TRIM25 Antibody

What is TRIM25 and what is its molecular structure?

TRIM25 is an E3 ubiquitin ligase and RNA-binding protein that plays a critical role in the innate immune response against RNA viruses. Structurally, TRIM25 contains multiple functional domains: a RING domain responsible for E3 ubiquitin ligase activity, a coiled-coil (CC) domain important for dimerization, and a C-terminal PRY/SPRY region that functions as an RNA-binding domain (RBD) . The protein has a molecular weight of approximately 72 kDa and is predominantly expressed in human cells . TRIM25's structural complexity enables it to participate in multiple cellular processes, particularly in antiviral defense mechanisms where it regulates RNA stability and protein ubiquitination.

How does TRIM25 contribute to antiviral immunity?

TRIM25 contributes to antiviral immunity through multiple mechanisms:

These diverse mechanisms make TRIM25 a crucial component of the cellular defense against viral infections.

Why is RNA binding important for TRIM25 function?

RNA binding is critical for TRIM25's antiviral functions for several reasons:

• RNA binding appears to be crucial for TRIM25's E3 ubiquitin ligase activity. Studies have shown that deletion of the RNA-binding domain reduces TRIM25 ubiquitination in vitro .

• TRIM25 binds promiscuously to positive strand influenza A virus (IAV) RNAs, while binding to negative strand RNAs is much weaker and confined to segment ends. This binding pattern suggests a role in directly targeting viral RNA species .

• The RNA-dependent interaction enables TRIM25 to co-localize with stress granules during viral infection, which dramatically increases its ubiquitination activity toward substrates localized in these granules .

• TRIM25's RNA binding capability is essential for its subcellular redistribution to viral replication organelles (ROs), allowing it to directly interfere with viral replication processes .

The experimental evidence demonstrates that RNA binding is not merely an additional function of TRIM25 but is integral to its role in antiviral immunity.

What are the challenges in detecting TRIM25 with antibodies?

Detecting TRIM25 with antibodies presents several research challenges:

• Protein expression levels: While TRIM25 is endogenously expressed in human cells, its expression level may vary across cell types and can be altered during viral infection, making consistent detection challenging .

• Subcellular localization changes: Upon viral infection or poly(I:C) treatment, TRIM25 redistributes to stress granules and other subcellular compartments, which may affect antibody accessibility in fixed samples .

• Potential cross-reactivity: Due to the presence of multiple TRIM family proteins with similar domains, ensuring antibody specificity is critical to avoid cross-reactivity with related proteins.

• Post-translational modifications: As an E3 ubiquitin ligase, TRIM25 undergoes auto-ubiquitination and other post-translational modifications that might mask antibody epitopes or alter protein mobility on gels .

• RNA-protein interactions: TRIM25's interaction with RNA may shield certain epitopes, particularly in techniques that preserve RNA-protein complexes, requiring careful antibody selection for different experimental approaches.

Researchers should validate TRIM25 antibodies thoroughly in their specific experimental system and select antibodies targeting epitopes appropriate for their research questions.

How can TRIM25 antibodies be used to study its interaction with stress granules?

TRIM25 antibodies can be instrumental in investigating its association with stress granules (SGs) through several sophisticated approaches:

• Co-immunofluorescence microscopy: Antibodies against TRIM25 and SG markers (such as G3BP1) can be used to quantify co-localization under various stressors. Recent research has shown that TRIM25 exhibits strong co-localization with both canonical stress granules and RNase L-dependent bodies (RLBs) upon poly(I:C) treatment, with stronger association with SGs than with RLBs .

• Proximity-dependent biotinylation assays: A G3BP1 proximity-dependent biotinylation assay revealed that TRIM25 was enriched 130 times in stress granules upon poly(I:C) treatment, despite only a 2-fold increase in total TRIM25 protein levels . Similar approaches with TRIM25 antibodies can identify proteins that associate with TRIM25 specifically in the context of SGs.

