EIF1AX Human

What is the structure and function of human EIF1AX?

Human EIF1AX is a 16-17 kDa protein encoded by the EIF1AX gene located on the X chromosome. The protein contains several key structural domains:

• An RNA-binding domain (amino acids 32-95) that shares homology with bacterial IF1

• A helical domain adjacent to the RNA-binding fold

• Unstructured N-terminal tail (NTT) and C-terminal tail (CTT)

• The CTT features primarily negative charges with hydrophobic residues at the terminus involved in protein-protein interactions

Functionally, EIF1AX serves as an essential eukaryotic translation initiation factor that promotes 43S pre-initiation complex (PIC) formation by stabilizing the binding of the ternary complex eIF2-GTP-methionyl-initiator tRNA to the 40S ribosomal subunit . It enhances ribosome dissociation into subunits and plays a critical role in the scanning mechanism that accurately locates the proper start codon on mRNA in eukaryotes .

How can researchers detect and analyze EIF1AX expression in human tissue samples?

Detection of EIF1AX in human tissues can be accomplished through several validated methods:

Western Blot Analysis:

• Recommended antibody dilution: 1:500-1:1000

• Expected molecular weight: 16-23 kDa (observed range)

• Positive controls: HepG2, A375, HeLa, and PC-3 cell lines have confirmed expression

Immunofluorescence/Immunocytochemistry:

• Recommended antibody dilution: 1:50-1:500

• Validated in HepG2 cells

Protocol Considerations:

• Storage of antibodies at -20°C maintains stability for one year after shipment

• For western blot applications, using PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) as a storage buffer is recommended

Researchers should titrate antibody concentrations in each testing system to achieve optimal signal-to-noise ratios for their specific experimental conditions.

What experimental models are most suitable for studying EIF1AX function?

When designing experiments to study EIF1AX function, researchers should consider:

Cell Line Models:

• HepG2, A375, HeLa, and PC-3 cell lines have confirmed EIF1AX expression and are suitable for in vitro studies

• Thyroid cancer cell lines are particularly valuable for investigating EIF1AX's role in tumorigenesis given the recurrent mutations observed in thyroid cancers

Functional Assays:

• Translation efficiency assays to measure the impact of EIF1AX variants on protein synthesis rates

• Ribosome profiling to assess alterations in start codon selection

• 43S and 48S complex formation assays to evaluate pre-initiation complex assembly

• Protein-protein interaction studies to investigate binding with other translation initiation factors

Technical Considerations:

• For mutation studies, targeting exons 2, 5, and 6 is critical as these regions contain the most frequently observed mutations

• When studying protein interactions, consider techniques that preserve the native conformation of the unstructured N- and C-terminal tails

What is the spectrum of EIF1AX mutations in human cancers and their functional implications?

EIF1AX mutations display distinct patterns across cancer types with significant functional implications:

Mutation Hotspots and Distribution:

Specific Mutations and Functional Impact:

• Codon 9 mutations (24% of cases): Substitution of glycine by arginine, aspartic acid, or valine

• Codon 13 mutations (12% of cases): Substitution of arginine to leucine, aspartic acid, or proline

• Codon 113 splice-site mutations affect consensus intronic nucleotides (c.338-1A and c.338-2G)

Functional Consequences:

• NTT mutations promote an open scanning-conducive conformation of PIC, potentially leading to leaky scanning

• CTT mutations may impair proper start codon recognition

• Both NTT and CTT mutations can disrupt preinitiation complex assembly

These mutations may contribute to carcinogenesis by altering translation initiation fidelity, potentially leading to dysregulated expression of oncogenes or tumor suppressors through mechanisms such as leaky scanning or alternative start codon usage.

What methodologies are most effective for detecting EIF1AX mutations in clinical samples?

Based on current research practices, the following methodological approaches are recommended for detecting EIF1AX mutations:

Sanger Sequencing:

• Particularly effective for targeted analysis of hotspot regions in exons

• Primary focus should be on exons 2, 5, and 6 which harbor most clinically relevant mutations

• Advantages: Accessible technology, straightforward interpretation

• Limitations: Lower sensitivity (approximately 15-20% mutant allele frequency required)

Next-Generation Sequencing:

• Preferred for comprehensive mutational profiling

• Panel-based approaches (e.g., ThyroSeq v2) that include EIF1AX alongside other cancer-associated genes provide contextual information

• Technical specifications:

  • Library preparation using amplicon-based approaches

  • Verification of library concentration and amplicon size using high-sensitivity DNA analysis

  • Sequencing on platforms such as Ion Torrent with appropriate template preparation

• Advantages: Higher sensitivity, ability to detect low-frequency variants, comprehensive genetic profiling

• Limitations: Higher cost, more complex bioinformatic analysis

Recommended Workflow:

• Initial screening with targeted NGS panels in tumors

• Confirmation of novel or unusual variants with Sanger sequencing

• Correlation with protein expression using immunohistochemistry or western blot

• Functional validation of novel variants using in vitro translation assays

How does EIF1AX interact with other components of the translation initiation machinery?

