Recombinant Human Probable E3 ubiquitin-protein ligase RNF144A (RNF144A)

What is the domain structure of RNF144A and how does it relate to its function?

RNF144A contains a characteristic RING1-IBR-RING2 (RBR) domain structure and a transmembrane (TM) domain that are essential for its E3 ubiquitin ligase activity. The RBR domain is responsible for the protein's catalytic function while the TM domain appears to regulate its activity through subcellular localization. Studies have shown that truncation of either the RBR or TM domain eliminates RNF144A's ubiquitination capability in cellular contexts, indicating these domains work cooperatively . The presence of the transmembrane tail anchor allows RNF144A to localize primarily to cytoplasmic vesicles and the plasma membrane, which is crucial for its interaction with cytosolic target proteins like DNA-PKcs .

What are the primary cellular functions of RNF144A?

RNF144A serves multiple important cellular functions:

• DNA damage response: RNF144A is induced in a p53-dependent manner following DNA damage and targets cytosolic DNA-PKcs for ubiquitination and degradation, thereby promoting apoptosis when necessary .

• Cytokine signaling modulation: RNF144A shapes the hierarchy of IL-2 receptor signaling by enhancing STAT5 activation while limiting RAF-ERK-MAPK output .

• Antiviral immunity: RNF144A positively regulates DNA virus- or cytosolic DNA-triggered innate immune responses by modulating STING (stimulator of interferon genes) activation .

These functions position RNF144A as a critical regulator at the intersection of cell survival, immune signaling, and antiviral defense mechanisms.

How is RNF144A expression regulated in cells?

RNF144A expression is dynamically regulated through multiple mechanisms:

• p53-dependent induction: During DNA damage responses, RNF144A is upregulated in a p53-dependent manner, suggesting its role in stress-induced cellular pathways .

• IL-2/STAT5-mediated induction: Transcriptome and ChIP-seq studies have identified RNF144A as one of the most highly induced genes regulated by IL-2-STAT5 signaling in primary T cells .

• Viral infection response: RNF144A expression appears to be modulated during viral infection, with its levels correlating inversely with disease severity in influenza patients .

This multi-layered regulation indicates RNF144A's importance in coordinating stress responses, immune signaling, and antiviral defense mechanisms.

What are the recommended methods for studying RNF144A's E3 ligase activity in vitro?

To study RNF144A's E3 ligase activity in vitro, researchers should consider the following methodological approach:

• Protein purification: Express and purify GST-tagged RNF144A from bacterial or mammalian expression systems. The GST tag facilitates purification while preserving enzymatic activity .

• In vitro ubiquitination assay components:

  • Purified E1 (ubiquitin-activating enzyme)

  • E2 conjugating enzyme (multiple E2s should be tested for optimal activity)

  • ATP regeneration system

  • Ubiquitin (wild-type or tagged versions for detection)

  • Purified substrate (e.g., DNA-PKcs) or RNF144A itself for autoubiquitination studies

• Detection methods:

  • Western blotting with anti-ubiquitin antibodies

  • Using tagged ubiquitin (HA-ubiquitin) for enhanced detection specificity

Note that in vitro conditions may yield primarily monoubiquitination patterns, while cellular environments often show polyubiquitination, suggesting additional factors present in vivo may enhance the reaction efficiency .

What cellular models are most appropriate for investigating RNF144A function?

Based on research findings, the following cellular models are valuable for RNF144A investigations:

• Cell lines:

  • HEK293T cells: Excellent for overexpression studies and protein interaction analysis

  • HaCaT cells: Useful for studying anti-viral responses and signaling pathways

  • Primary mouse embryonic fibroblasts (MEFs): Valuable for comparing wild-type and Rnf144a-deficient responses

• Primary cells:

  • T lymphocytes: Essential for investigating cytokine signaling, particularly IL-2/STAT5 pathway modulation

  • Bone marrow-derived macrophages (BMDMs): Ideal for studying antiviral responses

• Knockout models:

  • Rnf144a-knockout mice: Generated using CRISPR/Cas9 targeting the first coding exon, resulting in loss of the RING1 domain and a frameshift with premature stop codon

  • Knockdown approaches: siRNA targeting RNF144A is effective for transient reduction of expression

These models enable comprehensive investigation of RNF144A's diverse functions across immune regulation and cellular stress responses.

