Recombinant Mouse E3 ubiquitin-protein ligase RNF133 (Rnf133)

What is the function of E3 ubiquitin-protein ligase RNF133 in mice?

RNF133 is a testis-specific E3 ubiquitin ligase that plays a critical role in male fertility and spermiogenesis. It functions as part of the endoplasmic reticulum (ER) quality control system that recognizes and degrades proteins through the ubiquitin-proteasome pathway. Knockout studies have demonstrated that RNF133 is essential for normal sperm morphology, motility, and function, with male mice lacking RNF133 displaying severe subfertility .

What is the structural composition of RNF133?

RNF133 contains one transmembrane region and one RING finger domain positioned after the transmembrane region, with this domain localized in the cytoplasm. In silico protein structure prediction confirms these domains, and immunostaining studies have shown that recombinantly expressed human RNF133 localizes to the endoplasmic reticulum (ER) . This structural arrangement is consistent with its proposed function in ER-associated degradation (ERAD) .

What is the expression pattern of RNF133 during spermatogenesis?

RT-PCR analysis of mouse testes at different postnatal days reveals that RNF133 expression begins around postnatal day 25, which corresponds to the period when round spermatids are transitioning to elongating spermatids. This specific temporal expression pattern suggests that RNF133 functions primarily during the later stages of spermiogenesis, particularly in sperm maturation and cytoplasmic remodeling processes .

What experimental approaches are most effective for studying RNF133 function in vivo?

CRISPR/Cas9-mediated gene knockout in mice has proven to be an effective approach for studying RNF133 function. The generation of specific single-guide RNAs (sgRNAs) targeting the RNF133 gene, followed by phenotypic analysis of the resulting knockout mice, provides valuable insights into the physiological role of this protein. Recommended assessments include:

• Fertility testing through timed matings

• In vitro and in vivo fertilization assays

• Analysis of sperm parameters (count, morphology, motility)

• Ultrastructural examination of sperm using electron microscopy

Comparative analysis between RNF133 heterozygous and knockout mice has revealed significant differences in pregnancy success rates and number of pups per plug, demonstrating the crucial role of RNF133 in male fertility .

How should researchers design appropriate controls when studying RNF133?

When investigating RNF133 function, researchers should implement the following control strategy:

• Use heterozygous (HET) littermates as primary controls rather than wild-type mice

• Include both in vitro and in vivo fertility assessments to comprehensively evaluate sperm function

• Consider generating and analyzing RNF133/RNF148 double knockout (DKO) mice to assess potential functional redundancy

• Include temporal expression analysis across multiple developmental timepoints (P5-P60) to establish precise expression patterns

This approach has successfully demonstrated that while RNF133 KO males showed only 10.0 ± 10% pregnancy success compared to 100% in HET controls, combined deletion of paralogous RNF148 did not significantly worsen this phenotype, indicating limited functional compensation .

What methodologies are recommended for recombinant RNF133 protein expression and purification?

For effective recombinant RNF133 protein production, researchers should consider:

• Expression in mammalian cell systems (particularly HEK293 cells) rather than bacterial systems to ensure proper protein folding and post-translational modifications

• Inclusion of appropriate epitope tags (His, Avi, or Fc) to facilitate purification without disrupting protein function

• Verification of proper subcellular localization to the ER membrane through immunofluorescence

• Careful buffer optimization during purification to maintain the integrity of the transmembrane domain

When expressing recombinant RNF133, it's important to verify that the protein localizes correctly to the ER, as mislocalization may lead to artifacts in functional studies .

How does RNF133 interact with the E2 ubiquitin-conjugating enzyme UBE2J1?

RNF133 has been identified as an interaction partner for UBE2J1, an ER-localized E2 ubiquitin-conjugating enzyme also essential for spermiogenesis. Both proteins contain transmembrane domains and localize to the ER, suggesting they function as a complex in ERAD during spermatogenesis. The RNF133-UBE2J1 interaction represents a crucial ubiquitin-regulatory system for protein quality control during sperm development .

To investigate this interaction further, researchers should consider:

• Co-immunoprecipitation studies to confirm physical interaction

• In vitro ubiquitination assays to demonstrate functional cooperation

• Identification of shared substrates through comparative proteomic analysis of single and double knockout models

What is the relationship between RNF133 and its paralog RNF148?