• Immunoprecipitation coupled with RNA analysis: TRIM25 antibodies can be used for RNA immunoprecipitation (RIP) to isolate and identify the RNA species that associate with TRIM25 in stress granules during viral infection.

• Live-cell imaging: For dynamic studies, antibody fragments or nanobodies against TRIM25 can be used to track its recruitment to stress granules in real-time during viral infection or stress induction.

• Super-resolution microscopy: Using highly specific TRIM25 antibodies with techniques like STORM or PALM can reveal the precise spatial organization of TRIM25 within stress granules and its relationship with other SG components.

These techniques provide valuable insights into how TRIM25 contributes to the antiviral functions of stress granules during infection.

What is the relationship between TRIM25's E3 ubiquitin ligase activity and its RNA-binding function?

The relationship between TRIM25's E3 ubiquitin ligase activity and RNA-binding function is complex and still being fully elucidated:

• RNA binding enhances E3 ligase activity: Studies have shown that RNA binding appears to be crucial for TRIM25's E3 ubiquitin ligase activity, as deletion of the RNA-binding domain reduces TRIM25 ubiquitination in vitro . This suggests RNA may act as a cofactor or scaffold that promotes optimal E3 ligase function.

• Functional independence in antiviral activity: Surprisingly, both RNA-binding deficient mutant (TRIM25ΔRBD) and E3 ubiquitin ligase mutant (TRIM25ΔRING) still inhibited influenza A virus replication in certain experimental contexts . This indicates some level of functional independence between these domains in specific antiviral mechanisms.

• Synergistic effects in specific contexts: In stress granules, the co-condensation of TRIM25 with G3BP1 dramatically increases the ubiquitination activity of TRIM25 toward substrates localized in these granules . This suggests that RNA-dependent phase separation can enhance E3 ligase function in specific cellular compartments.

• Structural interplay: Biochemical studies have identified key residues in both the PRY/SPRY and CC domains responsible for RNA binding . The spatial arrangement of these domains likely facilitates communication between RNA binding and E3 ligase functions.

• Context-dependent requirements: RNA tethering experiments with MS2 stem loops indicated a need for both RING and RBD domains, unlike in IAV infections, suggesting that TRIM25 might have RING and RBD-independent functions that are crucial specifically for inhibiting viral infection .

Understanding this interplay is critical for developing strategies to modulate TRIM25 activity in antiviral therapeutic approaches.

How can TRIM25 antibodies help distinguish between TRIM25's different functional states?

TRIM25 antibodies can be strategically employed to distinguish between its different functional states through several specialized approaches:

• Phospho-specific antibodies: TRIM25 undergoes various post-translational modifications that correlate with different functional states. Phospho-specific antibodies can detect activation-specific modifications that occur during viral infection or stress responses.

• Conformation-specific antibodies: TRIM25 likely adopts different conformations when bound to RNA versus when free. Conformation-specific antibodies can differentiate between these states, providing insight into the proportion of TRIM25 actively engaged with RNA in different cellular contexts.

• Ubiquitination-state specific detection: As an E3 ligase that undergoes auto-ubiquitination, antibodies that specifically recognize ubiquitinated TRIM25 can help assess its catalytic activity state in various experimental conditions.

• Subcellular distribution analysis: Antibodies can track TRIM25's redistribution during viral infection, particularly its recruitment to stress granules, which represents a distinct functional state. The search results indicate that endogenous TRIM25 forms puncta and exhibits strong co-localization with G3BP1 puncta upon poly(I:C) treatment .

• Co-immunoprecipitation studies: TRIM25 antibodies can be used to identify different protein interaction partners associated with specific functional states, such as its interaction with viral components or stress granule proteins.

• Combined CLIP and immunoprecipitation: By coupling crosslinking immunoprecipitation (CLIP) techniques with specific TRIM25 antibodies, researchers can identify the RNA binding profile of TRIM25 in different functional contexts, as demonstrated in studies using iCLIP2 in virus-infected and uninfected cells .

