Recombinant Human E3 ubiquitin-protein ligase RNF133 (RNF133)

What is the structural characterization of human RNF133?

RNF133 is characterized by its distinct protein domain architecture that includes one transmembrane region and one RING finger domain positioned after the transmembrane region . The protein is primarily localized in the cytoplasm, specifically in the endoplasmic reticulum (ER) as confirmed by immunostaining of recombinantly expressed human RNF133 . The transmembrane domain suggests that RNF133 functions as an ER-associated E3 ubiquitin ligase, which is critical for its role in protein quality control mechanisms.

In silico prediction and structural analysis reveals that RNF133 shares approximately 54.9% identity with its paralog RNF148 in humans, based on pairwise sequence analysis . This similarity in structure suggests potential evolutionary conservation of function, though experimental evidence indicates limited functional redundancy between these paralogs.

What is the tissue-specific expression pattern of RNF133?

RT-PCR analysis of mouse and human reproductive and non-reproductive tissues confirms that RNF133 is predominantly testis-specific in both species . Within testicular tissue, expression analysis during postnatal development indicates that RNF133 is expressed abundantly around day 15, corresponding to the end of meiosis in the mouse . This temporal expression pattern suggests that RNF133 likely functions during spermiogenesis or in sperm formation and/or function rather than during earlier stages of spermatogenesis.

The testis-specific nature of RNF133 makes it a potential target for non-hormonal male contraception research, as its specialized expression pattern minimizes the risk of systemic effects when targeting this protein .

What is the physiological function of RNF133 based on knockout studies?

Knockout studies in mice demonstrate that RNF133 plays a critical role in male fertility. RNF133 KO males display severe subfertility compared to heterozygous controls . Specifically, homozygous KO males produced an average of only 2.3 ± 1.6 pups per litter compared to 8.6 ± 0.7 pups per litter for heterozygous males .

The subfertility phenotype is attributed to defects in sperm function rather than sperm production, as evidenced by:

• Only 10.0 ± 10% of copulation plugs from RNF133 KO males resulted in successful pregnancies compared to 100% for heterozygous males

• Significantly reduced fertilization rates both in vivo and in vitro fertilization assays (IVF)

• Normal sperm counts but significantly decreased motility and progressive motility in RNF133 KO sperm

• Abnormal sperm morphology, characterized by retention of cytoplasmic droplets and excess cytoplasm surrounding sperm nuclei

What are the optimal experimental approaches for studying RNF133 function in vitro?

When investigating RNF133 function in vitro, researchers should consider a multi-faceted approach that accounts for its transmembrane nature and tissue-specific expression:

1. Protein-Protein Interaction Analysis:

• Co-immunoprecipitation assays to identify binding partners, particularly focusing on known E2 ubiquitin-conjugating enzymes such as UBE2J1, which has been confirmed to interact with RNF133

• Proximity labeling techniques (BioID or APEX) to identify proximally associated proteins in the ER membrane context

• Yeast two-hybrid screens with the cytoplasmic domains of RNF133 to identify additional interacting proteins

2. Ubiquitination Assays:

• In vitro ubiquitination reconstitution assays using purified recombinant RNF133, E1, E2 (preferably UBE2J1), and potential substrate proteins

• Cell-based ubiquitination assays with epitope-tagged ubiquitin and RNF133 to detect ubiquitinated substrates

• Mass spectrometry-based approaches to identify the ubiquitinated proteome in RNF133-expressing versus RNF133-depleted cells

3. Localization Studies:

• Subcellular fractionation followed by immunoblotting to confirm ER localization

• Fluorescence microscopy with co-staining of ER markers to visualize RNF133 localization

• Live-cell imaging with fluorescently tagged RNF133 to monitor dynamic localization during spermatogenesis

4. Functional Assays in Cell Models:

• Use of testicular cell lines or primary spermatogenic cells for functional studies

• CRISPR/Cas9-mediated knockout or knockdown studies to assess cellular phenotypes

• Rescue experiments with wild-type or mutant (RING domain mutations) RNF133 to confirm specificity

How should researchers design experiments to identify substrates of RNF133?

