Recombinant Xenopus laevis Eukaryotic translation initiation factor 3 subunit M (eif3m)

What is eIF3m and what is its functional role in Xenopus laevis?

eIF3m is one of the thirteen subunits that compose the eukaryotic initiation factor 3 (eIF3) complex. In Xenopus laevis, as in other eukaryotes, eIF3m contributes to the assembly of the translation initiation complex. The eIF3 complex associates with the 40S ribosomal subunit in the 43S pre-initiation complex (PIC) that binds to the 5′ proximal region of mRNAs . This interaction is critical for proper scanning and recognition of the start codon during translation initiation.

Recent research indicates that eIF3m serves as an integral component of a larger "translasome" supercomplex in cells, which contains elongation factors, tRNA-synthetases, 40S and 60S ribosomal proteins, chaperones, and even components of the proteasome . This suggests that eIF3m may play roles beyond the canonical functions in translation initiation, potentially linking protein synthesis to other cellular processes including protein folding and degradation.

The highly conserved nature of eIF3m across species indicates its evolutionary importance, with Xenopus laevis eIF3m sharing significant sequence homology with human eIF3m, making it a valuable model for translational studies with biomedical relevance.

Why is Xenopus laevis an ideal model organism for studying translation factors like eIF3m?

Xenopus laevis offers several unique advantages as a model system for studying translation initiation factors such as eIF3m:

• Physiological synchronicity: Oocytes are naturally blocked in phase G2 of the cell cycle, providing a homogeneous population for experimental studies .

• Exceptional protein synthesis capacity: Each oocyte can produce 200-400 ng of protein per day, making it an efficient system for studying translation .

• Experimental abundance: A single female can provide 800-1,000 oocytes, allowing for robust experimental designs with multiple conditions and replicates .

• Cellular accessibility: The large cell size (1.2-1.4 mm in diameter) facilitates microinjection and manipulation techniques .

• Transcriptional separation: During meiotic maturation, oocytes are transcriptionally repressed, meaning all necessary proteins are translated from preexisting, maternally derived mRNAs. This separation of transcription and translation processes makes it easier to study translation mechanisms in isolation .

• Efficient experimental timeline: Given the speed of meiosis progression and translation after mRNA microinjection (~24 hours), Xenopus oocytes represent a fast system compared to reconstituted cellular systems .

These characteristics make Xenopus laevis an excellent model for studying the function of eIF3m and other translation factors, particularly for examining their roles in maternal mRNA translation and early development.

What are the recommended protocols for cloning and expressing recombinant Xenopus laevis eIF3m?

Based on established methodologies for other eIF3 subunits, the following protocol can be adapted for eIF3m:

• Cloning procedure:

  • Extract total RNA from Xenopus laevis embryos or oocytes using standard TRIzol-based methods

  • Perform RT-PCR using primers specific to the Xenopus laevis eIF3m ORF

  • Clone the amplified fragment into an appropriate expression vector with a suitable tag (His, GST, or FLAG) for purification

• Expression systems:

  • Bacterial expression: E. coli BL21(DE3) strain with pET-derived vectors

  • Insect cell expression: Baculovirus-insect cell system, which has been successfully used for other Xenopus translation factors

  • Cell-free wheat germ extract system, which has been employed for translation studies with other eIF3 components

• Purification approach:

  • Affinity column chromatography using the tag incorporated in the expression construct

  • Gel permeation chromatography for further purification and assessment of complex formation

  • Ion exchange chromatography to separate different forms of the protein

For optimal results with the baculovirus-insect cell system, which has proven effective for Xenopus laevis translation factors, infection at a multiplicity of infection (MOI) of 5-10 and protein expression for 48-72 hours at 27°C is recommended .

How can researchers effectively analyze interactions between eIF3m and other components of the translation machinery?