These approaches provide multidimensional information about TRIM25's functional state during viral infections and other cellular stress conditions.

What techniques can be combined with TRIM25 antibodies to study its RNA-binding properties?

Researchers can combine various sophisticated techniques with TRIM25 antibodies to comprehensively characterize its RNA-binding properties:

• RNA immunoprecipitation (RIP): TRIM25 antibodies can be used to immunoprecipitate TRIM25-RNA complexes, followed by RNA sequencing to identify bound RNA species. This approach has revealed that TRIM25 binds promiscuously to positive strand IAV RNAs, while binding to negative strand RNAs is much weaker and confined to segment ends .

• Cross-linking immunoprecipitation (CLIP): More advanced than standard RIP, CLIP techniques involve UV cross-linking of RNA-protein complexes before immunoprecipitation with TRIM25 antibodies. Studies have utilized iCLIP2 with size-matched inputs in virus-infected and uninfected cells to identify host and viral RNA motifs that TRIM25 binds to and determine its RNA structure and sequence specificity .

• Proximity RNA labeling: TRIM25 antibodies can be conjugated to RNA-modifying enzymes to label RNAs in close proximity to TRIM25 in living cells, providing spatial information about TRIM25-RNA interactions.

• In vitro RNA binding assays: Purified TRIM25 (potentially isolated using antibodies) can be used in electrophoretic mobility shift assays (EMSAs) or filter binding assays to determine binding affinities and specificities for different RNA structures or sequences.

• RNA-protein reconstitution experiments: TRIM25 mutants can be immunoprecipitated with antibodies to assess their RNA binding capabilities. This approach helped determine that RNA binding-deficient mutant TRIM25ΔRBD could still bind viral RNAs through an unknown mechanism .

• Fluorescence microscopy with RNA probes: Combining TRIM25 antibody staining with fluorescent RNA probes allows visualization of co-localization between TRIM25 and specific RNA species in fixed cells.

These combined approaches provide complementary information about TRIM25's RNA binding preferences, helping to unravel its complex role in antiviral immunity.

What are the optimal protocols for using TRIM25 antibodies in Western Blotting?

For optimal Western blotting results with TRIM25 antibodies, researchers should consider the following protocol recommendations:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for cell lysis

  • Include phosphatase inhibitors if phosphorylation states are relevant

  • Sonicate briefly to shear nucleic acids that might affect TRIM25 detection

  • Denature samples at 95°C for 5 minutes in reducing sample buffer

• Gel electrophoresis parameters:

  • Use 8-10% SDS-PAGE gels to properly resolve TRIM25 (72 kDa)

  • Run the gel at moderate voltage (100-120V) to ensure proper separation

  • Include positive controls from cell lines known to express TRIM25 (e.g., HEK293)

• Transfer conditions:

  • Use wet transfer systems for optimal transfer of the 72 kDa protein

  • Transfer at 100V for 1 hour or 30V overnight at 4°C

  • PVDF membranes are preferable over nitrocellulose for TRIM25 detection

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

  • Use primary TRIM25 antibody at manufacturer's recommended dilution (typically 1:1000)

  • Incubate with primary antibody overnight at 4°C for best results

  • Use gentle rocking during antibody incubations

• Detection considerations:

  • Enhanced chemiluminescence (ECL) detection provides good sensitivity

  • TRIM25 may appear as multiple bands due to post-translational modifications

  • Expected molecular weight is 72 kDa

  • Include appropriate loading controls (e.g., β-actin, GAPDH)

• Troubleshooting tips:

  • If detecting multiple bands, verify specificity using TRIM25 knockdown/knockout controls

  • For viral infection studies, include both infected and uninfected samples to observe potential mobility shifts

  • When studying TRIM25 in stress granules, consider using phosphorylation-specific antibodies as TRIM25 may undergo modifications during stress

Following these recommendations will help ensure specific and reliable detection of TRIM25 in Western blotting applications.

