EIF3I Human, Sf9

What is EIF3I and what role does it play in protein synthesis?

EIF3I (Eukaryotic translation initiation factor 3 subunit I) is a critical component of the eukaryotic translation initiation factor 3 (eIF-3) complex, which orchestrates numerous steps in protein synthesis initiation. The eIF-3 complex functionally links with the 40S ribosomal subunit and facilitates the recruitment of other key initiation factors including eIF-1, eIF-1A, eIF-2:GTP:methionyl-tRNAi and eIF-5 to form the 43S pre-initiation complex (43S PIC) .

This complex plays several essential roles in translation:

• Stimulates mRNA recruitment to the 43S PIC

• Enables scanning of mRNA for AUG recognition

• Facilitates disassembly and recycling of post-termination ribosomal complexes

• Prevents premature joining of 40S and 60S ribosomal subunits

What is the molecular structure and characteristics of EIF3I Human protein?

EIF3I Human recombinant protein produced in Sf9 Baculovirus cells has the following structural characteristics:

• Single glycosylated polypeptide chain containing 331 amino acids (amino acids 1-325 of the native sequence)

• Molecular mass of 37.3 kDa theoretically, but migrates at 40-57 kDa on SDS-PAGE under reducing conditions due to glycosylation

• Features a 6 amino acid His-tag at the C-terminus for purification purposes

• Specific amino acid sequence beginning with MKPILLQGHE and ending with His-tag

The protein contains the WD40 domain, which is critical for protein-protein interactions and has been identified as a potential site for post-translational modifications including acetylation and ubiquitination, particularly at position K185 .

How evolutionarily conserved is EIF3I across species?

Interestingly, despite being evolutionarily conserved between humans and the yeast Saccharomyces cerevisiae, EIF3I appears to be dispensable for active mammalian eIF3 complex formation. This represents a significant divergence in functional requirements across evolution. While yeast eIF3 comprises only five subunits (eIF3a, eIF3b, eIF3c, eIF3g, and eIF3i), mammalian eIF3 is much larger with 10-13 different polypeptide subunits .

Extensive deletion analyses suggest that three evolutionarily conserved subunits (eIF3a, eIF3b, and eIF3c) and three non-conserved subunits (eIF3e, eIF3f, and eIF3h) constitute the functional core of mammalian eIF3. This indicates that while EIF3I is preserved across species, its functional necessity has evolved differently between yeast and mammals .

What is the recommended storage and handling protocol for EIF3I Human Sf9 protein?

For optimal stability and activity maintenance of EIF3I protein:

• Store at 4°C if the entire vial will be used within 2-4 weeks

• For longer storage periods, keep frozen at -20°C

• For long-term storage, addition of a carrier protein (0.1% HSA or BSA) is recommended

• Avoid multiple freeze-thaw cycles which can compromise protein integrity

• The protein is typically provided in a solution (0.25mg/ml) containing 20mM Tris-HCl buffer (pH 8.0), 0.1M NaCl, 40% Glycerol and 1mM DTT

What role does EIF3I play in cancer biology and angiogenesis?

EIF3I has emerged as a critical regulator in cancer progression through multiple mechanisms:

Cancer Cell Survival: Recent research demonstrates EIF3I promotes colorectal cancer cell survival through translational regulation of specific mRNAs. Knockdown of eIF3i results in significant transcriptomic changes, with 224 genes downregulated and 161 genes upregulated, while 15,184 genes remain stable. Gene Ontology analysis reveals that these differentially expressed genes are primarily involved in blood vessel development and large ribosome subunit biogenesis .

Tumor Angiogenesis: EIF3I is highly expressed in endothelial cells during both embryonic and tumor angiogenesis. Its expression is dynamically regulated by extracellular signals:

• In HUVECs, treatment with mouse melanoma B16 cell conditioned medium (B16-CM) increases eIF3i protein expression to 114% compared to control

• Conditioned medium from hypoxic B16 cells (B16-HCM) further increases eIF3i protein expression to 167%

• VEGF-A treatment significantly upregulates eIF3i protein expression in endothelial cells

Molecular Mechanism: EIF3I promotes endothelial cell growth and migration by selectively upregulating ERK and VEGFR2 translation. Knockdown of eIF3i significantly attenuates the response of endothelial cells to induction signals from tumor cells, effectively blocking tumor angiogenesis .

How can ribosome profiling techniques be utilized to study EIF3I-mediated translational control?

Ribosome profiling (Ribo-seq) offers a powerful approach to investigate EIF3I's role in selective translation:

• Technique Overview: Ribo-seq utilizes deep sequencing to analyze ribosome-protected mRNA fragments, providing a comprehensive view of actively translating ribosomes at specific time points .