What are the key readouts for assessing RNF144A function in antiviral immunity?

To assess RNF144A's function in antiviral immunity, researchers should employ multiple complementary readouts:

• Viral infection quantification:

  • Plaque assays for measuring viral titers

  • Real-time PCR for viral genome quantification

  • Fluorescence microscopy for visualizing viral infection

  • Immunoblot assays for viral protein detection

• Antiviral signaling pathway activation:

  • Phosphorylation of STING, TBK1, IRF3, and p65 by western blotting

  • Formation of IRF3 dimers

  • Nuclear translocation of IRF3 and p65 by immunofluorescence or subcellular fractionation

• Cytokine and interferon production:

  • mRNA expression of Ifnb, Il-6, Ccl5, and Ifit1 by RT-qPCR

  • Protein levels of IFN-β, IL-6, and TNF-α by ELISA

• Functional antiviral activity:

  • Culture supernatant transfer experiments to assess protective capacity

  • In vivo infection studies measuring survival rate, body weight, and tissue viral loads

These comprehensive readouts provide a multi-dimensional assessment of RNF144A's role in antiviral immunity.

How does RNF144A modulate the balance between STAT5 and RAF-ERK signaling pathways?

RNF144A orchestrates a precise balance between STAT5 and RAF-ERK signaling through dual regulatory mechanisms:

• STAT5 pathway enhancement:

  • RNF144A increases the interaction between IL-2Rβ and STAT5

  • This enhanced receptor-transcription factor interaction promotes STAT5 phosphorylation and activation

  • The mechanism appears to involve scaffold/adaptor functions rather than ubiquitination

• RAF-ERK pathway suppression:

  • RNF144A directly polyubiquitinates RAF1, targeting it for proteasomal degradation

  • This prevents formation of the potent RAF1/BRAF kinase complex

  • Reduced RAF1 levels limit downstream ERK1/2 activation

This dual regulation by RNF144A is essential for proper T cell function, as evidenced by the fact that Rnf144a-deficient CD8+ T cells show impaired IL-2 induction of effector genes (Tnf, granzymes) and increased susceptibility to viral infections . The coordinated enhancement of STAT5 with concurrent limitation of RAF-ERK signaling appears to establish the appropriate threshold for immune activation while preventing excessive inflammatory responses.

What are the molecular mechanisms by which RNF144A enhances STING-mediated antiviral responses?

RNF144A enhances STING-mediated antiviral responses through several molecular mechanisms:

• STING translocation facilitation:

  • RNF144A promotes the translocation of STING from the endoplasmic reticulum (ER) to perinuclear compartments upon DNA virus infection or cytosolic DNA stimulation

  • This translocation is a critical step in STING activation

• STING dimerization and aggregation enhancement:

  • Coimmunoprecipitation assays demonstrate that RNF144A enhances the dimerization of STING

  • RNF144A promotes STING aggregation in response to HSV-1 infection, as shown by SDD-AGE (semi-denaturing detergent agarose gel electrophoresis) assays

• STING-TBK1-IRF3 complex formation:

  • RNF144A increases the affinity of STING for TBK1 and IRF3 upon HSV-1 infection

  • This enhanced complex formation facilitates downstream signaling activation

• ER-specific regulation:

  • RNF144A enhances activation of wild-type STING but not SAVI mutants (V147L, N154S, V155M) that localize to perinuclear compartments

  • This suggests RNF144A's contribution to STING activation occurs primarily at the ER rather than post-ER compartments

These mechanisms collectively explain how RNF144A deficiency impairs DNA virus- or cytosolic DNA-triggered innate immune responses and increases susceptibility to DNA virus infections.

How does the subcellular localization of RNF144A influence its substrate specificity?