Multiple complementary approaches can be employed to study eIF3m interactions:

• Affinity purification coupled with mass spectrometry:

  • Create Xenopus laevis cell lines expressing tagged eIF3m at endogenous levels

  • Purify eIF3m complexes using epitope tags with cleavable linkers (e.g., protein A tag with TEV protease cleavage site)

  • Analyze purified complexes using liquid chromatography-tandem mass spectrometry (LC-MS/MS) on a high-sensitivity mass spectrometer

  • Compare results from purifications with RNase treatment to distinguish RNA-dependent from RNA-independent interactions

• Co-immunoprecipitation assays:

  • Express myc-tagged eIF3m with FLAG-tagged potential binding partners

  • Perform immunoprecipitation with anti-myc antibodies

  • Detect co-precipitated proteins via western blot using anti-FLAG antibodies

• Surface plasmon resonance (SPR) analysis:

  • Immobilize purified eIF3m on a sensor chip

  • Flow potential binding partners over the chip at varying concentrations

  • Measure association and dissociation kinetics to determine binding constants

• Sucrose gradient analysis:

  • Prepare cellular extracts from Xenopus embryos at different developmental stages

  • Separate complexes by centrifugation through a sucrose gradient

  • Collect fractions and analyze by western blot for co-migration of eIF3m with other proteins

  • Immunoprecipitate eIF3m from specific fractions to confirm direct interactions

These methodologies can reveal both stable interactions within the core eIF3 complex and more transient interactions with regulatory factors or substrate mRNAs.

What techniques are most effective for studying the function of eIF3m in Xenopus laevis oocytes and embryos?

Several approaches have proven effective for studying eIF3 subunit function in Xenopus:

• Microinjection of mRNA encoding wild-type or mutant eIF3m:

  • Synthesize capped mRNA using mMESSAGE mMACHINE Kit (Ambion)

  • Inject 100-200 pg of mRNA into stage VI oocytes or fertilized eggs

  • Assess effects on translation of endogenous maternal mRNAs or co-injected reporter mRNAs

• Depletion studies:

  • Design antisense oligonucleotides targeting eIF3m mRNA

  • Use phosphorothioate-modified antisense oligonucleotides (AS-oligos) for increased stability

  • Inject 7-10 ng of AS-oligo into de-folliculated oocytes

  • Culture injected oocytes for 24 hours before maturation with progesterone

  • Transfer to host females for fertilization if studying embryonic effects

• Rescue experiments:

  • Co-inject AS-oligos with mRNA encoding resistant forms of eIF3m to confirm specificity

  • Use Xenopus tropicalis eIF3m that lacks the AS-oligo binding sequence for cross-species rescue

• Phenotypic analysis:

  • Assess effects on oocyte maturation by monitoring germinal vesicle breakdown (GVBD)

  • Evaluate translation of specific maternal mRNAs by western blot or reporter assays

  • Analyze embryonic development following fertilization

  • Perform in situ hybridization to assess effects on specific cell populations

• Biochemical assays:

  • Analyze polyadenylation of specific mRNAs following manipulation of eIF3m levels

  • Examine phosphorylation states of translation-dependent signaling factors

  • Perform in vitro translation assays using wheat germ extracts supplemented with recombinant eIF3m

These approaches allow for comprehensive analysis of eIF3m's role in regulating translation during oocyte maturation and early embryonic development in Xenopus laevis.

How does eIF3m contribute to the formation and function of the "translasome" supercomplex?

The eIF3 complex, including eIF3m, assembles into a large supercomplex termed the "translasome," which integrates multiple aspects of protein synthesis and quality control. Proteomic analysis using affinity-purified eIF3 complexes identified approximately 230 associated proteins, including components from diverse cellular pathways .

The translasome composition includes:

eIF3m likely serves as a scaffolding component that helps maintain the integrity of this supercomplex. The translasome concept suggests that translation does not occur in isolation but is physically coupled to downstream processes like protein folding and degradation, with eIF3m potentially mediating some of these connections .

Interestingly, genetic data indicate that the binding of eIF3 to importins-β is essential for cell growth, suggesting that proper nuclear-cytoplasmic shuttling of translation components is critical for function . This represents a previously underappreciated aspect of translation regulation that may involve eIF3m.

What is known about the regulatory functions of eIF3m in selective mRNA translation?

While direct evidence for eIF3m-mediated selective translation is limited in the search results, insights can be drawn from studies of other eIF3 subunits. For instance, the eIF3f subunit has been shown to function as a translation repressor for specific mRNAs such as nanos1 . This repression is relieved through interaction with the RNA-binding protein Dead-end 1 (Dnd1) .

