Recombinant Mouse mRNA export factor (Rae1)

What is the structural and functional characterization of mouse RAE1?

Mouse RAE1 contains four WD40 motifs and a C-terminal non-WD-repeat extension that facilitate binding to the GLEBS-like motif in NUP98. This interaction is critical for mRNA export, as demonstrated by studies showing that overexpression of the GLEBS-like motif inhibits nuclear envelope binding of RAE1 and induces nuclear accumulation of poly(A)+ RNA . RAE1 shuttles between the nucleus and cytoplasm in a temperature-dependent and RanGTP-independent manner . The docking of RAE1 to the nuclear envelope is highly dependent on new mRNA synthesis, indicating dynamic regulation by transcriptional processes . Beyond mRNA export, RAE1 functions as a mitotic checkpoint regulator essential for chromosomal stability and early embryogenesis, as evidenced by the embryonic lethality of RAE1-null mice .

How should recombinant mouse RAE1 be reconstituted and stored for optimal experimental use?

Recombinant Mouse Rae-1 epsilon Fc Chimera Protein is typically provided in lyophilized form from a 0.2 μm filtered solution in PBS. For optimal reconstitution, researchers should:

• Reconstitute at a concentration of 100 μg/mL in sterile PBS

• Use a manual defrost freezer for storage

• Avoid repeated freeze-thaw cycles to maintain protein integrity

For ELISA kits detecting mouse RAE1, storage recommendations include:

• Shipping at 4°C

• Upon receipt, store according to the kit's manual

• Typical validity period of 6 months

• Stability determined by activity loss rate (less than 5% within expiration date under appropriate storage conditions)

When handling recombinant RAE1 during purification, protease inhibitors (2 mM PMSF, 1 mM leupeptin, 2 mM aprotinin, and 1 mM pepstatin) should be incorporated to protect against proteolytic degradation .

What expression systems are most effective for producing functional recombinant mouse RAE1?

Several expression systems have been successfully employed for producing recombinant mouse RAE1, each with specific advantages depending on experimental requirements:

How can researchers experimentally distinguish between RAE1's role in mRNA export versus mitotic checkpoint regulation?

Distinguishing between RAE1's dual functions presents several methodological challenges requiring strategic experimental approaches:

• Temporal separation strategies: Utilize synchronized cell populations to isolate interphase (mRNA export) versus mitotic (checkpoint) functions. Cell cycle inhibitors can help maintain cells in specific phases for isolated functional analysis.

• Domain-specific mutations: Based on structural data of the RAE1-NUP98 interaction, create targeted mutations that specifically disrupt one function while preserving the other. For example, mutations in the GLEBS-like binding region would primarily affect mRNA export .

• Cell cycle-dependent localization analysis: Track RAE1 localization throughout the cell cycle using fluorescence microscopy. During interphase, functional RAE1 localizes to the nuclear envelope, while during mitosis, it associates with spindle apparatus components .

• Function-specific readouts:

  • For mRNA export: Poly(A)+ RNA in situ hybridization using FITC-coupled oligo-(dT) 50 mer probes

  • For mitotic checkpoint: Chromosome spreads analyzing aneuploidy rates (RAE1+/- MEFs show 20 ± 2% versus wild-type 9 ± 1% aneuploid metaphases)

• Rescue experiments with selective complementation: Introduce specific RAE1 mutants into RAE1-deficient backgrounds to determine which domains rescue particular phenotypes.

These approaches collectively enable separation of RAE1's distinct cellular functions while minimizing confounding variables from interconnected cellular processes.

What molecular mechanisms underlie RAE1's contribution to cancer progression?

RAE1 promotes cancer progression through multiple interconnected mechanisms, as evidenced by studies across cancer types:

• Genomic amplification driving overexpression: In colorectal cancer, RAE1 is amplified in 76.1% of tumors with copy numbers positively correlated with mRNA expression levels (R = 0.78, P < 0.001) .

• Anti-apoptotic effects: RAE1 inhibits apoptosis in cancer cells, conferring survival advantages and chemoresistance. In vitro and in vivo analyses confirmed this anti-apoptotic function as a key driver of tumor growth .

• Cell cycle dysregulation: As a mitotic checkpoint regulator, RAE1 promotes cell cycle progression in cancer cells. This effect appears linked to decreased proportions of multipolar spindle cells in colorectal cancer, potentially preventing mitotic catastrophe despite genomic instability .