RNF144A's subcellular localization plays a critical role in determining its substrate specificity and functional outcomes:

• Transmembrane domain influence:

  • RNF144A contains a transmembrane tail anchor domain that localizes it primarily to cytoplasmic vesicles and the plasma membrane

  • Truncation of this domain eliminates RNF144A's ubiquitination capability in cells, suggesting its positioning is crucial for substrate access

• Distinct subcellular substrate pools:

  • Cytoplasmic localization enables RNF144A to target cytosolic DNA-PKcs while nuclear DNA-PKcs remains protected

  • This compartmentalization allows selective regulation of DNA-PKcs pro-survival function without compromising its nuclear DNA repair activity

• Membrane-associated signaling complexes:

  • RNF144A associates with IL-2Rβ, STAT5, and RAF1 at the plasma membrane

  • This co-localization facilitates the dual regulation of cytokine signaling pathways

• ER-specific STING regulation:

  • RNF144A enhances wild-type STING activation (ER-localized) but not SAVI mutants (perinuclear-localized)

  • This indicates RNF144A's regulation of STING occurs specifically at the ER

The subcellular positioning of RNF144A therefore creates distinct regulatory zones, allowing differential access to substrate pools and enabling coordinated but compartmentalized control of multiple cellular processes.

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

Clinical studies have revealed a significant inverse relationship between RNF144A expression and viral infection severity in humans:

• Expression pattern in influenza patients:

  • RNF144A mRNA levels are significantly lower in patients with severe influenza compared to healthy controls or those with moderate disease

  • Analysis of whole blood transcriptomes from a large dataset (GSE101702) demonstrated this clear association

• ERK pathway activation correlation:

  • ERK-regulated genes are significantly enriched in transcriptomes of patients with severe influenza

  • These ERK-regulated genes show strong negative correlation with RNF144A expression

  • This is consistent with RNF144A's role in suppressing RAF-ERK signaling through RAF1 ubiquitination and degradation

• Biomarker potential:

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

  • When combined with downstream ERK targets like FOS and JUN (AP-1 subunits), RNF144A provides robust discriminatory power in receiver operating characteristic (ROC) analyses

These findings suggest that diminished RNF144A expression may contribute to dysregulated inflammatory responses during severe viral infections by allowing unchecked RAF-ERK signaling, potentially offering both prognostic value and therapeutic insight.

How does RNF144A deficiency affect in vivo responses to viral infection?

RNF144A deficiency significantly impairs in vivo antiviral responses as demonstrated in multiple infection models:

• HSV-1 infection outcomes in Rnf144a-deficient mice:

  • Greater body weight loss and lower survival rates

  • More severe lung tissue destruction

  • Higher viral loads in lung, liver, and spleen

  • Impaired production of type I interferons and proinflammatory cytokines

• Influenza infection susceptibility:

  • Rnf144a-deficient mice show increased susceptibility to influenza

  • CD8+ T cells from these mice have impaired IL-2-induced expression of effector genes (Tnf, granzymes)

  • This results in widespread inflammation and more severe disease manifestation

• Organ-specific effects:

  • Comprehensive examination across multiple organs shows consistent patterns of increased viral replication

  • Impaired antiviral signaling as evidenced by reduced phosphorylation of key signaling molecules (STING, TBK1, IRF3)

  • Diminished production of protective cytokines and chemokines

These findings demonstrate that RNF144A plays an essential protective role during viral infections in vivo, coordinating appropriate immune responses while preventing excessive inflammation.

What are the challenges in expressing and purifying functional recombinant RNF144A?