Despite sharing 58.9% and 54.9% sequence identity in mouse and human respectively, RNF133 and RNF148 display limited functional redundancy. While RNF133 knockout males exhibit severe subfertility, RNF148 knockout males retain normal fertility. Both proteins contain similar domain structures (transmembrane domain and RING finger domain) and are chromosomally linked, suggesting they arose from gene duplication .

This presents an interesting evolutionary question regarding their functional divergence. Analysis of their expression patterns shows they are both expressed around postnatal day 25, corresponding to the round-to-elongating spermatid transition phase, yet they appear to have distinct functions in spermatogenesis .

How does deletion of RNF133 affect sperm ultrastructure and function at the molecular level?

Ultrastructural analysis of RNF133 knockout sperm reveals retention of cytoplasmic droplets, suggesting defects in the final remodeling steps of spermiogenesis. These structural abnormalities likely contribute to the observed reduced motility and fertilization capacity .

At the molecular level, this phenotype is consistent with impaired ERAD function, similar to that observed in UBE2J1 knockout mice. The retention of cytoplasmic components suggests a failure to properly eliminate unnecessary proteins and organelles during sperm maturation, a process that typically relies on ubiquitin-mediated degradation .

How should researchers interpret fertility data from RNF133 knockout studies?

When analyzing fertility data from RNF133 knockout mice, researchers should consider:

This dramatic reduction in fertility parameters indicates that RNF133 is crucial for sperm function, with its absence creating defects that cannot be compensated by other ubiquitin ligases, including its paralog RNF148 .

What contradictions exist in the current understanding of RNF133 function?

Several research questions remain unresolved regarding RNF133 function:

• Despite structural similarities between RNF133 and RNF148, they exhibit remarkably different physiological requirements, with only RNF133 being essential for fertility

• The specific substrates of RNF133 during spermatogenesis remain unidentified, making it difficult to establish direct molecular mechanisms

• The retention of cytoplasmic droplets in knockout sperm suggests defects in cytoplasmic elimination, yet the specific ubiquitinated proteins involved in this process are unknown

These contradictions highlight the need for further research into the specific molecular mechanisms and substrates of RNF133 .

What are the optimal approaches for identifying RNF133 substrates during spermatogenesis?

To identify physiological substrates of RNF133, researchers should consider a multi-faceted approach:

• Comparative proteomic analysis of testicular tissue from wild-type and RNF133 knockout mice at specific developmental stages

• Proximity-based labeling techniques (BioID or TurboID) using RNF133 as bait to identify proteins in close proximity

• Ubiquitin remnant profiling to identify ubiquitination sites that are reduced in RNF133 knockout tissues

• In vitro ubiquitination assays with recombinant RNF133, UBE2J1, and candidate substrates

These approaches would help identify proteins that accumulate in the absence of RNF133-mediated ubiquitination and degradation, providing insights into its role in sperm development .

How can researchers effectively target RNF133 for contraceptive development?

Given the testis-specific expression of RNF133 and its essential role in male fertility, it represents a potential non-hormonal male contraceptive target. Research approaches should include:

• Development of small-molecule inhibitors of the RNF133 RING domain activity

• Exploration of PROTACs (Proteolysis Targeting Chimeras) technology to degrade RNF133

• Assessment of reversibility by evaluating sperm function after drug withdrawal

• Comparative analysis with other testis-specific E3 ligases (RNF148, RNF151, ZSWIM2) to identify the most promising targets

This approach aligns with the emerging interest in developing non-hormonal male contraceptives that specifically target testis-expressed proteins essential for fertility .

What techniques should be employed to study the evolutionary conservation of RNF133 across species?

RNF133 is conserved across mammalian species, including mice, humans, and domestic cats. To study its evolutionary significance:

• Perform comparative sequence analysis across species to identify conserved domains and motifs

• Analyze expression patterns in reproductive tissues of different mammals

• Determine if the RNF133-UBE2J1 interaction is conserved across species

• Investigate whether the male fertility requirement is maintained in other animal models

Understanding the evolutionary conservation of RNF133 would provide insights into its fundamental role in mammalian reproduction and help identify functionally critical regions of the protein .