• Epithelial-mesenchymal transition (EMT) induction: In breast cancer models, RAE1 enhances aggressive phenotypes by inducing EMT signals that facilitate invasion and metastasis .

• Clinical implications: High RAE1 expression correlates with:

  • Increased distant metastasis

  • Poor survival outcomes (multivariate Cox: HR = 8.61, 95% CI = 2.41-30.7, P < 0.001)

  • Independent prognostic value in multivariate models

These findings collectively position RAE1 as a multifunctional oncogenic driver affecting both cell survival and cell division processes, with potential as a therapeutic target, particularly for overcoming chemoresistance in colorectal cancer .

What are the most effective methodologies for studying RAE1-dependent mRNA export?

Multiple complementary approaches can effectively measure and characterize RAE1-dependent mRNA export:

• Combined immunofluorescence and RNA FISH: This two-step approach allows simultaneous visualization of:

  • RAE1 protein localization (using affinity-purified antibodies)

  • poly(A)+ RNA distribution (using FITC-coupled oligo-dT probes)
    Followed by confocal microscopy analysis to determine spatial relationships between RAE1 and exported mRNAs .

• Functional perturbation of RAE1-NUP98 interaction: Overexpression of the GLEBS-like motif serves as a competitive inhibitor that:

  • Disrupts RAE1 docking to the nuclear envelope

  • Induces nuclear accumulation of poly(A)+ RNA

  • Can be reversed by point mutations in the GLEBS-like motif or RAE1 overexpression
    This approach provides strong functional evidence for RAE1's direct role in mRNA export .

• Temperature-dependent shuttling assays: Leveraging RAE1's temperature-dependent nucleocytoplasmic shuttling properties allows controlled modulation of export dynamics. Unlike many export factors, RAE1 shuttling is RanGTP-independent, making it distinctly amenable to temperature manipulation .

• Transcription-dependent analyses: Since RAE1's nuclear envelope docking depends on active mRNA synthesis, transcription inhibitors can be used to differentiate RAE1-specific export from other pathways .

• Viral inhibition models: The ORF10 protein from gammaherpesviruses selectively inhibits RAE1-dependent mRNA export, providing a useful tool for studying the specifics of this pathway. Structural studies of the ORF10-RAE1-NUP98 complex have revealed mechanistic details about how this process functions .

These methodologies collectively provide robust approaches for characterizing RAE1's role in mRNA export across different experimental contexts.

How does the RAE1-NUP98 interaction specifically mediate mRNA export and how can this interaction be manipulated experimentally?

The RAE1-NUP98 interaction forms a critical functional unit in mRNA export with several key features:

• Structural basis: RAE1 binds to a GLEBS-like NUP98 motif at the nuclear pore complex through:

  • Multiple WD-repeat domains

  • C-terminal non-WD-repeat extension
    This interaction is direct, as confirmed by in vitro binding studies and chemical cross-linking .

• Functional significance: The RAE1-NUP98 interaction serves as an anchoring mechanism for mRNA export, with experimental evidence showing that:

  • Overexpression of the GLEBS-like motif inhibits RAE1 binding to the nuclear envelope

  • This inhibition causes nuclear accumulation of poly(A)+ RNA

  • Both effects are reversed by point mutations in the GLEBS-like motif or RAE1 overexpression

• Experimental manipulation approaches:

  Manipulation Method	Technique	Outcome	Reference
  Competitive inhibition	Overexpression of GLEBS-like motif	Blocks RAE1-NUP98 interaction
  Mutational analysis	Point mutations in GLEBS-like motif	Abrogates inhibitory effect
  Rescue experiments	RAE1 overexpression	Restores mRNA export despite GLEBS inhibition
  Biochemical disruption	In vitro binding with recombinant proteins	Characterizes interaction parameters
  Viral interference	ORF10 from gammaherpesviruses	Reveals structural basis of selective inhibition

• Differential regulation: The RAE1-NUP98 interaction is dynamically regulated, with:

  • Strong dependence on new mRNA synthesis

  • Temperature-dependent but RanGTP-independent shuttling properties

  • Potential for selective inhibition by viral factors

These characteristics make the RAE1-NUP98 interaction a valuable target for studying mRNA export mechanisms and potentially developing therapeutic interventions for conditions involving aberrant mRNA export.

