EIF4A3 Antibody

What is EIF4A3 and what are its primary cellular functions?

EIF4A3 (eukaryotic translation initiation factor 4A, isoform 3) is a DEAD-box RNA helicase and a core component of the exon junction complex (EJC) . Despite its name suggesting involvement in translation initiation (like its family members eIF4A1 and eIF4A2), EIF4A3 is functionally distinct . It plays crucial roles in post-splicing events including mRNA export, cytoplasmic localization, and nonsense-mediated decay (NMD) . The protein is predominantly localized in the nucleus, unlike the cytoplasmic eIF4A1 and eIF4A2 variants .

Recent research has uncovered unexpected non-canonical functions of EIF4A3 beyond RNA processing. Studies have demonstrated that EIF4A3 can directly bind to microtubules independent of its EJC and RNA interactions, enabling it to promote microtubule polymerization and neuronal growth . This dual functionality makes EIF4A3 a fascinating protein that links RNA metabolism with cytoskeletal regulation.

How does EIF4A3's structure relate to its function?

EIF4A3 is a 47 kDa protein that functions as an ATP-dependent RNA clamp within the EJC . Its structure enables it to serve as a nucleation center for recruiting other EJC components . The protein's RNA binding and ATPase activities are essential for its function in the EJC, but these activities can be regulated differently depending on interacting partners:

This regulatory mechanism ensures that EIF4A3 can be stabilized on RNA in specific contexts, particularly after splicing events .

What techniques are most effective for detecting EIF4A3 in experimental settings?

Based on empirical evidence, researchers have successfully detected EIF4A3 using several complementary approaches:

When designing experiments to detect EIF4A3, it's important to note that the antibody reactivity has been validated with human and mouse samples . For optimal results, sample-dependent titration is recommended to determine ideal conditions for your specific experimental system.

How does EIF4A3 contribute to axon development and neuronal growth?

Recent studies have revealed a surprising non-canonical role for EIF4A3 in promoting axon development. Unlike its EJC partners MAGOH and RBM8A, EIF4A3 uniquely influences axonal growth through direct interaction with the cytoskeleton . In conditional knockout models (Eif4a3 cKO), axon length was reduced by approximately 35% compared to controls, and the proportion of polarized neurons was reduced by 38% .

The mechanism appears to be independent of EIF4A3's role in the EJC. When researchers depleted other EJC components (RBM8A and MAGOH) using shRNAs, they observed no impact on axonal growth, further supporting the unique requirement of EIF4A3 for axonal development .

Most significantly, rescue experiments demonstrated that EIF4A3 controls axonal and neurite outgrowth in an EJC- and RNA-independent manner . This finding represents a fundamental mechanism by which neurons repurpose core gene expression machinery to directly control cytoskeletal dynamics.

What methods can be used to study EIF4A3's microtubule-binding properties?

To investigate EIF4A3's interaction with microtubules, researchers can employ several approaches:

• Biochemical assays: Direct binding assays and competition experiments can demonstrate that EIF4A3 binds to microtubules in a manner that is mutually exclusive with its EJC binding .

• In vitro reconstitution assays: These can show that EIF4A3 is sufficient to promote microtubule polymerization .

• Live imaging of growing neurons: This technique reveals EIF4A3's essential role in microtubule dynamics during neuronal development .

• Rescue experiments: Introducing wild-type or mutant EIF4A3 into Eif4a3-deficient neurons can determine which domains are critical for rescuing axonal growth phenotypes .

When designing experiments to study this interaction, it's important to distinguish between EIF4A3's canonical RNA-binding functions and its direct effects on the cytoskeleton.

What is the significance of EIF4A3 as a cancer biomarker?

Pan-cancer analysis has revealed that EIF4A3 emerges as a promising biomarker with potential to enhance tumor screening, prognostic evaluation, and design of individualized treatment strategies . Studies have shown significantly increased expression levels of EIF4A3 in most cancer types compared to normal tissue .

Particularly in bladder cancer, EIF4A3 expression was considerably higher in tumor tissue compared to para-cancer tissue. Validation experiments confirmed that bladder cancer patients with higher EIF4A3 expression had significantly worse prognostic outcomes . This pattern was observed across multiple cancer types, where high expression levels of EIF4A3 were negatively associated with patient prognosis .