How can TRIM25 antibodies be used effectively in immunoprecipitation studies?

To effectively use TRIM25 antibodies in immunoprecipitation (IP) studies, particularly when investigating its interactions with RNA and other proteins, researchers should implement the following strategies:

• Selection of lysis conditions:

  • For protein-protein interactions: Use gentle lysis buffers (e.g., 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 5% glycerol) with protease inhibitors

  • For RNA-protein complexes: Consider non-denaturing conditions that preserve RNA-protein interactions

  • When studying ubiquitination: Include deubiquitinase inhibitors (e.g., N-ethylmaleimide) to preserve ubiquitinated species

• Pre-clearing step:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • For RNA-IP studies, include an RNase inhibitor in all buffers

• Antibody binding strategies:

  • Direct approach: Incubate cell lysates with TRIM25 antibody followed by protein A/G beads

  • Indirect approach: Pre-couple TRIM25 antibodies to protein A/G beads before incubation with lysates

  • Consider cross-linking antibodies to beads with dimethyl pimelimidate (DMP) to prevent antibody co-elution

• Optimizing immunoprecipitation conditions:

  • For protein interactions: Overnight incubation at 4°C with gentle rotation

  • For RNA-protein complexes: Shorter incubation times (2-4 hours) to minimize RNA degradation

  • Include appropriate controls (IgG control, TRIM25 knockout/knockdown samples)

• Washing and elution considerations:

  • For high stringency: Increase salt concentration in wash buffers

  • For maintaining weak interactions: Use gentler washing conditions

  • For RNA-IP: Include RNase inhibitors in all wash buffers

  • Elution can be performed with SDS sample buffer for Western blot analysis or with milder conditions for functional studies

• Specialized applications:

  • For studying TRIM25 in stress granules: Lysates from cells treated with poly(I:C) show TRIM25 enrichment in G3BP1-positive structures

  • For viral RNA interactions: Consider crosslinking approaches (e.g., UV crosslinking) before lysis to preserve transient interactions

  • For detecting ubiquitination: Perform TRIM25 IP followed by ubiquitin Western blot or vice versa

• Verification strategies:

  • Reciprocal IP for protein-protein interactions

  • RT-PCR or RNA-seq of immunoprecipitated material to identify bound RNAs

  • Mass spectrometry analysis of TRIM25 immunoprecipitates to identify novel interacting partners

These protocols can be adapted based on the specific research question and cellular context being investigated.

What controls should be used when studying TRIM25 with antibodies in virus-infected cells?

When studying TRIM25 with antibodies in virus-infected cells, implementing proper controls is crucial for obtaining reliable and interpretable results:

• Antibody specificity controls:

  • TRIM25 knockout or knockdown cells to confirm antibody specificity

  • Isotype control antibodies to identify non-specific binding

  • Peptide competition assays using the immunizing peptide to verify epitope specificity

  • Multiple TRIM25 antibodies targeting different epitopes to confirm observations

• Infection controls:

  • Mock-infected cells processed in parallel with infected samples

  • Time-course samples to track TRIM25 changes throughout infection

  • UV-inactivated virus controls to distinguish between viral binding and active replication effects

  • When using influenza virus, consider NS1 mutant strains (e.g., PR8 R38K41A) that cannot inhibit TRIM25, which provides a good platform to study TRIM25's functions

• Subcellular localization controls:

  • Co-staining with organelle markers to track TRIM25 redistribution during infection

  • Markers for stress granules (e.g., G3BP1) or RLBs to confirm TRIM25 association with these structures

  • In cells with G3BP1/2 double knockout, TRIM25 puncta formation can be assessed to determine dependence on stress granule formation

• Functional validity controls:

  • TRIM25 mutants that lack specific domains (e.g., TRIM25ΔRBD, TRIM25ΔRING) to dissect domain-specific functions