• Application to EIF3I Research:

  • Ribosome-protected RNA fragments typically measure 25-32 nucleotides in length

  • These fragments are predominantly distributed in the coding sequence region of the genome

  • By comparing polysome profiles between control and eIF3i-depleted cells, researchers can identify specific mRNAs whose translation is regulated by eIF3i

• Methodological Approach for EIF3I Studies:

  • Fractionate cell extracts using 10-50% sucrose gradient to isolate polysome-bound mRNA

  • Analyze the levels of target mRNAs (e.g., VEGFR2, ERK) in each fraction by RT-PCR

  • A shift of specific mRNAs from polysome fractions toward monosome fractions following eIF3i depletion indicates eIF3i-dependent translational control

This approach has revealed that eIF3i depletion selectively reduces translation of VEGFR2 and ERK mRNAs without affecting housekeeping genes like β-actin and GAPDH in HUVECs .

What is known about post-translational modifications of EIF3I and their functional significance?

Post-translational modifications of EIF3I appear to be critical regulatory mechanisms:

• K185N Mutation: A K185N mutation in the WD40 domain of eIF3i has been identified as a potential acetylation and ubiquitination modification site. This domain is critical for protein-protein interactions within translation complexes .

• Impact of Genetic Alterations:

  • EIF3I expression associates with different types of copy number alterations including deep deletion, shallow deletion, and diploid states

  • Patients with shallow deletions show reduced levels of eIF3i

  • In colorectal cancer patients, genetic alterations in EIF3I are predominantly distributed in stage III (71.43%)

  • Notably, genetic alterations are not associated with better prognosis

These findings suggest that both the expression level and post-translational modifications of EIF3I are important determinants of its function in normal and pathological conditions.

How does EIF3I interact with other factors in translation pre-initiation complexes?

EIF3I functions within complex macromolecular assemblies during translation initiation:

• Within the eIF3 Complex: EIF3I is part of the so-called Yeast-Like-Core (YLC) subcomplex, which includes the C-terminal part of eIF3a, eIF3b, and eIF3i. This subcomplex binds to the solvent-exposed side in the human 48S pre-initiation complex (h-48S) .

• Structural Position: In reconstituted human 48S pre-initiation complexes assembled on capped mRNA in the presence of eIF4B and eIF4F, the eIF3 PCI/MPN core and the YLC subcomplex (including eIF3i) are found bound to the solvent-exposed side of the initiation complex .

• Functional Interactions: EIF3I contributes to multiple aspects of translation initiation through its interactions:

  • Facilitates recruitment of other initiation factors including eIF-1, eIF-1A, eIF-2:GTP:methionyl-tRNAi and eIF-5

  • Contributes to mRNA recruitment and scanning

  • Participates in disassembly and recycling of post-termination complexes

What experimental strategies can be used to study the specific translational targets of EIF3I?

Several complementary approaches can be employed to identify and characterize specific mRNAs whose translation is regulated by EIF3I:

• Polysome Profiling with eIF3i Knockdown:

  • Transfect cells with eIF3i-specific siRNAs (using at least two non-overlapping sequences to confirm specificity)

  • Fractionate cell extracts using 10-50% sucrose gradient

  • Analyze distribution of target mRNAs across monosome and polysome fractions by RT-PCR

  • A shift from polysome to monosome fractions indicates reduced translation efficiency

• Integrated Transcriptomic and Proteomic Analysis:

  • Compare RNA-seq data with proteomics data following eIF3i modulation

  • Identify mRNAs whose protein levels change disproportionately to mRNA levels

  • This approach can distinguish translational regulation from transcriptional effects

• Ribosome Profiling (Ribo-seq):

  • Isolate and sequence ribosome-protected mRNA fragments

  • Compare ribosome occupancy patterns between control and eIF3i-depleted cells

  • Analyze distribution of fragment lengths (typically 25-32 nt) and their genomic location

  • Focus on the coding sequence regions where most fragments are distributed

These techniques have successfully identified VEGFR2 and ERK as specific translational targets of eIF3i in endothelial cells, demonstrating the selectivity of eIF3i-mediated translational control .

What are the key quality control parameters for EIF3I Human Sf9 recombinant protein?

When working with EIF3I Human recombinant protein produced in Sf9 Baculovirus cells, researchers should consider the following quality control parameters:

Researchers should also verify functionality through binding assays with known interaction partners within the eIF3 complex .

How can researchers effectively reconstitute functional eIF3 complexes containing EIF3I for in vitro studies?

