RNF34 Human

What is the molecular structure of RNF34 and what functional domains does it contain?

RNF34 is an E3 ubiquitin-protein ligase containing a RING finger motif, which is crucial for its protein-protein and protein-DNA interactions . Unlike the IAP family proteins which contain BIR domains, RNF34 features an FYVE domain with phospholipid-binding activity . This domain is thought to mediate the partial plasma membrane localization of the protein . RNF34 also contains a caspase-interacting domain that enables it to bind to and ubiquitinate caspases 8 and 10 .

To investigate RNF34 structure-function relationships, researchers typically employ:

• Deletion mutant construction to identify key functional domains

• Site-directed mutagenesis (e.g., the H342A mutation creates an E3 ligase-dead variant)

• Yeast two-hybrid screening to map protein interaction domains

• Recombinant protein expression and purification for in vitro functional assays

What is the subcellular localization of RNF34 and how can it be detected in different cellular compartments?

Immunohistochemical studies reveal that RNF34 is present in multiple cellular compartments including the nucleus, cytoplasm, and cell membrane . In clear cell renal cell carcinoma (ccRCC) samples, all three localizations have been observed, and each pattern can have distinct clinical correlations .

For studying RNF34 subcellular localization, researchers should:

• Use immunohistochemistry with specific anti-RNF34 antibodies for tissue samples

• Employ cellular fractionation followed by Western blotting for biochemical analysis

• Consider using affinity-purified antibodies (e.g., Rb anti-RNF34 antibody) that have been validated for developmental studies

• Apply confocal microscopy with appropriate markers to confirm mitochondrial localization when studying its interaction with MAVS

What are the most effective knockdown strategies for studying RNF34 function?

Based on the research literature, several effective RNF34 knockdown strategies have been developed:

• siRNA approach: Multiple siRNA sequences targeting different regions of RNF34 mRNA have been validated. In particular, siRNF34-1 and siRNF34-3 showed substantial knockdown efficiency and functional effects in THP-1 cells .

• shRNA stable knockdown: Researchers have generated stable knockdown cell lines using shRNAs targeting:

  • The coding region (nucleotides 587-607) of rat RNF34 (designated Sh1)

  • The 3′-UTR (nucleotides 1623-1643) of rat RNF34 (designated Sh2)

• Rescue experiments: For validation of target specificity, construct rescue vectors lacking the 3'-UTR to restore RNF34 expression in cells treated with UTR-targeting shRNAs .

The research evidence shows that different knockdown efficiencies may yield varying functional outcomes. For instance, in antiviral studies, shRNF34-1 and shRNF34-3 clones (with >90% reduction in RNF34 expression) displayed lower VSV titers than control cells, while shRNF34-2 (with less knockdown efficiency) did not show significant effects .

What techniques are most suitable for studying RNF34's E3 ubiquitin ligase activity?

To effectively study RNF34's E3 ubiquitin ligase activity, researchers should consider the following methodological approaches:

• Mutational analysis: The RNF34 H342A mutant serves as an E3 ligase-dead control, showing compromised inhibition of VSV-mediated IFN-β and NF-κB activation compared to wild-type RNF34 .

• Co-immunoprecipitation: To detect protein-protein interactions between RNF34 and potential substrates, such as PGC-1α or caspases 8 and 10 .

• Ubiquitination assays: In vitro and cell-based assays to detect ubiquitin transfer to substrates. For example, research has shown that RNF34 promotes the ubiquitination of unfolded proteins by directly recognizing NBD1 domains .

• Functional readouts: Measure the degradation of known substrates (e.g., PGC-1α) in the presence and absence of RNF34, or with wild-type versus mutant RNF34 .

• Proteasome inhibition experiments: To determine whether RNF34-mediated ubiquitination leads to proteasomal degradation (K48-linked) or serves signaling functions (K63-linked) .

How does RNF34 expression correlate with clinical outcomes in cancer patients?

Extensive research using The Cancer Genome Atlas (TCGA) data has established strong correlations between RNF34 expression and clinical outcomes in cancer patients, particularly in clear cell renal cell carcinoma (ccRCC):

This multivariate analysis confirms RNF34 expression as an independent prognostic parameter, even after adjusting for TNM stage parameters and grading .

What molecular mechanisms explain RNF34's role in cancer progression?

Research has identified several key molecular mechanisms through which RNF34 contributes to cancer progression:

• Inhibition of apoptosis: RNF34 exhibits antiapoptotic properties by ubiquitinating caspases 8 and 10, leading to their proteasomal degradation. This inhibits death receptor-mediated apoptosis, allowing cancer cells to evade programmed cell death .

• Degradation of tumor suppressors: RNF34 participates in the proteasomal degradation of the tumor suppressor protein p53 and phosphorylated p53, further promoting oncogenic potential .

• Cell death pathway regulation: In colorectal carcinogenesis, RNF34 overexpression exerts a negative impact on cell death signaling pathways .

• Subcellular localization effects: Different subcellular localizations of RNF34 (nuclear, cytoplasmic, and membranous) have distinct prognostic implications in ccRCC, suggesting compartment-specific functions .

To investigate these mechanisms, researchers typically employ:

• Protein-protein interaction studies between RNF34 and its targets

• Ubiquitination assays with specific substrates

• Cell viability and apoptosis assays following RNF34 manipulation

• Immunohistochemistry to determine subcellular localization patterns in patient samples

How does RNF34 contribute to neurological deficits following intracerebral hemorrhage?