Expressing and purifying functional recombinant RNF144A presents several technical challenges that researchers should address:

• Transmembrane domain complications:

  • The transmembrane tail anchor domain of RNF144A can cause aggregation and insolubility

  • Solution: Consider generating truncation constructs that preserve the RBR domain but remove the TM domain for initial functional studies

  • Alternative approach: Use detergent-based extraction methods optimized for membrane proteins

• Maintaining E3 ligase activity:

  • RNF144A's E3 ligase activity is highly regulated and can be lost during purification

  • Solution: GST-tagging has been successfully used to purify active RNF144A

  • Ensure reducing conditions are maintained throughout purification to prevent oxidation of catalytic cysteine residues

• Expression system selection:

  • Bacterial systems may lack post-translational modifications needed for full activity

  • Mammalian expression systems better preserve regulatory modifications but yield lower protein amounts

  • Solution: Compare both systems to balance quantity and quality of purified protein

• Activity verification:

  • In vitro conditions often yield primarily monoubiquitination while cellular environments show polyubiquitination

  • Solution: Validate purified protein function through autoubiquitination assays before attempting substrate ubiquitination

These approaches help overcome the inherent challenges in working with this complex membrane-associated E3 ligase.

What controls are essential when studying RNF144A's impact on signaling pathways?

When investigating RNF144A's effects on signaling pathways, several critical controls must be included:

• Domain mutant controls:

  • Include RBR domain mutants that abolish E3 ligase activity

  • Use TM domain deletion mutants to assess the importance of subcellular localization

  • These controls help distinguish between catalytic and scaffolding functions of RNF144A

• Pathway specificity controls:

  • Examine effects on multiple pathways (e.g., STAT5 vs. RAF-ERK)

  • Test response to various stimuli (e.g., IL-2 for cytokine signaling, DNA vs. RNA ligands for innate immunity)

  • RNF144A specifically affects DNA-triggered but not RNA-triggered responses

• Genetic model validation:

  • Compare findings between siRNA knockdown and CRISPR knockout models

  • Use rescue experiments with wild-type RNF144A to confirm specificity of observed phenotypes

  • Include littermate controls when using knockout mice to minimize genetic background effects

• Substrate specificity verification:

  • Confirm direct interaction with proposed substrates through co-immunoprecipitation

  • Demonstrate ubiquitination of specific substrates both in vitro and in vivo

  • Utilize ubiquitin mutants (e.g., K48-only or K63-only) to determine ubiquitin chain topology

These controls ensure robust and reproducible findings when investigating RNF144A's complex roles in cellular signaling networks.

What are the unexplored aspects of RNF144A function in immune regulation?

Despite significant progress in understanding RNF144A biology, several important aspects remain unexplored:

• Immune cell type-specific functions:

  • While roles in T cells and macrophages have been partially characterized, RNF144A's function in B cells, dendritic cells, and natural killer cells remains largely unknown

  • Investigation of cell type-specific expression patterns, regulatory mechanisms, and functional consequences would provide a more comprehensive understanding of RNF144A in immune regulation

• Cytokine response specificity:

  • RNF144A has been identified as an IL-2/STAT5-induced gene in T cells

  • Potential roles in other cytokine signaling pathways (IL-7, IL-15, IL-21) that also utilize STAT5 merit investigation

  • The molecular basis for cytokine response specificity remains to be elucidated

• Regulation of adaptive immunity:

  • Current research has focused primarily on innate immune responses and early T cell activation

  • RNF144A's potential roles in memory formation, B cell responses, and adaptive immune regulation represent important areas for future study

• Autoimmunity implications:

  • The balance between STAT5 and RAF-ERK signaling influenced by RNF144A may have implications for autoimmune diseases

  • Investigation of RNF144A expression and function in autoimmune contexts could reveal new regulatory mechanisms and therapeutic targets

These unexplored areas represent significant opportunities for advancing our understanding of RNF144A's role in comprehensive immune regulation.

How might targeting RNF144A be developed as a therapeutic approach for viral infections?