What experimental controls are essential when studying RAE1 haplo-insufficiency phenotypes?

• Genotype controls:

  • Wild-type cells/animals (RAE1+/+) as baseline control

  • Complete knockout (if viable in the system) or strong knockdown to establish a phenotypic spectrum

  • Chromosome spreads should include analysis from both RAE1+/+ and RAE1+/- cells (expected aneuploidy rates: 9 ± 1% vs. 20 ± 2%)

• Functional rescue controls:

  • Re-expression of wild-type RAE1 in haplo-insufficient cells should restore normal phenotype

  • Domain-specific RAE1 mutants can identify which functions are critical for specific phenotypes

  • Titrated expression levels to establish dose-dependency

• Temporal and developmental controls:

  • Stage-specific analyses (RAE1-null causes embryonic lethality while haplo-insufficiency permits development with specific defects)

  • Cell cycle phase-specific measurements to differentiate interphase versus mitotic phenotypes

• Specificity controls:

  • Parallel analysis of haplo-insufficiency for related proteins (other WD40 repeat proteins or mRNA export factors)

  • Analysis of both mRNA export and chromosome segregation phenotypes to distinguish primary from secondary effects

• Methodological controls:

  • For chromosome spreads: properly synchronized populations, multiple time points after synchronization

  • For mRNA export: both nuclear accumulation and cytoplasmic depletion measurements

  • For spindle morphology: multiple spindle markers and quantitative analysis methods

Proper implementation of these controls helps distinguish direct consequences of RAE1 haplo-insufficiency from indirect effects or experimental artifacts, enabling more reliable interpretation of the complex phenotypes associated with reduced RAE1 levels.

How can viral interactions with RAE1 be leveraged to understand mRNA export mechanisms?

Viral interactions with RAE1, particularly through ORF10 from gammaherpesviruses, provide unique insights into mRNA export mechanisms:

• Selective inhibition model: ORF10 inhibits mRNA export in a transcript-selective manner by forming a complex with RAE1 and NUP98, unlike most viral inhibitors that block export non-selectively .

• Structural insights: The resolved structure of the ORF10-RAE1-NUP98 ternary complex reveals:

  • Detailed intermolecular interactions

  • Two highly conserved residues of ORF10 (L60 and M413) that are critical for complex assembly and export inhibition

  • Mechanistic basis for selective transcript inhibition

• RNA binding characteristics: Although ORF10 occupies the RNA-binding groove of RAE1-NUP98, the ternary complex maintains RNA-binding ability through ORF10-RNA direct interaction, revealing a sophisticated mechanism of selective inhibition without complete functional blockade .

• Experimental applications:

  • Using ORF10 mutations to map critical interaction surfaces for mRNA export

  • Developing ORF10-derived tools to selectively inhibit export of specific transcripts

  • Utilizing viral inhibition as a temporal control system for RAE1 function

These viral interactions provide a natural "molecular scalpel" for dissecting RAE1 function with greater precision than traditional genetic knockdown approaches, offering unique opportunities to understand the specificity and regulation of mRNA export pathways.

What technical challenges arise when measuring RAE1-dependent phenotypes in cancer models?

Investigating RAE1 in cancer models presents several technical challenges that require careful experimental design:

• Copy number variation complexities:

  • RAE1 is amplified in 76.1% of colorectal cancer tissues

  • Copy numbers correlate with expression (R = 0.78, P < 0.001), requiring normalization strategies when comparing across samples

• Distinguishing driver from passenger effects:

  • Despite strong correlation with poor prognosis (HR = 8.61), establishing causality requires:

    • Carefully controlled overexpression and knockdown experiments

    • Rescue experiments with wild-type and mutant constructs

    • Analysis of downstream pathways to establish mechanism

• Context-dependent function detection:

  • RAE1 localizes differently in normal versus cancer cells:

    • Normal cells: weak to moderate staining

    • Cancer cells: strong cytoplasmic and membrane staining

  • Immunohistochemical protocols must be optimized for this range of expression

• Measuring dual functionality:

  • Simultaneous assessment of:

    • mRNA export (using RNA FISH)

    • Mitotic checkpoint function (using chromosome spreads)

    • Apoptosis inhibition (using appropriate markers)

    • Cell cycle effects (using flow cytometry)

• Technical considerations for specific assays:

  • Immunohistochemistry requires careful optimization for membrane versus cytoplasmic staining

  • RNA export assays need nuclear-cytoplasmic fractionation quality controls

  • Spindle morphology analysis requires careful quantification of multipolar versus bipolar spindles

These challenges require integrated experimental approaches that account for the pleiotropic effects of RAE1 in cancer cells while maintaining technical rigor in quantitative assessments.