Identifying E3 ligase substrates remains challenging but can be approached systematically:

1. Proteomic Approaches:

• Quantitative proteomics comparing protein levels in RNF133 knockout versus wild-type testicular cells to identify upregulated proteins (potential substrates)

• Diglycine remnant profiling to identify changes in the ubiquitinated proteome

• Protein stability profiling using methods such as Global Protein Stability (GPS) or Tandem Fluorescent Protein Timer (tFT) systems

2. Candidate Approach:

• Focus on proteins involved in spermiogenesis, particularly those that participate in cytoplasmic removal during sperm maturation, given the retention of cytoplasm phenotype observed in RNF133 KO sperm

• Test proteins involved in sperm motility, as RNF133 KO sperm show reduced motility

• Examine ER-associated degradation (ERAD) substrates specifically in testicular cells

3. Validation Experiments:

• Direct binding assays between RNF133 and candidate substrates

• In vitro and in vivo ubiquitination assays to confirm substrate modification

• Half-life analysis of candidate proteins in the presence or absence of RNF133

• Mutational analysis of substrate ubiquitination sites to confirm functional relevance

4. Functional Correlation:

• Phenotypic analysis of substrate knockout/knockdown to determine if they recapitulate aspects of the RNF133 KO phenotype

• Rescue experiments in RNF133 KO background by modulating substrate levels or activity

What controls are essential when analyzing RNF133 knockout phenotypes?

1. Genetic Controls:

• Use of littermate controls, preferably heterozygous mice rather than wild-type, to minimize genetic background effects

• Inclusion of multiple independent knockout lines to rule out off-target CRISPR effects

• Rescue experiments with wild-type RNF133 to confirm that phenotypes are directly caused by loss of RNF133

2. Functional Redundancy Controls:

• Analysis of RNF148 expression in RNF133 knockout tissues to assess potential compensatory upregulation

• Creation and characterization of RNF133/RNF148 double knockout mice to assess functional redundancy

• Comparison of single versus double knockout phenotypes at molecular and physiological levels

3. Physiological Assays:

• Time-controlled mating studies to distinguish between fertilization defects and embryonic development issues

• Both in vivo and in vitro fertilization assessments to isolate the source of fertility defects

• Comprehensive sperm parameter analysis (count, morphology, motility) using computer-assisted sperm analysis (CASA)

4. Developmental Controls:

• Temporal analysis of phenotype manifestation during spermatogenesis

• Stage-specific analysis of spermatogenic tubules to identify the earliest point of defect

• Comparison with phenotypes of other ER quality control machinery knockouts, such as UBE2J1 KO

How do researchers reconcile the overlapping yet distinct functions of RNF133 and RNF148?

The paralogous relationship between RNF133 and RNF148 presents an intriguing research challenge. Despite sharing approximately 58.9% and 54.9% sequence identity in mouse and human respectively , knockout studies reveal distinct functional roles:

Analytical Approaches:

• Comparative Expression Analysis:

  • Perform detailed spatiotemporal expression profiling of both proteins during testicular development

  • Use single-cell RNA sequencing to identify potential cell type-specific expression differences

• Structural Biology Approaches:

  • Determine crystal structures of both proteins to identify structural differences that may explain functional divergence

  • Create chimeric proteins swapping domains between RNF133 and RNF148 to identify functionally critical regions

• Substrate Specificity Analysis:

  • Perform comparative ubiquitome analyses to identify unique and shared substrates

  • Develop in vitro assays to compare substrate binding affinities and ubiquitination efficiencies

• Evolutionary Analysis:

  • Examine evolutionary conservation patterns to identify functionally divergent regions under different selective pressures

  • Perform phylogenetic analysis across species to determine when functional divergence may have occurred

Despite their similarities, RNF133 knockout mice show severe subfertility, while RNF148 knockout mice retain normal fertility . The limited functional redundancy suggests that these proteins have evolved distinct functions or target different substrates despite their structural similarities.

What are the main technical challenges in producing functional recombinant RNF133 for in vitro studies?