Developing therapeutic approaches targeting RNF144A for viral infections presents both promising opportunities and important considerations:

• Potential therapeutic strategies:

  • RNF144A enhancement: Given that reduced RNF144A expression correlates with severe viral disease, approaches to increase RNF144A expression or activity could strengthen antiviral responses

  • Small molecule activators: Compounds that enhance RNF144A's E3 ligase activity toward RAF1 could help limit excessive inflammatory responses during severe infections

  • Stabilization approaches: Interventions that prevent RNF144A degradation might prolong its beneficial effects on immune signaling

• Therapeutic contexts:

  • Early intervention: RNF144A enhancement might be most beneficial early in infection to establish appropriate immune responses

  • Severe disease: In cases of severe influenza or other viral infections where RNF144A expression is diminished, restoration could help rebalance inflammatory responses

  • Personalized approach: RNF144A expression levels could serve as a biomarker to identify patients most likely to benefit from targeted therapy

• Delivery challenges and solutions:

  • Cell-specific targeting: Develop approaches to enhance RNF144A specifically in relevant immune cell populations

  • Temporal control: Design interventions with appropriate timing to match the dynamic nature of antiviral responses

  • Combination therapy: Pair RNF144A-targeting approaches with direct antivirals for synergistic benefit

• Safety considerations:

  • Potential effects on DNA damage responses given RNF144A's role in regulating DNA-PKcs

  • Possible impacts on growth and development based on the growth-deficient phenotype of knockout mice

  • Need for careful tissue-specific targeting to avoid unwanted effects in non-immune tissues

These considerations provide a framework for developing RNF144A-targeted therapeutic approaches for viral infections.

What are the optimized protocols for studying RNF144A-substrate interactions?

To effectively study RNF144A-substrate interactions, researchers should consider these optimized protocols:

• Co-immunoprecipitation (Co-IP) approaches:

  • Cell lysis conditions: Use mild detergents (0.5% NP-40 or Triton X-100) to preserve membrane-associated interactions

  • Cross-linking step: Consider mild cross-linking (0.5-1% formaldehyde) to capture transient E3-substrate interactions

  • Antibody selection: Use tag-specific antibodies (FLAG, HA, GST) for recombinant proteins or validated RNF144A-specific antibodies for endogenous studies

• Proximity labeling methods:

  • BioID or TurboID fusion constructs with RNF144A to identify proximal interacting proteins

  • APEX2-based proximity labeling for temporal resolution of interaction dynamics

  • These approaches are particularly valuable for identifying membrane-proximal interactions of RNF144A

• Ubiquitination assays:

  • In vivo ubiquitination: Transfect HA-ubiquitin with RNF144A and potential substrates, followed by denaturing immunoprecipitation to eliminate non-covalent interactions

  • In vitro reconstitution: Use purified components (E1, E2, ATP, ubiquitin) with GST-RNF144A and purified substrate to demonstrate direct ubiquitination

  • Chain-specific antibodies or ubiquitin mutants to determine ubiquitin chain topology

• Proteomic approaches:

  • Quantitative proteomics comparing wild-type and Rnf144a-deficient cells to identify stabilized substrates

  • Ubiquitin remnant profiling to identify specific ubiquitination sites on substrates

  • Interaction proteomics under various stimulation conditions (DNA damage, cytokine treatment, viral infection)

These optimized protocols enable comprehensive characterization of RNF144A's substrate interactions across various cellular contexts.

What are the most effective gene editing approaches for studying RNF144A function?

Effective gene editing approaches for studying RNF144A function include:

• CRISPR/Cas9 knockout strategies:

  • Target early exons to ensure complete functional disruption

  • Previous successful approach: Targeting the first coding exon, resulting in loss of the RING1 domain and a frameshift with premature stop codon

  • Generate multiple independent clones to control for off-target effects

  • Validate knockout at both mRNA and protein levels to confirm complete elimination

• Domain-specific editing:

  • Generate precise mutations in key catalytic residues of the RBR domain to disrupt E3 ligase activity while preserving protein structure

  • Create TM domain deletions to study the importance of subcellular localization

  • Engineer phosphorylation site mutants to investigate regulatory mechanisms

• Knockin approaches:

  • Endogenous tagging of RNF144A for visualization and biochemical studies

  • Fluorescent protein fusions for live-cell imaging of dynamics

  • Introduce specific disease-associated variants to study their functional consequences

• Inducible systems:

  • Doxycycline-inducible expression systems for controlled restoration of RNF144A in knockout backgrounds

  • CRISPR interference (CRISPRi) or activation (CRISPRa) for temporal regulation of endogenous RNF144A expression

  • Degron-based approaches for rapid protein depletion to study acute loss of function

• Tissue-specific strategies:

  • Conditional knockout using Cre-lox systems with tissue-specific promoters

  • AAV-delivered CRISPR for in vivo targeting in specific tissues or cell types

  • These approaches help overcome the growth deficiency observed in germline knockout mice

These gene editing approaches provide complementary strategies for comprehensive functional characterization of RNF144A.