By analogy, eIF3m may similarly participate in selective mRNA translation through:

• mRNA-specific recruitment: eIF3m could interact with RNA-binding proteins that recognize specific cis-elements in target mRNAs.

• Competitive binding: eIF3m might compete with inhibitory factors for binding to other eIF3 subunits, thereby regulating the activity of the complex on specific mRNAs.

• Conditional activation: Post-translational modifications of eIF3m could alter its activity in response to cellular signaling, leading to differential translation of specific mRNA subsets.

• Developmental regulation: The composition of eIF3 complexes, including the presence or activity of eIF3m, may change during development to accommodate stage-specific translation requirements.

For instance, maternal mRNAs in Xenopus oocytes require specific translational activation during maturation and early embryogenesis. eIF3m might contribute to this regulation, potentially in concert with other factors like Dnd1 that have been shown to promote translation of specific maternal mRNAs through interaction with the eIF3 complex .

How do post-translational modifications affect eIF3m function?

Post-translational modifications (PTMs) are likely important regulators of eIF3m function, though specific data on eIF3m modifications in Xenopus laevis are not directly presented in the search results. Based on studies of other eIF3 subunits and translation factors, several types of modifications may regulate eIF3m activity:

• Phosphorylation: Likely the most common regulatory modification, phosphorylation could alter eIF3m's:

  • Binding affinity for other eIF3 subunits

  • Interaction with mRNAs or regulatory proteins

  • Subcellular localization

  • Stability and turnover

• Ubiquitination: May regulate eIF3m levels through proteasomal degradation or affect its activity in non-proteolytic ways.

• Methylation and acetylation: Could fine-tune protein-protein or protein-RNA interactions.

• SUMOylation: May influence nuclear-cytoplasmic distribution, given the presence of importins in the eIF3 interactome .

To study these modifications in Xenopus laevis eIF3m, researchers could employ:

• Phosphoproteomic analysis of affinity-purified eIF3m at different developmental stages

• Site-directed mutagenesis of predicted modification sites followed by functional assays

• Pharmacological inhibitors of specific modifying enzymes to assess effects on eIF3m function

• In vitro modification assays using purified enzymes and recombinant eIF3m

Understanding the PTM landscape of eIF3m could reveal mechanisms by which translation is regulated during oocyte maturation and early embryonic development in response to hormonal or developmental signals.

What are common challenges in expressing and purifying recombinant Xenopus laevis eIF3m?

Researchers working with recombinant eIF3m should anticipate several technical challenges:

• Solubility issues:

  • eIF3m may form inclusion bodies when expressed in bacterial systems

  • Solution: Lower expression temperature (16-18°C), use solubility-enhancing tags, or switch to eukaryotic expression systems like insect cells

• Protein stability:

  • As part of a multi-subunit complex, isolated eIF3m may be unstable

  • Solution: Co-express with interacting partners, optimize buffer conditions (add glycerol, reduce salt concentration), use stabilizing additives

• Post-translational modifications:

  • Bacterial systems lack many eukaryotic PTMs that may be essential for function

  • Solution: Use baculovirus-insect cell expression systems that can provide more appropriate modifications

• Functional activity:

  • Recombinant eIF3m may not fold properly or lack essential binding partners

  • Solution: Verify activity through binding assays with known interactors, compare with native eIF3 complex purified from Xenopus oocytes

• RNA contamination:

  • Purified eIF3m may contain bound RNA that affects its properties

  • Solution: Include RNase treatment steps, followed by size exclusion chromatography

Researchers have successfully addressed similar challenges with other translation factors by using the baculovirus-insect cell system, which provides a eukaryotic environment conducive to proper folding and modification while allowing high-level expression .

How can researchers distinguish between direct and indirect effects when manipulating eIF3m levels?