Producing functional recombinant transmembrane E3 ligases like RNF133 presents several technical challenges:

1. Expression System Selection:

• Mammalian expression systems are preferable for proper folding and post-translational modifications but yield lower protein amounts

• Insect cell systems offer a compromise between proper folding and yield

• E. coli systems provide high yield but may require refolding procedures that are particularly challenging for transmembrane proteins

2. Solubilization Strategies:

• Use of detergents that maintain the native conformation while efficiently solubilizing the protein

• Detergent screening panel (DDM, LMNG, MNG-3, etc.) to identify optimal solubilization conditions

• Consider nanodiscs or amphipols as alternative membrane mimetics for functional studies

3. Purification Optimization:

• Two-step affinity chromatography using tags at both N and C termini to ensure full-length protein isolation

• Size exclusion chromatography to separate monomeric protein from aggregates

• Avoid harsh elution conditions that may denature the transmembrane domain

4. Activity Verification:

• Development of robust in vitro ubiquitination assays with known E2 partners like UBE2J1

• Structural integrity assessment using circular dichroism or limited proteolysis

• Thermal stability assays to ensure the purified protein remains folded in experimental conditions

5. Domain-Based Approach:

• Express the RING domain separately for interaction and activity studies

• Use of the cytoplasmic portion for substrate identification

• Reconstitution experiments combining individually purified domains

How should researchers interpret conflicting data on RNF133 function across different species?

When encountering species-specific differences in RNF133 function, researchers should adopt a systematic comparative approach:

1. Sequence and Structure Analysis:

• Perform detailed sequence alignment and conservation analysis across species

• Identify species-specific post-translational modification sites

• Examine regulatory regions (promoters, enhancers) for divergence in expression control

2. Functional Conservation Testing:

• Cross-species complementation studies (e.g., human RNF133 in mouse knockout models)

• Creation of humanized mouse models for RNF133

• Direct comparative functional assays using proteins from different species

3. Experimental Variables to Control:

• Ensure comparable developmental stages when comparing across species

• Account for differences in spermatogenesis timing and regulation

• Normalize for differences in protein expression levels when comparing functions

4. Evolutionary Context Consideration:

• Analyze selection pressure on different domains of RNF133 across species

• Consider the evolutionary history of RNF133 in the context of reproductive biology differences

• Examine co-evolution patterns with interacting partners and substrates

What experimental approaches can assess RNF133 as a potential non-hormonal male contraceptive target?

RNF133's testis-specific expression and critical role in male fertility make it a promising non-hormonal male contraceptive target . Research approaches should include:

1. Target Validation Studies:

2. Inhibitor Development Pipeline:

• Structure-based drug design targeting the RING domain

• Development of PROTACs (Proteolysis Targeting Chimeras) specifically targeting RNF133

• High-throughput screening assays using recombinant RNF133 activity as readout

3. Efficacy and Safety Assessment:

• Dose-response studies correlating RNF133 inhibition with fertility effects

• Assessments of reversibility upon discontinuation of treatment

• Comprehensive toxicology evaluation focusing on reproductive and non-reproductive tissues

4. Delivery Strategy Development:

• Testis-specific drug delivery systems to enhance target specificity

• Formulation studies to optimize bioavailability in testicular tissue

• Duration of effect studies to determine appropriate dosing regimens

How can researchers effectively study the interaction between RNF133 and UBE2J1 in the context of ER quality control?

The interaction between RNF133 and UBE2J1 in ER quality control represents a critical aspect of their function in spermatogenesis . To effectively study this interaction:

1. Biochemical Interaction Studies:

• Surface plasmon resonance or isothermal titration calorimetry to determine binding kinetics and affinity

• Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

• Mutagenesis studies targeting predicted interaction sites to identify critical residues

2. Functional Assays:

• Reconstituted ubiquitination assays with purified components

• Cellular depletion of UBE2J1 in RNF133-expressing cells to assess functional dependency

• Comparative analysis of UBE2J1 KO and RNF133 KO phenotypes to identify shared versus unique functions

3. Structural Biology Approaches:

• Co-crystallization or cryo-EM studies of the RNF133-UBE2J1 complex

• NMR studies of domain interactions between the proteins

• Molecular dynamics simulations to understand the dynamic nature of the interaction

4. Cellular Context Studies:

• Proximity ligation assays to visualize and quantify interactions in situ

• FRET-based interaction studies in living cells

• Co-localization studies during different stages of spermatogenesis

5. Substrate Processing Analysis:

• Identification of ERAD substrates processed by the RNF133-UBE2J1 complex

• Kinetic analysis of substrate degradation dependent on both proteins

• Comparative ubiquitination profiles of wild-type, RNF133 KO, and UBE2J1 KO cells

What methodological considerations are important when analyzing RNF133 expression in human patient samples?