  • Rescue experiments in TRIM25 knockout cells by reintroducing wild-type or mutant TRIM25

  • Include controls for RNA degradation when studying TRIM25's effect on viral RNA stability

• Technical controls:

  • Input samples (pre-immunoprecipitation) for IP experiments

  • Loading controls appropriate for the subcellular fraction being analyzed

  • RNase treatment controls when studying RNA-dependent interactions

  • Non-crosslinked samples as controls for crosslinking experiments

• Virus-specific controls:

  • For influenza studies, use NS1 mutants that cannot bind TRIM25 (like R38K41A mutations) to observe TRIM25's unrestricted activity

  • Include viral protein expression analysis to correlate with TRIM25 activity

  • Consider using multiple virus strains to identify strain-specific differences in TRIM25 interactions

These comprehensive controls help distinguish genuine TRIM25-related phenomena from artifacts and provide mechanistic insights into TRIM25's role during viral infection.

How can TRIM25 antibodies be used to investigate its role in liquid-liquid phase separation?

TRIM25 antibodies can be powerful tools for investigating its role in liquid-liquid phase separation (LLPS) through several specialized approaches:

• Immunofluorescence microscopy techniques:

  • Fixed-cell analysis: Use TRIM25 antibodies in combination with markers of phase-separated compartments (e.g., G3BP1 for stress granules) to quantify co-localization

  • Super-resolution microscopy: Apply TRIM25 antibodies with techniques like STORM or PALM to examine the internal structure of TRIM25-containing condensates at nanoscale resolution

  • Live-cell imaging: Combine TRIM25 antibody fragments or nanobodies with fluorescent tags to track dynamic LLPS events in real-time

• Biochemical characterization of LLPS:

  • Differential centrifugation: Fractionate cells and use TRIM25 antibodies to detect its distribution between soluble and condensate fractions

  • Antibody-based pulldown of phase-separated structures: Use mild lysis conditions to preserve condensates, followed by immunoprecipitation with TRIM25 antibodies

  • Sequential extraction protocols: Extract cells with increasing detergent strengths and use TRIM25 antibodies to track its partitioning behavior

• Stimulus-dependent LLPS studies:

  • Poly(I:C) treatment: TRIM25 undergoes LLPS and co-condenses with G3BP1 in a dsRNA-dependent manner

  • RNA virus infection: TRIM25 antibodies can track its redistribution during infection

  • Comparison between different stressors: Use TRIM25 antibodies to compare its LLPS behavior under viral infection versus other cellular stresses

• Mechanistic investigations:

  • Domain-specific analysis: Compare wild-type TRIM25 with mutants lacking specific domains (e.g., PRY/SPRY domain) to determine their contribution to LLPS

  • RNA dependency: Combine RNase treatment with TRIM25 immunostaining to assess the role of RNA in maintaining phase-separated structures

  • Post-translational modification analysis: Use modification-specific antibodies to determine how phosphorylation or ubiquitination affects TRIM25's LLPS properties

• Functional implications of LLPS:

  • Activity assays: Measure TRIM25's ubiquitination activity within condensates using antibodies against ubiquitinated substrates

  • Client recruitment: Use TRIM25 antibodies alongside antibodies against potential client proteins to assess recruitment into condensates

  • Antiviral activity correlation: Correlate the formation of TRIM25-containing condensates with antiviral outcomes using virus-specific antibodies

Research has shown that TRIM25's condensation with G3BP1 dramatically increases its ubiquitination activity toward substrates in stress granules and is critical for activating the RIG-I signaling pathway and restricting RNA virus infection . These approaches can further elucidate the molecular mechanisms and functional significance of TRIM25's phase separation behavior.

What approaches can resolve contradictory data when studying TRIM25 with different antibodies?

