Recombinant Mouse Probable E3 ubiquitin-protein ligase RNF144A (Rnf144a)

What is the domain organization of RNF144A and how does it influence its function?

RNF144A contains four key domains: RING1, In Between Ring (IBR), RING2, and a transmembrane (TM) domain with a tail anchor (TA) . The RING domains are cytoplasmically oriented, allowing interactions with intracellular proteins . The TM domain plays a dual regulatory role through:

• Membrane localization: The C-terminal membrane tail-anchor domain localizes RNF144A to the plasma membrane

• E3 ligase activation: The GXXXG motif (G252XXXG256) within the TM domain mediates self-association and is critical for ubiquitin ligase activity

Deletion of the TM domain abolishes membrane localization and significantly reduces ubiquitin ligase activity . Mutations in the GXXXG motif (G252L/G256L) preserve membrane localization but are defective in self-association and ligase activity .

How does RNF144A function as an E3 ubiquitin ligase?

RNF144A functions as a RING-HECT hybrid E3 ubiquitin ligase belonging to the RBR (RING1-IBR-RING2) family . It can operate with multiple E2 enzymes including UbcH7 and UbcH5a . Key functional characteristics include:

• Requires both RING1 domain and TM domain for autoubiquitination activity

• Exhibits both monoubiquitination in vitro and heavy ubiquitination in vivo

• Unlike other RBR family members, RNF144A is specifically induced by IL-2 in a JAK-dependent and STAT5-regulated manner

• Targets specific proteins (like RAF1) for proteasomal degradation to regulate signaling pathways

What are effective strategies for generating RNF144A knockout models?

Researchers have successfully generated RNF144A knockout models using CRISPR/Cas9 technology, with two documented approaches:

Approach 1: Target the first coding exon, resulting in loss of the RING1 domain and a frameshift with premature translation stop codon in all transcript isoforms . This approach produced viable but growth-deficient mice.

Approach 2: Complete knockout of RNF144A expression at the protein level in both cells and organs . This model showed increased susceptibility to viral infections and impaired immune responses.

When designing knockout experiments, consider:

• Confirming knockout efficiency at both mRNA and protein levels

• Examining multiple tissues/cell types due to differential expression

• Accounting for potential compensatory mechanisms from related proteins like RNF144B

• Including proper controls as RNF144A-deficient mice show growth deficiency that might influence experimental outcomes

What methodologies are effective for studying RNF144A subcellular localization and membrane association?

To analyze RNF144A's membrane association and subcellular localization, researchers have successfully employed:

• Membrane flotation sucrose gradient assays: Used to confirm the requirement of the TA domain for plasma membrane localization

• Immunofluorescence studies: Applied to visualize subcellular distribution and confirm membrane association

• Deletion and mutation analysis: Generating constructs like:

  • GST-RNF144A-WT (a.a. 1–292)

  • GST-RNF144A-RING1 mutant (C20A/C23A)

  • GST-RNF144A-ΔTM (Δa.a. 250–270)

  • GST-RNF144A-RBR mutant (a.a. 1–229)

• Site-directed mutagenesis: Particularly of the GXXXG motif (G252L/G256L) to study the role of self-association while preserving membrane localization

These approaches allow researchers to distinguish between effects caused by altered localization versus effects on protein-protein interactions or enzymatic activity.

How does RNF144A regulate IL-2 signaling pathways in T cells?

RNF144A orchestrates IL-2 receptor (IL-2R) signaling through dual mechanisms that create a signaling hierarchy :

• Enhancement of JAK-STAT5 signaling:

  • Promotes the association between IL-2Rβ and STAT5

  • Increases STAT5 phosphorylation and activation

  • Upregulates IL-2-induced effector genes including TNF and granzymes

• Restriction of RAF-ERK-MAPK signaling:

  • Directly targets RAF1 for polyubiquitination and proteasomal degradation

  • Prevents formation of the potent RAF1/BRAF kinase complex

  • Limits IL-2-induced RAF-ERK1/2 activation

This dual regulation creates a balanced output from the IL-2R, which is critical for proper T cell function. RNF144A knockout mice exhibit dysregulated signal output with diminished STAT5 and elevated ERK phosphorylation .

What is the role of RNF144A in antiviral signaling pathways?

RNF144A positively regulates DNA virus-triggered or exogenous cytosolic DNA-triggered innate immune responses through several mechanisms :

• Upregulation of antiviral gene expression:

  • Enhances expression of IFNB, CXCL10, CCL5, and TNF in response to HSV-1 infection

  • Increases production of IFN-β and TNF-α at protein levels

• Promotion of STING pathway activation:

  • Facilitates phosphorylation of STING, IRF3, and p65 upon DNA virus infection

  • Shows specificity for cytosolic DNA sensing pathways (not RNA sensing)

• Restriction of viral replication:

  • RNF144A overexpression restricts HSV-1 infection

  • RNF144A knockdown promotes HSV-1 infection

Importantly, RNF144A appears to be induced by HSV-1 infection or viral DNA stimulation, suggesting a positive feedback mechanism in antiviral defense .

What are the consequences of RNF144A deficiency in vivo?