What approaches can effectively isolate RAE1's direct effects on the mitotic checkpoint from its mRNA export functions?

Isolating RAE1's mitotic checkpoint role from its mRNA export functions requires strategic experimental approaches:

• Acute versus chronic depletion comparisons:

  • Inducible degradation systems to assess immediate mitotic effects (6-12 hours)

  • Stable knockdown for long-term consequences

  • This temporal separation helps distinguish direct mitotic functions from indirect effects mediated through altered gene expression

• Structure-function dissection:

  • Engineer domain-specific RAE1 mutations based on structural data

  • Test these mutants for selective rescue of either mRNA export or mitotic checkpoint defects

  • Focus on regions that interact with mitotic checkpoint proteins versus NUP98

• Cell cycle-specific functional assays:

  • Mitotic checkpoint strength measurements:

    • Time from nuclear envelope breakdown to anaphase onset

    • Response to spindle poisons

    • Measurement of chromosome missegregation rates (20 ± 2% in RAE1+/- versus 9 ± 1% in wild-type)

  • mRNA export efficiency in the same cells:

    • Nuclear/cytoplasmic ratio of poly(A)+ RNA

    • Export kinetics of reporter mRNAs

• Genetic separation strategies:

  • Compensate for mRNA export defects using alternative export pathways

  • Use fast-acting chemical inhibitors of specific RAE1 interactions

  • Employ separation-of-function alleles in complementation experiments

• Biochemical isolation approaches:

  • Identify and characterize RAE1-containing protein complexes specific to:

    • Interphase (mRNA export)

    • Mitosis (checkpoint regulation)

  • Determine which protein interactions are essential for each function

These approaches collectively enable researchers to dissect RAE1's complex phenotypes and establish mechanistic relationships between its diverse cellular roles.

How do RAE1-NUP98 and RAE1-viral protein interactions differ structurally and functionally?

Structural and functional comparisons between RAE1-NUP98 and RAE1-viral protein interactions reveal important mechanistic distinctions:

This comparative analysis highlights how viral proteins have evolved to selectively target and modulate RAE1 function in ways that benefit viral replication, while simultaneously revealing fundamental aspects of RAE1's normal cellular functions in mRNA export.

How might RAE1 function as a therapeutic target in cancer treatment strategies?

RAE1's multifaceted roles in cancer progression suggest several promising therapeutic approaches:

• Targeting RAE1 overexpression: High RAE1 expression correlates with poor prognosis (HR = 8.61) and distant metastasis in colorectal cancer, making it a potential marker for aggressive disease requiring intensive treatment .

• Exploiting anti-apoptotic mechanisms: RAE1 inhibits apoptosis and promotes chemoresistance, suggesting that RAE1 inhibition could sensitize resistant tumors to conventional therapies .

• Leveraging RAE1's role in EMT: In breast cancer, RAE1 enhances aggressive phenotypes by inducing epithelial-mesenchymal transition signals. Targeting this pathway could potentially reduce invasive and metastatic potential .

• Therapeutic strategies:

  • Small molecule inhibitors of RAE1-NUP98 interaction

  • Peptide-based inhibitors derived from the GLEBS-like motif

  • RNA interference approaches to reduce RAE1 expression

  • Viral-derived proteins (like modified ORF10) that selectively inhibit RAE1 function

• Biomarker applications:

  • RAE1 expression as a prognostic marker (particularly in estrogen receptor-positive breast cancers)

  • RAE1 amplification for patient stratification

  • Monitoring RAE1-dependent pathways to assess treatment response

These approaches would need to carefully balance inhibition of RAE1's oncogenic functions against potential adverse effects from disrupting its normal cellular roles in mRNA export and mitotic checkpoint regulation.

What is the relationship between RAE1's embryonic lethality in knockout models and its role in cancer progression?