When faced with contradictory data from different TRIM25 antibodies, researchers can implement several systematic approaches to resolve discrepancies:

• Comprehensive antibody validation:

  • Epitope mapping: Determine the exact epitopes recognized by each antibody to understand potential interference from protein interactions or modifications

  • Western blot analysis in TRIM25 knockout cells: Confirm specificity and rule out cross-reactivity with other TRIM family proteins

  • Immunoprecipitation followed by mass spectrometry: Identify all proteins captured by each antibody

  • Peptide competition assays: Verify epitope specificity by competing with immunizing peptides

• Context-dependent analysis:

  • Domain accessibility assessment: Different antibodies may have variable access to epitopes based on TRIM25's conformation or interaction partners

  • Post-translational modification interference: Test whether modifications like phosphorylation or ubiquitination affect antibody recognition

  • RNA-binding effects: Determine if RNA binding to TRIM25 masks certain epitopes, as TRIM25 is known to bind RNA through its PRY/SPRY domain

• Technical optimization:

  • Fixation method comparison: Different fixatives (paraformaldehyde, methanol, etc.) can affect epitope availability

  • Buffer condition optimization: Test multiple lysis and immunoprecipitation buffers to ensure optimal conditions for each antibody

  • Sample preparation variations: Compare native versus denaturing conditions to identify conformation-dependent recognition

• Parallel methodological approaches:

  • Genetic tagging: Introduce epitope tags (FLAG, HA, etc.) to TRIM25 as independent detection methods

  • Orthogonal techniques: Combine antibody-based methods with other approaches like mass spectrometry or RNA-seq

  • Alternative detection strategies: Use aptamers or nanobodies as alternatives to conventional antibodies

• Biological validation:

  • Functional correlation: Determine which antibody results correlate with known TRIM25 functions (e.g., antiviral activity)

  • Structure-function analysis: Use TRIM25 mutants with defined functional alterations to validate antibody specificity

  • Cross-species comparison: Test antibodies against TRIM25 from different species with known sequence variations

• Data integration framework:

  • Weighted evidence approach: Assign confidence levels to data based on antibody validation metrics

  • Consensus analysis: Focus on findings that are consistent across multiple antibodies

  • Contextual interpretation: Consider that seemingly contradictory results may reflect different functional states of TRIM25

Recent studies have shown that TRIM25 exhibits complex behaviors, such as undergoing redistribution during viral infection and having multiple RNA-binding domains beyond the previously characterized PRY/SPRY domain . These complexities may explain why different antibodies yield varying results depending on the experimental context.

What emerging techniques might enhance TRIM25 antibody applications in research?

Several cutting-edge techniques are poised to revolutionize TRIM25 antibody applications in research:

• Proximity labeling approaches:

  • TurboID or APEX2 fusions with TRIM25 combined with antibody-based detection can map the dynamic TRIM25 interactome during viral infection

  • RNA-protein proximity labeling using TRIM25 antibodies could identify transient RNA interactions that are difficult to capture with traditional methods

  • G3BP1 proximity-dependent biotinylation assays have already revealed TRIM25's enrichment in stress granules , and similar approaches can be expanded

• Single-molecule techniques:

  • Single-molecule pull-down (SiMPull) using TRIM25 antibodies can provide insights into stoichiometry and heterogeneity of TRIM25 complexes

  • Single-molecule FRET with antibody fragments can monitor conformational changes in TRIM25 during RNA binding or E3 ligase activation

  • Single-molecule tracking in living cells can reveal the dynamics of TRIM25 recruitment to viral replication sites

• Advanced imaging methods:

  • Expansion microscopy with TRIM25 antibodies can provide super-resolution insights into its localization within stress granules or viral replication organelles

  • Correlative light and electron microscopy (CLEM) can connect TRIM25 immunofluorescence with ultrastructural features

  • Live-cell super-resolution with anti-TRIM25 nanobodies can track dynamic phase separation events in real-time

• High-throughput screening approaches:

  • TRIM25 antibody-based FACS sorting coupled with single-cell transcriptomics can identify cellular states associated with different TRIM25 expression or localization patterns