Reconstituting functional eIF3 complexes provides a powerful approach to study the specific role of EIF3I within the translation machinery:

• Baculovirus-Based Coexpression System:

  • This system has been successfully used to reconstitute a human eIF3 complex consisting of 11 subunits

  • The reconstituted complex successfully promotes the recruitment of the 40S ribosomal subunit to mRNA

• Core Complex Generation:

  • Focus on including the essential subunits: eIF3a, eIF3b, eIF3c (evolutionarily conserved) and eIF3e, eIF3f, eIF3h (non-conserved)

  • Interestingly, despite evolutionary conservation, eIF3g and eIF3i are dispensable for active mammalian eIF3 complex formation

• Functional Validation:

  • Test the reconstituted complex for its ability to promote 40S ribosomal subunit recruitment to mRNA

  • Assess formation of 43S pre-initiation complexes

  • Evaluate mRNA scanning capacity and AUG recognition

• Structural Characterization:

  • Use cryo-EM approaches to analyze structural organizations of the complexes

  • Focus on the interaction between the eIF3 YLC subcomplex (containing eIF3i) and the solvent-exposed side of the 48S pre-initiation complex

What strategies can be employed to study EIF3I's role in tumor angiogenesis?

Based on research findings, several experimental approaches can be implemented to investigate EIF3I's role in tumor angiogenesis:

• In Vitro Endothelial Cell Models:

  • Culture human umbilical vein endothelial cells (HUVECs)

  • Manipulate eIF3i expression using siRNA knockdown (using two non-overlapping siRNAs for specificity)

  • Verify knockdown efficiency by qPCR and western blot with eIF3i-specific antibodies

  • Treat cells with tumor-derived conditional medium (e.g., from B16 melanoma cells) or VEGF-A

  • Assess effects on endothelial cell proliferation, migration, and tube formation

• Molecular Signaling Analysis:

  • Evaluate VEGFR/ERK signaling pathway after eIF3i knockdown

  • Analyze protein expression of VEGFR2 and ERK through western blotting

  • Assess VEGFR2 and ERK mRNA levels through qPCR

  • Perform polysome analysis to determine translational efficiency of these mRNAs

• In Vivo Tumor Angiogenesis Models:

  • Establish tumor xenografts in appropriate animal models

  • Develop endothelial-specific eIF3i knockdown or knockout models

  • Analyze tumor growth, vascular density, and endothelial cell infiltration

  • Evaluate effects on tumor angiogenesis through immunohistochemistry for endothelial markers

Through these approaches, researchers have demonstrated that eIF3i plays a critical role in promoting endothelial cell survival, proliferation, and migration by selectively enhancing translation of VEGFR2 and ERK proteins, ultimately facilitating tumor angiogenesis .

What are the potential therapeutic implications of targeting EIF3I in cancer?

EIF3I presents a promising therapeutic target for cancer treatment based on its dual role in both cancer cells and tumor vasculature:

• Dual Targeting Strategy:

  • EIF3I is critical for the proliferation and survival of cancer cells

  • It is simultaneously required for the translational activation of VEGF-A

  • It promotes endothelial cell survival via augmented translation of key angiogenic factors

  • This dual role makes it a potentially powerful target for comprehensive cancer therapy

• Potential Approaches:

  • Development of small molecule inhibitors targeting the WD40 domain of eIF3i

  • Exploration of specific siRNA or antisense oligonucleotide delivery systems

  • Investigation of compounds that may disrupt eIF3i's interaction with other components of the translation machinery

• Clinical Context:

  • The association of eIF3i genetic alterations with stage III colorectal cancer (71.43%) suggests potential stage-specific therapeutic windows

  • The observation that genetic alterations are not associated with better prognosis indicates complex underlying mechanisms that require further investigation

How might technological advances in structural biology further illuminate EIF3I function?

Advanced structural biology techniques offer significant opportunities to enhance our understanding of EIF3I:

• Cryo-Electron Microscopy:

  • High-resolution cryo-EM has already enabled visualization of the human 48S pre-initiation complex at 6.3Å

  • Further advances could provide atomic-level details of eIF3i interactions within the complex

  • Understanding the precise positioning of eIF3i within the YLC subcomplex bound to the solvent-exposed side of the initiation complex

• Integrative Structural Approaches:

  • Combining cryo-EM with cross-linking mass spectrometry to map interaction networks

  • Employing hydrogen-deuterium exchange mass spectrometry to identify dynamic binding interfaces

  • Using single-molecule FRET to track conformational changes during translation initiation

• Structural Impact of Post-Translational Modifications:

  • Investigating how the K185N mutation and other modifications in the WD40 domain affect the structural properties of eIF3i

  • Determining how these structural changes impact protein-protein interactions and translation regulation

These structural insights would provide a foundation for rational drug design targeting eIF3i in cancer therapy and other potential therapeutic applications.