Recombinant Rat E3 ubiquitin-protein ligase RNF133 (Rnf133)

What is E3 ubiquitin-protein ligase RNF133?

RNF133 is a testis-specific E3 ubiquitin ligase containing a RING finger domain, a motif involved in protein-protein and protein-DNA interactions. The RNF133 gene has no introns and encodes a 376 amino acid protein with a molecular weight of approximately 42.3 kDa. This protein plays a critical role in the ubiquitination pathway, which tags proteins for degradation or modification during spermatogenesis .

What is the molecular structure and domain organization of RNF133?

RNF133 contains a transmembrane domain followed by a RING finger domain that extends into the cytoplasm. In silico prediction analysis reveals that RNF133 is localized to the endoplasmic reticulum (ER), which has been confirmed through immunostaining of recombinantly expressed human RNF133. The protein contains a C3H2C3 RING finger domain characteristic of E3 ubiquitin ligases, allowing it to function in ER quality control during spermatogenesis .

How is the expression of RNF133 regulated during spermatogenesis?

RT-PCR analysis of mouse testes at different developmental stages reveals that RNF133 expression begins around postnatal day 25, corresponding to the transition of round spermatids to elongating spermatids. This temporal expression pattern suggests RNF133 functions specifically during the later stages of spermiogenesis. Both mouse and human RNF133 show strict testis-specific expression across reproductive and non-reproductive tissues, indicating its specialized role in male germ cell development .

What are the consequences of RNF133 deficiency on male fertility?

Studies utilizing CRISPR/Cas9-generated RNF133 knockout mice demonstrate that RNF133 deficiency leads to severe subfertility in males. RNF133 knockout males produced significantly fewer offspring (2.3 ± 1.6 pups per litter) compared to heterozygous controls (8.6 ± 0.7 pups per litter). More critically, the pregnancy success rate for RNF133 knockout males was dramatically reduced to 10.0 ± 10% compared to 100% for heterozygous males, with knockout males fathering significantly fewer pups per copulation plug (0.15 ± 0.15 vs. 8.4 ± 0.24) .

How does RNF133 affect sperm morphology and function?

RNF133 deficiency results in abnormal sperm morphology and significantly reduced motility. Ultrastructural analysis reveals that cytoplasmic droplets are retained in RNF133 knockout spermatozoa, indicating defects in cytoplasmic removal during sperm maturation. These morphological abnormalities directly impact sperm function, as demonstrated by fertilization experiments. While sperm from heterozygous controls fertilized 83.9 ± 8.2% of oocytes in vitro, sperm from RNF133 knockout mice only fertilized 6.8 ± 3.0% of oocytes, resulting in dramatically fewer 2-cell stage embryos (1.4 ± 0.7% vs. 73.1 ± 10.0%) .

What specific defects in sperm motility are observed in RNF133-deficient mice?

RNF133 knockout sperm exhibit significant defects in hyperactivation, a specialized motility pattern essential for successful fertilization. When incubated in capacitation medium, sperm from heterozygous controls showed a tenfold increase in hyperactivation from 0.5 ± 0.3% at 15 minutes to 5.2 ± 1.7% after 120 minutes. In contrast, RNF133 knockout sperm showed minimal change in hyperactivation (0.7 ± 0.4% at 15 minutes to 1.3 ± 0.4% at 120 minutes). This defect in acquiring hyperactivated motility likely contributes substantially to the reduced fertilization capacity observed in RNF133 knockout males .

How does RNF133 function in the ubiquitin-proteasome pathway?

As an E3 ubiquitin ligase, RNF133 facilitates the transfer of ubiquitin to target proteins, marking them for degradation via the proteasome. RNF133 is specifically involved in endoplasmic reticulum (ER) quality control, participating in the recognition and degradation of proteins no longer needed in the ER. Its transmembrane domain anchors it to the ER membrane, allowing it to access both ER lumen and cytosolic compartments to regulate protein homeostasis during spermatogenesis. This positioning is crucial for its role in maintaining proper protein turnover during the dramatic cellular remodeling that occurs during sperm development .

What proteins interact with RNF133 and how do these interactions affect its function?

RNF133 has been identified to interact with UBE2J1, an E2 ubiquitin-conjugating enzyme that is also essential for spermiogenesis. Both proteins are ER transmembrane proteins, suggesting they function cooperatively in ER quality control during spermatogenesis. UBE2J1 knockout mice display defects in the dislocation step of ER quality control, and electron microscopy analysis reveals excess residual cytoplasm in the head and midpiece of sperm, similar to the phenotype observed in RNF133 knockout mice. This interaction between RNF133 (an E3 ligase) and UBE2J1 (an E2 enzyme) represents a critical functional pairing within the ubiquitination machinery that regulates spermatid development .

What is the relationship between RNF133 and its paralog RNF148?