How should researchers interpret apparently contradictory data on RNF144A function?

When faced with seemingly contradictory data regarding RNF144A function, researchers should consider several interpretative frameworks:

• Context-dependent activities:

  • RNF144A functions in multiple pathways (DNA damage response, cytokine signaling, antiviral immunity)

  • Apparent contradictions may reflect genuine biological differences in these distinct contexts

  • Carefully document experimental conditions, cell types, and stimuli when comparing results

• Dual regulatory mechanisms:

  • RNF144A can function both as an E3 ligase and as a scaffold/adaptor protein

  • Some effects may be independent of its catalytic activity (e.g., STAT5 pathway enhancement)

  • Others rely on ubiquitination activity (e.g., RAF1 degradation)

  • Use catalytically inactive mutants to distinguish between these modes of action

• Subcellular compartmentalization:

  • RNF144A's transmembrane domain creates distinct regulatory zones

  • Effects may differ between cytosolic, membrane-associated, and vesicular compartments

  • Use subcellular fractionation and localization studies to resolve apparent contradictions

• Methodological considerations:

  • Overexpression vs. knockout approaches may yield different results due to stoichiometric effects

  • Acute vs. chronic loss of function may reveal different aspects of RNF144A biology

  • Validate key findings using complementary approaches (e.g., pharmacological and genetic)

• Quantitative analysis:

  • Some contradictions may reflect quantitative rather than qualitative differences

  • Use dose-response relationships and temporal dynamics to fully characterize RNF144A's effects

  • Consider potential feedback mechanisms that may compensate for RNF144A manipulation

This multifaceted interpretative approach helps reconcile apparent contradictions and develop a more nuanced understanding of RNF144A's complex biology.

What bioinformatic approaches are most useful for analyzing RNF144A-related datasets?

Several bioinformatic approaches are particularly valuable for analyzing RNF144A-related datasets:

• Expression correlation analysis:

  • Examine correlation between RNF144A and potential target genes across various tissues and conditions

  • Example: Negative correlation between RNF144A and ERK-regulated genes in influenza patients

  • Tools: WGCNA (Weighted Gene Co-expression Network Analysis), GSEA (Gene Set Enrichment Analysis)

• Pathway enrichment analysis:

  • Identify significantly enriched pathways in RNF144A-associated gene sets

  • Compare transcriptomes of wild-type and Rnf144a-deficient cells under various stimulation conditions

  • Tools: KEGG, Reactome, Gene Ontology enrichment analysis

• Protein-protein interaction prediction:

  • Predict potential RNF144A substrates and interacting partners

  • Integrate ubiquitinome and proteome data to identify proteins regulated by RNF144A

  • Tools: STRING, BioGRID, UbiBrowser for E3-substrate prediction

• Structural modeling:

  • Model RNF144A's RBR domain based on related E3 ligases with known structures

  • Predict substrate binding sites and catalytic mechanisms

  • Tools: AlphaFold2, I-TASSER, molecular dynamics simulations

• Clinical data integration:

  • Correlate RNF144A expression with disease outcomes in patient cohorts

  • Develop predictive models using RNF144A as a biomarker

  • Example: ROC analysis of RNF144A expression for distinguishing severe from moderate influenza

  • Tools: Survival analysis packages, machine learning approaches for biomarker validation

• Cross-species conservation analysis:

  • Identify evolutionarily conserved regulatory elements and functional domains

  • Compare RNF144A function across model organisms

  • Tools: Ensembl Compara, UCSC Genome Browser conservation tracks