  • Automated high-content imaging with TRIM25 antibodies can screen for modulators of its antiviral activity or stress granule association

• Structural biology integration:

  • Combining cryo-electron tomography with TRIM25 immunogold labeling can visualize TRIM25 in its native cellular context

  • Hydrogen-deuterium exchange mass spectrometry with TRIM25 antibodies can map conformational changes upon RNA binding or during viral infection

• In situ analysis:

  • Proximity ligation assays with TRIM25 antibodies can detect protein-protein interactions in fixed cells with high sensitivity

  • In situ sequencing of TRIM25-bound RNAs using antibody-based capture can map the spatial distribution of these interactions

  • CODEX or other highly multiplexed imaging approaches can simultaneously visualize TRIM25 alongside dozens of other proteins in tissues

These emerging techniques, when combined with traditional antibody applications, will provide unprecedented insights into TRIM25's complex roles in antiviral immunity and RNA metabolism. For example, they could help elucidate how TRIM25 targets viral mRNAs for degradation or how it regulates the RIG-I signaling pathway in different cellular contexts .

How might TRIM25 antibodies help resolve current controversies in innate immunity research?

TRIM25 antibodies can serve as critical tools for resolving several ongoing controversies in innate immunity research:

• The precise role of TRIM25 in RIG-I activation:

  • Controversy: While TRIM25 was initially described as essential for RIG-I activation through K63-linked ubiquitination , recent studies suggest it may be redundant in human cells, with RIPLET playing the critical role .

  • Resolution approach: TRIM25 antibodies can be used in careful time-course studies with cell-type specific knockouts to delineate the temporal and contextual requirements for TRIM25 in RIG-I activation across different cell types and species.

• RNA-binding versus E3 ligase activity in antiviral functions:

  • Controversy: Studies show that both RNA-binding deficient mutant (TRIM25ΔRBD) and E3 ubiquitin ligase mutant (TRIM25ΔRING) can still inhibit IAV replication, raising questions about which activity is essential .

  • Resolution approach: Domain-specific TRIM25 antibodies can track the recruitment and activity of different TRIM25 mutants during infection, helping to determine which functions are critical in different viral contexts.

• Cell-type specificity of TRIM25 functions:

  • Controversy: TRIM25 knockout affects RIG-I response in mouse embryonic fibroblasts (MEFs) but not in human HEK293 cells , suggesting species or cell-type specific roles.

  • Resolution approach: Comparative immunoprecipitation studies with TRIM25 antibodies across different cell types can identify cell-specific interaction partners that might explain these functional differences.

• Mechanism of TRIM25-mediated viral RNA degradation:

  • Controversy: TRIM25 has been shown to destabilize viral mRNAs , but the exact mechanism and whether this requires cofactors remains unclear.

  • Resolution approach: TRIM25 antibodies can be used to isolate TRIM25-containing ribonucleoprotein complexes from infected cells to identify the complete machinery involved in viral RNA degradation.

• Functional significance of TRIM25 in stress granules:

  • Controversy: TRIM25 strongly associates with antiviral stress granules , but whether this is a cause or consequence of its antiviral activity is debated.

  • Resolution approach: Using TRIM25 antibodies in live-cell imaging coupled with functional assays can establish the temporal relationship between TRIM25 recruitment to stress granules and inhibition of viral replication.

• Multiple RNA-binding domains controversy:

  • Controversy: While the PRY/SPRY domain was initially identified as TRIM25's RNA-binding domain, additional domains have been found , and TRIM25ΔRBD can still bind viral RNAs through unknown mechanisms .

  • Resolution approach: Domain-specific TRIM25 antibodies combined with RNA crosslinking studies can map the complete spectrum of RNA-binding regions and their relative contributions.

By applying TRIM25 antibodies in these carefully designed experimental approaches, researchers can help resolve these fundamental controversies in innate immunity research and advance our understanding of host-virus interactions.