As a novel m6A suppressor, EIF4A3 controls global m6A mRNA modification levels, influencing gene expression patterns relevant to cancer progression . Interestingly, it was found to be correlated with numerous RNA modification genes across multiple cancer types .

How does EIF4A3 relate to cancer immunotherapy?

Analysis of publicly available databases has revealed that EIF4A3 expression is significantly related to immune score and immune cell levels in most cancer types . Intriguingly, EIF4A3 was identified as a superior immunotherapy biomarker when compared to several traditional immunotherapy biomarkers .

This finding suggests potential applications in predicting immunotherapy response and possibly developing new therapeutic approaches targeting EIF4A3. Researchers investigating cancer immunology should consider examining EIF4A3 expression patterns in their specific cancer models to determine potential relevance to immune response mechanisms.

How does EIF4A3 function in nonsense-mediated decay (NMD)?

EIF4A3 serves as a critical component of the nonsense-mediated decay (NMD) pathway, which is a surveillance mechanism that recognizes mRNAs containing premature termination codons to prevent the accumulation of truncated proteins . As a core component of the EJC, EIF4A3 acts as an ATP-dependent RNA clamp that serves as a nucleation center to recruit other EJC components .

Research has demonstrated that siRNA-mediated knockdown of EIF4A3 leads to a defect in NMD . This makes it an excellent target for studying NMD mechanisms. Notably, selective inhibitors of EIF4A3 have been developed that show significant NMD inhibitory activity in cellular contexts .

When studying NMD, it's important to design appropriate reporter systems that can distinguish between different steps of the process. Monitoring the levels of known NMD targets after EIF4A3 manipulation can provide insights into the efficiency of the pathway.

What is known about EIF4A3's role in viral RNA processing?

Studies investigating human papillomavirus (HPV) have examined EIF4A3's potential role in processing viral transcripts. Interestingly, depletion of EIF4A3 did not alter the splicing profile of HPV16 early transcripts but did induce an increase in E7 oncoprotein levels .

These findings indicate that while EIF4A3 does not influence the localization of these viral mRNAs, it does regulate their cellular levels . This suggests a potential role for EIF4A3 in controlling viral gene expression through mechanisms distinct from its canonical functions in RNA splicing and export.

What controls should be included when studying EIF4A3 knockdown effects?

When designing experiments to study the effects of EIF4A3 knockdown, several controls should be implemented:

• Positive controls for knockdown efficiency: Include known targets of EIF4A3 function. For example, researchers have used KPNA1 as a positive control since EIF4A3 depletion leads to an increase in exon skipping of the constitutive exon 11 of KPNA1 .

• Rescue experiments: To confirm that observed phenotypes are specifically due to EIF4A3 loss, include conditions where wild-type EIF4A3 is reintroduced to knockdown cells .

• Alternative splicing controls: When studying EIF4A3's role in RNA processing, include a panel of transcripts with documented EIF4A3-dependent splicing patterns.

• Cell viability assessment: Since EIF4A3 loss can induce apoptosis in some contexts, include measures to distinguish between direct molecular effects and secondary consequences of cell death. In some studies, researchers generated a double conditional knockout of both Eif4a3 and p53 to assess axonal tract development in the absence of apoptosis .

How can I distinguish between EJC-dependent and EJC-independent functions of EIF4A3?

Distinguishing between EIF4A3's canonical role in the EJC and its emerging non-canonical functions requires careful experimental design:

• Comparative knockdown studies: Deplete other EJC components (MAGOH, RBM8A) alongside EIF4A3 and compare phenotypes. Functions that are uniquely affected by EIF4A3 depletion but not by depletion of other EJC components are likely EJC-independent .

• Domain-specific mutations: Use EIF4A3 mutants that selectively disrupt either its RNA-binding, ATP-binding, or protein-protein interaction capabilities to determine which domains are essential for the function being studied .

• Rescue experiments with specific variants: Test whether wild-type EIF4A3 or mutant versions with compromised EJC or RNA binding can rescue phenotypes in EIF4A3-deficient models .

• Co-localization studies: Determine whether EIF4A3 co-localizes with EJC components or with other cellular structures (like microtubules) in the context being studied .

In neuronal development studies, researchers demonstrated EIF4A3's EJC-independent function by showing that a mutant form of EIF4A3 deficient in RNA binding could still rescue axonal growth defects .