RNF133 and RNF148 are paralogous genes that are chromosomally linked and encode putative ER transmembrane E3 ubiquitin-protein ligases with similar domain structures. They share significant sequence identity (58.9% in mouse and 54.9% in human) and both contain a transmembrane region followed by a RING finger domain. Despite their structural similarity, studies with double knockout mice (RNF133/RNF148 DKO) have demonstrated limited functional redundancy. The subfertility phenotype of double knockout males remained similar to that of RNF133 single knockout males (2.5 ± 0.15 pups per litter), suggesting that RNF148 cannot compensate for the loss of RNF133 despite their structural similarities .

What experimental approaches are most effective for studying RNF133 function?

Multiple complementary approaches have proven effective for studying RNF133 function:

• Gene knockout studies: CRISPR/Cas9-generated knockout mice provide a powerful system for analyzing RNF133's role in fertility in vivo.

• Expression analysis: RT-PCR across tissues and developmental stages reveals tissue specificity and temporal expression patterns critical for understanding when and where RNF133 functions.

• Fertility assessment: Detailed mating studies including pregnancy rate, litter size, and timed matings provide quantitative measures of fertility impacts.

• Sperm functional analysis: Computer-assisted sperm analysis (CASA) allows precise measurement of motility parameters and hyperactivation rates.

• Fertilization assays: Both in vitro and in vivo fertilization studies provide direct evidence of sperm fertilization capacity.

• Ultrastructural analysis: Electron microscopy enables examination of sperm morphological defects at the subcellular level .

How can recombinant RNF133 be optimally produced for functional studies?

For functional studies of recombinant RNF133, several expression systems can be employed, each with specific advantages:

• Mammalian cell expression: HEK293 cells provide the most physiologically relevant system for expressing full-length RNF133 with proper post-translational modifications and membrane insertion. This system is particularly important for studying the intact transmembrane protein.

• Bacterial expression: E. coli systems can be used for producing the soluble cytoplasmic domains of RNF133, particularly the RING finger domain for structural studies and in vitro ubiquitination assays.

• Purification strategies: Affinity tags such as His-tags, Avi-tags, or Fc-fusion tags facilitate purification and detection of recombinant RNF133. For the full-length transmembrane protein, detergent-based purification protocols are necessary to maintain protein stability and functionality .

What techniques are available for identifying RNF133 substrates during spermatogenesis?

Identifying the physiological substrates of RNF133 is crucial for understanding its role in spermatogenesis. Several approaches can be employed:

• Proximity labeling: BioID or TurboID fusion proteins can identify proteins in close proximity to RNF133 within the cellular environment.

• Ubiquitinome analysis: Comparative proteomic analysis of ubiquitinated proteins in wild-type versus RNF133 knockout testes can reveal potential substrates.

• Co-immunoprecipitation: Antibody-based pulldown of RNF133 followed by mass spectrometry can identify interacting proteins, including potential substrates.

• Yeast two-hybrid screening: This approach can identify direct protein-protein interactions between RNF133 and potential substrates.

• In vitro ubiquitination assays: Reconstituted systems using purified components can verify direct ubiquitination of candidate substrates by RNF133 .

What are the implications of RNF133 research for male contraceptive development?

RNF133's testis-specific expression and critical role in male fertility position it as a promising target for non-hormonal male contraceptive development. The severe subfertility observed in RNF133 knockout males (90% reduction in fertility) suggests that pharmacological inhibition of its function could effectively reduce male fertility without affecting hormone levels, potentially avoiding side effects associated with hormonal contraceptives. Additionally, RNF133 could potentially serve as a platform for targeted protein degradation approaches like PROTACs (Proteolysis-Targeting Chimeras) to selectively degrade other testis-specific proteins essential for fertility, as indicated by its classification as a potential contraceptive target in recent research .

How might RNF133 dysfunction contribute to specific forms of male infertility?

Understanding RNF133's role in spermatogenesis provides insights into specific forms of male infertility characterized by:

• Morphological abnormalities: Particularly retention of cytoplasmic droplets, which is observed in both RNF133 knockout mice and some cases of human male infertility.

• Motility defects: The significant impairment of hyperactivation in RNF133-deficient sperm suggests that mutations or dysregulation of RNF133 could contribute to asthenozoospermia (reduced sperm motility) in humans.

• Failed capacitation: The inability of RNF133 knockout sperm to undergo proper capacitation suggests that RNF133 dysfunction could be involved in cases where sperm fail to acquire fertilization competence.

• ER stress-related infertility: Given RNF133's role in ER quality control, it may be implicated in cases of male infertility associated with ER stress during spermatogenesis .

What comparative insights can be gained from studying RNF133 across species?

Comparative studies of RNF133 across species provide valuable evolutionary and functional insights:

• Evolutionary conservation: The high conservation of RNF133 across mammals suggests its fundamental importance in reproduction and potential as a broadly applicable target for reproductive interventions.

• Functional divergence: Studying the functional relationship between RNF133 and its paralog RNF148 across species can reveal how gene duplication events have led to specialization or redundancy in reproductive processes.

• Species-specific adaptations: Variations in RNF133 structure and regulation across species with different reproductive strategies may highlight adaptive changes that could inform synthetic approaches to modulating fertility.

• Translational relevance: Similarities between rodent and human RNF133 function suggest that findings from mouse models may translate effectively to human reproductive biology, enhancing our understanding of male infertility mechanisms .