EIF1AX participates in a complex network of interactions within the translation initiation machinery:

Key Protein-Protein Interactions:

• EIF1AX interacts with IPO13 (Importin 13), suggesting a role in nuclear-cytoplasmic shuttling

• Within the 43S preinitiation complex, EIF1AX interacts with:

  • The 40S ribosomal subunit near the A-site

  • eIF2-GTP-methionyl-initiator tRNA ternary complex, stabilizing its binding

  • Other initiation factors involved in scanning and start codon recognition

Functional Dynamics:

• The NTT and CTT domains of EIF1AX act in opposite manners on scanning and start codon recognition

• The CTT contains hydrophobic residues at its terminus that likely mediate protein-protein interactions

• Mutations on the RNA-binding surface can disrupt proper 43S and 48S preinitiation complex formation

Methodological Approaches for Studying Interactions:

• Co-immunoprecipitation with other initiation factors

• Cryo-electron microscopy of ribosomal complexes

• Protein crosslinking followed by mass spectrometry

• Yeast two-hybrid or mammalian two-hybrid assays

• Surface plasmon resonance for quantitative binding kinetics

Understanding these interactions is crucial for interpreting how mutations may disrupt translation initiation and contribute to disease states.

What are the clinical and biological differences between EIF1AX mutations in benign versus malignant thyroid nodules?

EIF1AX mutations occur in both benign and malignant thyroid nodules, with distinct characteristics:

Mutation Patterns:

• Both benign nodules and carcinomas can harbor EIF1AX mutations

• Splice-site mutations affecting codon 113 (intron 5/exon 6 junction) were identified in 46% of EIF1AX-positive cases

• Codon 9 mutations were found in 24% of cases, while codon 13 mutations appeared in 12% of cases

Co-occurring Genetic Alterations:

• The mutational context differs between benign and malignant nodules

• In malignant thyroid cancers, EIF1AX mutations have been observed in conjunction with other oncogenic drivers

• Comprehensive mutational profiling using panels that assess other thyroid cancer-related genes (AKT1, BRAF, NRAS, HRAS, KRAS, etc.) provides crucial context for interpreting the significance of EIF1AX mutations

Biological Impact:

• EIF1AX mutations may represent early events in thyroid tumorigenesis

• Different mutations may have varying effects on translation initiation fidelity

• The specific impact on cellular phenotype likely depends on:

  • The exact mutation and domain affected

  • Co-occurring genetic alterations

  • Cell-type specific factors

Research Recommendations:

• Paired analysis of benign and malignant regions from the same patient

• Longitudinal studies of benign nodules with EIF1AX mutations to assess malignant transformation risk

• Functional studies comparing the effects of identical mutations in benign versus malignant cellular contexts

What approaches are recommended for functional validation of novel EIF1AX variants?

To functionally validate novel EIF1AX variants, researchers should implement a multi-faceted approach:

In Vitro Translation Assays:

• Cell-free translation systems to directly measure the impact on translation initiation efficiency

• Reporter constructs with various start codon contexts to assess scanning fidelity

• Measurement of 43S and 48S preinitiation complex formation using sucrose gradient centrifugation

Structural Biology Approaches:

• Cryo-electron microscopy to visualize the impact of mutations on ribosome binding

• NMR spectroscopy for studying effects on the unstructured NTT and CTT domains

• X-ray crystallography for detailed structural analysis of the RNA-binding domain

Cellular Models:

• CRISPR/Cas9-mediated introduction of specific mutations

• Isogenic cell lines with and without the variant of interest

• Polysome profiling to assess global translation effects

• Ribosome profiling to identify specific mRNAs affected by the variant

Protein Interaction Studies:

• Quantitative analysis of binding to known interaction partners (e.g., IPO13)

• Assessment of incorporation into the 43S preinitiation complex

• Protein stability and turnover analysis

Computational Approaches:

• Molecular dynamics simulations to predict structural consequences

• Conservation analysis across species to assess evolutionary constraints

• Integration with public domain data on similar variants

For comprehensive validation, researchers should aim to integrate findings across multiple experimental platforms to build a coherent model of how the variant impacts translation initiation and cellular phenotype.

How might EIF1AX mutations contribute to therapy resistance in cancer?

As a key regulator of translation initiation, EIF1AX mutations could contribute to therapy resistance through several mechanisms:

Altered Stress Response:

• Translation initiation is tightly regulated during cellular stress

• EIF1AX mutations might impair the normal downregulation of translation during stress conditions

• This could lead to continued protein synthesis and survival under therapeutic pressure

Selective Translation of Resistance Factors:

• Mutations affecting start codon selection could alter the translation efficiency of specific mRNAs

• Preferential translation of mRNAs encoding resistance factors might occur

• The "translational landscape" may be reprogrammed to favor survival pathways

Research Approaches:

• Comparison of translatomes (actively translated mRNAs) in treatment-sensitive versus resistant cells with EIF1AX mutations

• Evaluation of stress granule formation and composition in cells with EIF1AX variants

• Assessment of translation efficiency during drug treatment using puromycin incorporation assays

• Testing combination therapies targeting both the primary oncogenic driver and translation initiation

This area represents an exciting frontier in understanding treatment resistance mechanisms and developing new therapeutic strategies.

What is the potential role of EIF1AX in cancer immunotherapy response prediction?

The relationship between translation initiation factors like EIF1AX and immunotherapy response is an emerging area of investigation:

Potential Mechanisms:

• Altered translation could affect presentation of tumor antigens

• Changes in the cellular stress response might influence immune recognition

• Translation of specific immune modulatory factors could be affected

Research Considerations:

• Analysis of neoantigen loads in tumors with versus without EIF1AX mutations

• Correlation of EIF1AX mutational status with immunotherapy response metrics

• Investigation of tumor microenvironment characteristics in EIF1AX-mutant cancers

• Assessment of PD-L1 and other immune checkpoint molecule expression in relation to EIF1AX status

As immunotherapy becomes increasingly important in cancer treatment, understanding how fundamental cellular processes like translation initiation affect immune responses could provide valuable insights for patient stratification and combination therapy development.