When analyzing RNF133 expression in human clinical samples, researchers should consider several methodological aspects:

1. Sample Collection and Processing:

• Standardized protocols for testicular biopsy collection and preservation

• Rapid processing to minimize RNA and protein degradation

• Careful microdissection to isolate specific seminiferous tubule stages

2. Expression Analysis Methods:

• Quantitative RT-PCR with carefully validated reference genes for normalization

• Immunohistochemistry using validated antibodies with appropriate positive and negative controls

• In situ hybridization to detect mRNA at the cellular level

• Western blotting with recombinant RNF133 as a positive control

3. Patient Stratification:

• Detailed clinical information including fertility status and semen parameters

• Histological assessment of spermatogenesis in the same sample

• Age-matched controls to account for age-related changes in spermatogenesis

4. Interpretative Considerations:

• Correlation of RNF133 expression with specific infertility diagnoses

• Analysis of potential genetic variants in RNF133 that may affect expression or function

• Consideration of other ER quality control components in the same samples

5. Ethical and Practical Limitations:

• Limited availability of human testicular tissue necessitating careful experimental design

• Use of archived samples with consideration of preservation method effects on RNA/protein quality

• Development of non-invasive methods to infer RNF133 function, such as analysis of sperm phenotypes

What emerging technologies could advance our understanding of RNF133 function?

Several emerging technologies offer promising approaches to deepen our understanding of RNF133:

1. Advanced Genome Editing:

• Base editing or prime editing to create specific point mutations in RNF133 without disrupting the entire gene

• CRISPR activation/inhibition systems for temporal control of RNF133 expression

• CRISPR screens to identify synthetic lethal interactions with RNF133 in testicular cells

2. Single-Cell Technologies:

• Single-cell RNA sequencing to identify cell populations affected by RNF133 deletion

• Single-cell proteomics to analyze protein changes in specific spermatogenic cell types

• Spatial transcriptomics to map RNF133 expression within the architectural context of the testis

3. Advanced Imaging:

• Super-resolution microscopy to visualize RNF133 distribution in subcellular compartments

• Live-cell imaging of fluorescently tagged RNF133 during spermatogenesis

• Label-free imaging techniques to study RNF133 function in native contexts

4. Systems Biology Approaches:

• Multi-omics integration (transcriptomics, proteomics, metabolomics) to build comprehensive models of RNF133 function

• Network analysis to position RNF133 within the broader context of ER quality control

• Mathematical modeling of ubiquitination kinetics by the RNF133-UBE2J1 complex

5. Organoid and Advanced Cell Culture Models:

• Testicular organoids to study RNF133 function in a more physiologically relevant context

• Microfluidic testis-on-a-chip systems for dynamic studies of RNF133 function

• Co-culture systems to examine cell-cell interactions mediated by RNF133

How might research on RNF133 inform our broader understanding of ER-associated protein degradation in specialized cells?

Research on RNF133 provides a valuable model for understanding tissue-specific adaptations of general cellular processes:

Conceptual Framework:

• Tissue-Specific ERAD Components:

  • Investigation of how ubiquitous processes like ERAD are adapted for tissue-specific functions

  • Identification of other tissue-specific E3 ligases involved in specialized ERAD pathways

  • Comparative analysis of ERAD machinery across different specialized cell types

• Developmental Regulation:

  • Analysis of how ERAD components are regulated during cellular differentiation

  • Investigation of transcription factors controlling tissue-specific expression of ERAD components

  • Temporal coordination of ERAD machinery with developmental processes

• Substrate Specificity Determinants:

  • Identification of features that direct specific substrates to tissue-specific versus general ERAD pathways

  • Investigation of whether tissue-specific E3 ligases recognize unique degrons or structural features

  • Development of prediction algorithms for substrate-E3 ligase pairing in specialized cells

• Evolutionary Adaptations:

  • Comparative analysis of specialized ERAD components across species with different reproductive strategies

  • Investigation of whether RNF133-like adaptations exist in other specialized cell types

  • Understanding how general cellular processes diverge to support specialized functions during evolution

Data Table: Comparison of RNF133 and Related E3 Ubiquitin Ligases in Reproductive Biology