RNF144A-deficient mice display several significant phenotypes that reveal its physiological importance:

• Growth and development:

  • Runted appearance and growth deficiency

  • Normal fat and lean body composition (proportionally small)

• Immune function:

  • CD8+ T cell immunodeficiency

  • Diminished cytotoxic T lymphocyte (CTL) function

  • Impaired IL-2-induced effector gene expression

• Response to viral infection:

  • Increased susceptibility to HSV-1 infection

  • Greater body weight loss and lower survival rates

  • More severe lung damage after HSV-1 infection

  • Widespread inflammation in response to influenza infection

• Molecular signaling:

  • Decreased STAT5 activation

  • Increased ERK activation

  • Reduced antiviral immune responses in various organs

Interestingly, RNF144A-deficient mice show no gross phenotypes under sterile homeostatic conditions aside from the runted appearance, suggesting its functions become critical during immune challenge .

What is the relationship between RNF144A expression and disease severity in humans?

In human patients, RNF144A expression shows significant correlation with disease severity in viral infections:

• Influenza infection:

  • RNF144A expression correlates inversely with disease severity

  • Lower expression is associated with more severe disease

  • Expression levels of RNF144A and ERK target genes show inverse correlation

  • RNF144A mRNA expression performs well as a biomarker distinguishing severe from moderate cases of influenza

• Other conditions:

  • Mutations in the GXXXG motifs of RNF144A have been found in human cancers

  • The G252D mutation of RNF144A preserves self-association and ubiquitin ligase activity but loses membrane localization and turns over rapidly

  • RNF144A has been linked to neurological and inflammatory diseases

These findings suggest that RNF144A expression levels and functional mutations could serve as prognostic markers or therapeutic targets in various diseases.

How can researchers distinguish between the membrane localization and self-association functions of RNF144A?

To dissect these two independent functions, researchers can utilize specific mutants that separate membrane localization from self-association:

• GXXXG motif mutants (G252L/G256L):

  • Preserve membrane localization

  • Defective in self-association

  • Reduced ubiquitin ligase activity

• Membrane localization loss mutants:

  • Retain self-association capability

  • Maintain E3 ligase activity

  • This activity can be blocked by additional G252L/G256L mutations

• G252D cancer-associated mutant:

  • Preserves self-association and ubiquitin ligase activity

  • Loses membrane localization

  • Turns over rapidly in cells

Using these mutants in combination with functional assays allows researchers to attribute phenotypes to specific molecular functions of RNF144A.

What are the most effective methods to study RNF144A substrate specificity?

To identify and validate RNF144A substrates, researchers should employ complementary approaches:

• Protein-protein interaction studies:

  • Co-immunoprecipitation to identify binding partners

  • Proximity labeling techniques (BioID, APEX) to identify membrane-proximal interaction partners

  • Studies have identified IL-2Rβ, STAT5, and RAF1 as interaction partners

• Ubiquitination assays:

  • In vitro ubiquitination assays using purified components

  • In vivo ubiquitination assays with wild-type and mutant RNF144A

  • RAF1 has been confirmed as a direct ubiquitination target

• Proteomic approaches:

  • Stable isotope labeling with amino acids in cell culture (SILAC) to quantify protein abundance changes

  • Ubiquitinome analysis to identify proteins with altered ubiquitination profiles

  • Comparison between wild-type and RNF144A-deficient cells

• Functional validation:

  • Rescue experiments with substrate mutants lacking ubiquitination sites

  • Analysis of substrate protein levels, half-life, and activity in the presence/absence of RNF144A

Given that RBR family members like Parkin have between 90-1700 protein substrates, RNF144A likely has multiple biological functions and substrates beyond those currently identified .

What are unexplored aspects of RNF144A biology that warrant further investigation?

Several promising research directions remain to be fully explored:

• Tissue-specific functions:

  • While immune functions are being characterized, the role of RNF144A in other tissues remains poorly understood

  • The growth deficiency phenotype suggests functions beyond immune regulation

• Regulatory mechanisms:

  • Upstream regulators of RNF144A beyond IL-2/STAT5

  • Post-translational modifications that regulate RNF144A activity

  • Turnover and degradation mechanisms

• Complete substrate identification:

  • Comprehensive identification of ubiquitination substrates in different cell types

  • Identification of potential non-degradative ubiquitination targets

• Therapeutic applications:

  • Small molecule modulators of RNF144A activity

  • Potential for targeting RNF144A in viral infections or cancer

How might RNF144A research inform therapeutic strategies for viral infections and immune disorders?

The emerging understanding of RNF144A suggests several therapeutic applications:

• Biomarker development:

  • RNF144A expression levels as prognostic markers for influenza severity

  • Monitoring RNF144A expression to predict response to immunotherapy

• Therapeutic targeting:

  • Enhancing RNF144A activity to boost antiviral responses

  • Modulating RNF144A to fine-tune the balance between STAT5 and ERK signaling pathways

• Genetic screening:

  • Identifying RNF144A mutations in patients with immune disorders or increased susceptibility to viral infections

  • Personalized medicine approaches based on RNF144A status

Research suggests that even partial deficiency in RNF144A can significantly impact immune responses during infection, while homeostasis is maintained under sterile conditions . This characteristic makes RNF144A an attractive therapeutic target with potentially limited side effects.