EIF3J Human

Where is eIF3j located within the ribosomal complex?

Human eIF3j binds specifically to the aminoacyl (A) site and mRNA entry channel of the 40S ribosomal subunit, positioning it directly within the ribosomal decoding center . This strategic localization allows eIF3j to influence critical interactions between mRNA, tRNA, and the ribosome during translation processes. Due to this positioning, eIF3j can effectively regulate access to the mRNA-binding cleft in response to various initiation factors .

What are the primary functions of eIF3j in translation processes?

eIF3j demonstrates several distinct regulatory functions in translation:

How does eIF3j influence translation termination at the molecular level?

eIF3j stimulates peptidyl-tRNA hydrolysis induced by the eukaryotic release factor complex (eRF1-eRF3) . Using a reconstituted mammalian in vitro translation system, researchers have demonstrated that human eIF3j directly interacts with the pre-termination ribosomal complex, while eRF3 destabilizes this interaction . In solution, eIF3j binds to eRF1, eRF3, and PABP in the presence of GTP. Toe-printing assays have determined that eIF3j functions at the specific stage of binding release factors to the A-site of the ribosome before GTP hydrolysis occurs . Furthermore, when combined with the initiation factor eIF3, which also stimulates peptide release, eIF3j's activity in translation termination significantly increases .

What is the relationship between eIF3j and circular RNA translation?

eIF3j has been identified as a potent inhibitor of circular RNA (circRNA) translation through systematic RNAi screening of all 43 Drosophila eIFs . Mechanistically, eIF3j induces translation repression by promoting the dissociation of the eIF3 complex from circRNA templates . This binding interaction specifically requires the C-terminus of eIF3j . These findings reveal a critical regulatory mechanism whereby eIF3j selectively controls the translation of a specific subset of RNAs, suggesting a specialized role in post-transcriptional gene regulation that differs from its functions in canonical mRNA translation.

How does the presence or absence of eIF3j affect ribosomal affinity for mRNA?

eIF3j significantly reduces the 40S ribosomal subunit's affinity for mRNA when bound to the decoding center . Interestingly, this reduced affinity undergoes a dynamic shift upon recruitment of initiator tRNA - high affinity for mRNA is restored even though eIF3j remains present in the mRNA-binding cleft . This suggests eIF3j acts as a conditional regulator of mRNA accessibility, creating a checkpoint mechanism that ensures proper sequential assembly of the translation initiation complex. This function appears critical for preventing premature or inappropriate mRNA engagement with the ribosome before the complete initiation complex has formed.

What techniques are most effective for studying eIF3j-ribosome interactions?

Several complementary approaches have proven valuable for investigating eIF3j-ribosome interactions:

How can researchers effectively reconstitute the eIF3 complex for functional studies?

Successful reconstitution of the 13-subunit human eIF3 complex has been achieved using Escherichia coli expression systems . This approach revealed that the structural core of eIF3 consists of eight subunits with conserved orthologues in the proteasome lid complex and COP9 signalosome . This reconstituted core binds to the small (40S) ribosomal subunit, to translation initiation factors involved in mRNA cap-dependent initiation, and to the hepatitis C viral (HCV) internal ribosome entry site (IRES) RNA .

The addition of remaining eIF3 subunits enables the reconstituted complex to assemble intact initiation complexes with the HCV IRES . Importantly, negative-stain EM reconstructions further reveal how the approximately 400 kDa structural core organizes the highly flexible 800 kDa eIF3 complex and mediates translation initiation . When including eIF3j in these reconstitutions, researchers must account for its labile association with the core complex by adjusting experimental conditions accordingly.

What approaches should be used to investigate eIF3j's role in circRNA translation?

Investigation of eIF3j's impact on circRNA translation requires specialized approaches:

• RNAi screening: Systematic knockdown of eIF3j to evaluate effects on circRNA translation efficiency using model translatable circRNAs such as Drosophila circSfl .

• RNA-protein interaction assays: Techniques like RNA immunoprecipitation or crosslinking immunoprecipitation to determine direct binding between eIF3j and specific circRNA templates.

• C-terminal domain analysis: Since the C-terminus of eIF3j is required for circRNA binding , mutational analysis of this region can provide insights into binding specificity.

• Comparative translation assays: Assessing translation efficiency of linear versus circular RNA templates in the presence/absence of eIF3j to determine specificity of inhibition.

How does eIF3j contribute to translation factor coordination?

eIF3j functions as a coordination hub between various translation factors. It interacts with eIF1A and influences mRNA binding to the 40S subunit. During termination, eIF3j facilitates the binding of release factors eRF1 and eRF3 to the ribosomal A-site . Additionally, in combination with the initiation factor eIF3, which also stimulates peptide release, eIF3j's activity in translation termination increases . These interactions suggest eIF3j serves as a regulatory node that helps orchestrate the transitions between different phases of translation, ensuring proper sequential recruitment of factors at specific stages.

What are the species-specific differences in eIF3j function?

Comparative studies reveal both conserved and divergent functions of eIF3j across species:

These differences highlight the evolutionary adaptation of eIF3j to fulfill species-specific regulatory needs in translation.

What recent discoveries have changed our understanding of eIF3j function?

Recent research has substantially revised our understanding of eIF3j in several key areas:

• The identification of eIF3j as a potent inhibitor of circRNA translation represents a novel regulatory function that suggests specialized roles in post-transcriptional gene regulation .

• Evidence now supports that eIF3j functions in an eIF3-independent manner and is not a bona fide eIF3 subunit, contrary to earlier classifications .

• The discovery that eIF3j stimulates peptidyl-tRNA hydrolysis during termination, providing a mechanistic understanding of how it influences translation completion .

• The finding that the C-terminus of eIF3j is specifically required for binding to circRNA templates, suggesting structural determinants of RNA specificity .

What are promising future research directions for eIF3j studies?

Several promising avenues for future eIF3j research include:

• Structural investigations: Determining high-resolution structures of eIF3j in complex with the ribosome at different stages of translation to understand its dynamic interactions.

• Transcriptome-wide binding analysis: Identifying the complete repertoire of RNAs regulated by eIF3j beyond the currently known examples.

• Post-translational modifications: Investigating how modifications of eIF3j might regulate its various functions in different cellular contexts.

• Therapeutic potential: Exploring whether modulation of eIF3j activity could provide novel approaches for targeting dysregulated translation in disease states.

• Integrated multi-omics approaches: Combining proteomics, transcriptomics, and ribosome profiling to comprehensively map eIF3j's impact on the translatome.