Research using RNF34 transgenic mice has provided significant insights into RNF34's role in exacerbating neurological deficits following intracerebral hemorrhage (ICH):

• Aggravation of brain injury: RNF34 overexpression significantly worsened multiple aspects of ICH-induced brain damage, including:

  • Memory impairment

  • Brain edema

  • Infarction area

  • Hematoma volume

  • Loss of neuronal activity

• Temporal expression pattern: RNF34 and oxidative stress levels gradually increased from 6 to 48 hours after ICH challenge and were positively correlated, suggesting a time-dependent role in pathophysiology .

• Oxidative stress mechanisms: RNF34 upregulation exacerbated several aspects of oxidative stress:

  • Increased intracellular reactive oxygen species (ROS)

  • Enhanced superoxide anion generation

  • Elevated mitochondrial ROS (mROS) production

  • Decreased adenosine triphosphate (ATP) production

• Molecular pathway: The primary mechanism involves RNF34-mediated degradation of PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha), which leads to decreased expression of downstream protective factors:

  • UCP2 (uncoupling protein 2)

  • MnSOD (manganese superoxide dismutase)

These findings reveal that RNF34 exacerbates neurological deficits and brain injury by facilitating PGC-1α protein degradation and promoting mitochondrial dysfunction-mediated oxidative stress .

What experimental models and techniques are optimal for studying RNF34 in neurological contexts?

Based on the research literature, several experimental approaches have proven effective for studying RNF34 in neurological contexts:

• Animal models:

  • RNF34 transgenic mice to study gain-of-function effects

  • ICH model established via intracerebral injection of autologous blood into wild-type and RNF34 transgenic mice

• Neurological assessments:

  • Memory and cognitive function tests

  • Motor coordination evaluation

  • Quantification of brain edema, infarction, and hematoma volume

• Cellular studies:

  • Primary neuronal cultures from wild-type and RNF34 transgenic mice

  • Oxygen-glucose deprivation models to simulate ischemic conditions

• Molecular analyses:

  • Co-immunoprecipitation to detect RNF34-PGC-1α interaction

  • Ubiquitination assays to assess RNF34-mediated PGC-1α degradation

  • Western blotting for PGC-1α, UCP2, and MnSOD quantification

• Oxidative stress and mitochondrial function measurements:

  • Intracellular ROS detection assays

  • Superoxide anion quantification

  • ATP production measurement

  • Mitochondrial membrane potential analysis

These approaches allow comprehensive investigation of RNF34's role in neurological disorders, from behavioral phenotypes to underlying molecular mechanisms.

How does RNF34 regulate antiviral immune responses?

Research has identified RNF34 as a negative regulator of antiviral immune responses through its interaction with key components of the RIG-I-like receptor (RLR) signaling pathway:

• MAVS interaction: RNF34 binds to MAVS (mitochondrial antiviral signaling protein) in the mitochondrial compartment after viral infection .

• Suppression of interferon production:

  • VSV-induced IFN-β and IL6 secretion was substantially increased in THP-1 cells with RNF34 knockdown

  • RNF34 overexpression substantially decreased IFN-β and NF-κB promoter activity in response to VSV infection

  • These effects were dependent on RNF34's E3 ligase activity, as the H342A mutant showed compromised inhibition

• Impact on viral replication:

  • Stable RNF34 knockdown clones (shRNF34-1 and shRNF34-3) displayed lower VSV titers than control cells

  • NDV-GFP replication was substantially reduced in RNF34 knockdown cells

These findings suggest that RNF34 functions as a checkpoint in antiviral immunity, potentially limiting excessive inflammatory responses but potentially also being exploited by viruses to evade host defenses.

Methodologically, researchers investigating RNF34's role in immunity typically employ:

• Viral infection models (VSV, NDV-GFP)

• Measurements of cytokine production (IFN-β, IL-6)

• Reporter assays for signaling pathway activation (IFN-β and NF-κB promoters)

• Viral titer quantification

• MAVS-RNF34 interaction studies

How does RNF34 interact with other ubiquitin ligases in immune regulation?

While the search results don't provide comprehensive information on RNF34's interaction with other ubiquitin ligases in immune regulation, we can extract some relevant insights:

• Context within E3 ligase networks: The search results mention that "multiple RING-finger-containing E3 ligases have been reported to be involved in RLR signaling pathways" , suggesting RNF34 functions within a network of E3 ligases regulating antiviral immunity.

• Comparison with IAP family: RNF34 is described as an "IAP-like protein" but differs from the IAP family in key ways:

  • IAPs bind to and ubiquitinate caspase-3, caspase-7, and caspase-9 via the BIR domain

  • RNF34 mediates ubiquitination of caspases 8 and 10 via its caspase-interacting domain

  • Both act as antiapoptotic proteins but through different mechanisms

• Potential cooperative activity: In CFTR regulation, RNF34 works alongside another E3 ligase called RFFL:

  • Both are localized in the cytoplasm and endosomes

  • Their combined ablation dramatically increases functional PM ∆F508-CFTR by inhibiting ubiquitination in post-Golgi compartments

  • This suggests potential cooperative or complementary roles in certain contexts

For researchers investigating these interactions, recommended approaches include:

• Proteomic analysis of RNF34-containing complexes

• Co-immunoprecipitation studies with other known E3 ligases

• Dual knockdown/knockout experiments to identify synergistic effects

• Substrate specificity comparisons between RNF34 and other E3 ligases