Distinguishing direct from indirect effects is a critical challenge when studying translation factors like eIF3m:

• Temporal analysis:

  • Monitor changes immediately following eIF3m manipulation

  • Direct effects typically occur rapidly, while indirect effects emerge later

  • Use time-course experiments to establish the sequence of events

• Rescue experiments:

  • Test whether wild-type eIF3m can reverse phenotypes caused by depletion

  • Use structure-function analysis with mutant versions to identify critical domains

  • Cross-species rescue (e.g., using Xenopus tropicalis eIF3m resistant to antisense oligos targeting X. laevis eIF3m)

• Biochemical validation:

  • Demonstrate direct physical interactions with proposed targets using purified components

  • Use techniques like surface plasmon resonance to quantify binding affinities

  • Perform in vitro reconstitution experiments with defined components

• Reporter assays:

  • Design reporters specifically responsive to eIF3m-dependent mechanisms

  • Include appropriate controls with mutations in potential eIF3m-responsive elements

  • Test in cell-free translation systems where the components can be precisely controlled

• Comparative analysis:

  • Compare effects of eIF3m manipulation with those of other eIF3 subunits

  • Identify shared versus unique phenotypes to pinpoint eIF3m-specific functions

  • Use epistasis experiments to establish pathway relationships

These approaches, used in combination, can help establish causality and distinguish direct eIF3m functions from downstream consequences of translation dysregulation.

What controls should be included when studying the effects of eIF3m on specific mRNA translation?

Rigorous experimental design for studying eIF3m effects on translation should include several key controls:

Implementing these controls helps ensure that observed effects can be confidently attributed to specific functions of eIF3m rather than experimental artifacts or indirect consequences of manipulation.

What are promising areas for future research on Xenopus laevis eIF3m?

Several exciting research directions could advance our understanding of eIF3m function:

• Structural biology:

  • Cryo-EM structures of Xenopus laevis eIF3 complexes containing eIF3m

  • Comparative structural analysis of eIF3m in different functional states

  • Identification of critical interaction surfaces for binding partners

• Single-molecule studies:

  • Real-time visualization of eIF3m-containing complexes during translation initiation

  • FRAP (Fluorescence Recovery After Photobleaching) analysis to study dynamics of eIF3m in live Xenopus oocytes and embryos

  • Single-molecule tracking to determine the movement of eIF3m between different cellular compartments

• Translatomics:

  • Ribosome profiling following eIF3m manipulation to identify affected mRNAs

  • CLIP-seq (Cross-Linking Immunoprecipitation followed by sequencing) to identify directly bound mRNAs

  • Comparison of eIF3m-dependent translatome across developmental stages

• Systems biology:

  • Network analysis of the eIF3m interactome in different developmental contexts

  • Mathematical modeling of how eIF3m perturbations affect the broader translation system

  • Integration of transcriptomic, proteomic, and functional data to build predictive models

• Developmental regulation:

  • Spatial and temporal mapping of eIF3m expression and activity during embryogenesis

  • Analysis of eIF3m's role in developmental transitions requiring translational reprogramming

  • Investigation of potential functions in cell fate decisions and pattern formation

These research directions would contribute to a more comprehensive understanding of eIF3m's role in translation regulation and developmental biology.

How might CRISPR-Cas9 genome editing be applied to study eIF3m function in Xenopus laevis?

CRISPR-Cas9 technology offers powerful approaches for studying eIF3m in Xenopus laevis:

• Generation of eIF3m knockout or knockin lines:

  • Design sgRNAs targeting conserved regions of the eIF3m gene

  • Inject Cas9 protein with sgRNAs into fertilized eggs

  • Raise F0 mosaic animals and breed to establish stable lines

  • Create epitope-tagged versions at the endogenous locus for interaction studies

• Domain-specific mutations:

  • Introduce precise mutations in functional domains using homology-directed repair

  • Create structure-function series of mutations affecting specific interactions

  • Engineer conditional alleles to control eIF3m function at specific developmental stages

• Reporter knockins:

  • Insert fluorescent reporters under control of the endogenous eIF3m promoter

  • Generate translational fusions to study localization and dynamics

  • Create split fluorescent protein systems to visualize eIF3m interactions in vivo

• Regulatory element analysis:

  • Modify potential regulatory elements controlling eIF3m expression

  • Identify enhancers and repressors affecting tissue-specific or stage-specific expression

  • Create reporter constructs to monitor eIF3m transcriptional regulation

• High-throughput screening:

  • Develop pooled CRISPR screens targeting potential eIF3m interactors

  • Screen for modifiers of eIF3m-related phenotypes

  • Identify synthetic lethal or synthetic viable interactions

The tetraploid nature of Xenopus laevis creates both challenges and opportunities for CRISPR-based approaches, potentially allowing analysis of partial loss-of-function phenotypes when only some alleles are targeted.