The embryonic lethality of RAE1-null mice and RAE1's role in cancer progression share mechanistic connections with important implications:

• Essential developmental functions: RAE1-null mice exhibit embryonic lethality, indicating that RAE1 is essential for early development, likely due to its fundamental roles in:

  • Chromosome segregation during rapid embryonic cell divisions

  • mRNA export supporting developmental gene expression programs

• Dose-dependent phenotypes: Similar to the embryonic context, cancer cells often exhibit:

  • Increased proliferation rates requiring efficient chromosome segregation

  • Altered gene expression programs dependent on mRNA export

  • Sensitivity to gene dosage effects (76.1% of colorectal cancers show RAE1 amplification)

• Genomic instability connection:

  • RAE1+/- MEFs show increased chromosome missegregation (20 ± 2% vs. 9 ± 1% aneuploid metaphases)

  • Cancer cells often exhibit genomic instability but must avoid catastrophic levels

  • RAE1 overexpression may help cancer cells balance increased proliferation with sufficient mitotic fidelity

• Adaptation mechanisms:

  • Cancer cells adapt to rely on RAE1 overexpression

  • This creates a targetable dependency not present in normal differentiated cells

  • Potential therapeutic window between cancer cells and normal tissues

This relationship suggests that cancer cells co-opt RAE1's essential functions in development to support their own pathological growth, creating both vulnerabilities and resistance mechanisms that could be exploited therapeutically.

How does mouse RAE1 function compare with its human counterpart in experimental systems?

Comparative analysis of mouse and human RAE1 reveals important similarities and differences relevant to experimental design:

• Sequence and structural conservation:

  • Both contain four WD40 motifs critical for protein interactions

  • High degree of homology in GLEBS-binding domains

  • Similar localization patterns at the nuclear envelope and throughout the cytoplasm

• Functional conservation:

  • Both function in mRNA export through interaction with NUP98

  • Both serve as mitotic checkpoint regulators

  • Both implicated in cancer progression when overexpressed

• Experimental considerations:

  • Antibody cross-reactivity: Some antibodies may recognize epitopes common to both species, while others are species-specific

  • Recombinant protein production: Similar expression systems work for both human and mouse RAE1, though optimal conditions may differ

  • Interspecies complementation: Human RAE1 can often substitute for mouse RAE1 in functional assays, though with potential quantitative differences

• Distinct experimental tools:

  • Mouse models allow in vivo studies of RAE1 haplo-insufficiency and tissue-specific effects

  • Human cancer cell lines provide clinically relevant contexts for studying RAE1 in disease

  • Species-specific interaction partners may exist and should be considered when translating findings between systems

• RAE1 expression detection tools:

  • Mouse-specific ELISA kits are available for detecting native RAE1 in tissue homogenates, cell lysates and biological fluids

  • These detection methods may not readily transfer to human samples without validation

Understanding these similarities and differences is essential for designing experiments and interpreting results across species, particularly when translating findings from mouse models to human disease contexts.

What is the current understanding of RAE1's role in viral pathogenesis beyond mRNA export inhibition?

Beyond its well-established role in viral-mediated mRNA export inhibition, RAE1's interaction with viral proteins has broader implications for viral pathogenesis:

• Selective rather than global inhibition strategy:

  • Unlike many viral inhibitors that block export non-selectively, ORF10 from gammaherpesviruses targets RAE1 to inhibit export in a transcript-selective manner

  • This selective approach may allow viruses to modulate host gene expression patterns rather than completely shutting down cellular functions

• Potential impact on mitotic checkpoint:

  • Given RAE1's dual role in mRNA export and mitotic checkpoint regulation, viral interactions may inadvertently (or purposefully) affect chromosome segregation

  • This could contribute to genomic instability in chronically infected cells

• Competing mechanisms:

  • Although ORF10 occupies the RNA-binding groove of the RAE1-NUP98 complex, the ternary complex maintains RNA-binding ability through ORF10-RNA direct interaction

  • This reveals sophisticated viral adaptation to modulate rather than completely block host functions

• Evolutionary insights:

  • The targeted interaction between viral proteins and RAE1 suggests strong evolutionary pressure to develop specific inhibition mechanisms

  • This specificity indicates the particular importance of RAE1-mediated mRNA export for antiviral responses

• Potential therapeutic implications:

  • Understanding the molecular details of viral protein-RAE1 interactions could lead to the development of antiviral strategies

  • Structural insights from the ORF10-RAE1-NUP98 complex provide templates for designing inhibitors of these interactions

These broader roles in viral pathogenesis highlight RAE1 as a nexus of host-pathogen interactions with implications beyond simple mRNA export inhibition.