different E2s influence ubiquitin chain topology

  • Map interaction domains through mutagenesis and binding studies

• Deubiquitinating enzyme (DUB) counterregulation:

  • Identify DUBs that antagonize RNF19B activity

  • Characterize the dynamics between ubiquitination and deubiquitination

  • Investigate potential co-regulation mechanisms

• Scaffold and adaptor protein interactions:

  • Identify proteins that may recruit RNF19B to specific cellular locations

  • Map the interactome using techniques like BioID, yeast two-hybrid, or co-immunoprecipitation followed by mass spectrometry

• Regulatory post-translational modifications:

  • Characterize modifications (phosphorylation, SUMOylation) that regulate RNF19B activity

  • Identify enzymes responsible for these modifications

  • Determine how these modifications affect substrate specificity or catalytic activity

Understanding these interactions will provide a systems-level view of how RNF19B functions within the broader context of cellular protein homeostasis.

What role does RNF19B play in signaling pathways during Xenopus development and immune response?

Based on available data and knowledge of related proteins, RNF19B likely participates in multiple signaling pathways:

• TNF receptor signaling pathways:

  • Possible involvement in regulating receptor complex components

  • Potential contribution to NF-κB activation through linear or K63 ubiquitination

  • Regulation of signaling duration through targeted degradation

• Immune cell development and function:

  • Role in macrophage polarization (M1/M2 balance)

  • Potential regulation of cytokine production

  • Possible impact on immune cell differentiation during development

• Cell death pathways:

  • Regulation of proteins involved in apoptosis

  • Potential modulation of inflammatory cell death mechanisms

• Developmental signaling:

  • Possible roles in major developmental pathways (Wnt, Notch, TGF-β)

  • Temporal regulation of signaling components during specific developmental windows

Experimental approaches to elucidate these functions should include:

• Pathway-specific reporter assays in RNF19B-manipulated systems

• Phospho-proteomics to identify affected signaling nodes

• Developmental phenotyping following RNF19B manipulation

• Epistasis experiments with known pathway components

What are the major technical challenges in studying Xenopus laevis RNF19B and how can they be overcome?

Research on Xenopus laevis RNF19B faces several technical challenges:

• Genome complexity challenges:

  • The allotetraploid nature of X. laevis creates complications in genetic manipulation

  • Solution: Design strategies targeting conserved regions in both homeologs or use X. tropicalis as a diploid alternative

• Protein expression and purification difficulties:

  • RING finger proteins often show poor solubility and stability

  • Solution: Optimize expression conditions with solubility tags, lower temperatures, and specialized folding conditions including zinc supplementation

• Substrate identification complexity:

  • Transient enzyme-substrate interactions make identification challenging

  • Solution: Employ proximity labeling approaches, develop substrate traps through catalytic inactivation, and use systems biology approaches combining proteomics with bioinformatics

• Functional redundancy:

  • Other E3 ligases may compensate for RNF19B loss

  • Solution: Perform double knockdowns, detailed phenotypic analysis, and substrate-focused approaches rather than relying solely on knockout phenotypes

• Developmental stage specificity:

  • RNF19B functions may vary across developmental stages

  • Solution: Use inducible or tissue-specific manipulation approaches to target specific developmental windows

These methodological approaches represent emerging strategies to address the inherent challenges in studying E3 ubiquitin ligases in complex developmental systems.

What are promising future research directions for Xenopus laevis RNF19B studies?

Several high-potential research directions for Xenopus laevis RNF19B include:

• Single-cell analyses of RNF19B function:

  • Apply scRNA-seq to identify cell populations where RNF19B is dynamically regulated

  • Use cell-specific manipulation to determine tissue-autonomous vs. non-autonomous functions

  • Correlate with single-cell proteomics to examine cell-specific substrates

• Mechanistic studies of immune modulation:

  • Investigate RNF19B's role in amphibian-specific immune responses

  • Examine how RNF19B regulates macrophage polarization in the context of amphibian regeneration

  • Explore potential connections to immune checkpoint regulation as seen in mammalian systems

• Novel methodological approaches:

  • Develop degron-based systems to achieve rapid protein degradation in Xenopus

  • Apply advanced imaging techniques to visualize RNF19B activity in real-time

  • Use synthetic biology approaches to engineer RNF19B variants with novel substrate specificities

• Translational applications:

  • Explore whether insights from Xenopus RNF19B can inform therapeutic strategies targeting human RNF19B in diseases like hepatocellular carcinoma

  • Investigate the potential for RNF19B modulation in regenerative medicine contexts

• Comparative developmental studies:

  • Conduct detailed comparisons of RNF19B function across different amphibian species with varying regenerative capacities

  • Explore whether RNF19B contributes to species-specific locomotor adaptations

These research directions leverage the unique advantages of the Xenopus model system while addressing fundamental questions about ubiquitin ligase function in development, immunity, and evolution.