Recombinant Aspergillus niger Eukaryotic translation initiation factor 3 subunit J (hcr1)

Basic Research Questions

• What is the function of eIF3j/HCR1 in Aspergillus niger and how does it compare to other fungal species?

Methodologically, researchers investigating eIF3j function should use protein-protein interaction techniques (such as co-immunoprecipitation or pull-down assays) that can detect transient interactions, rather than relying solely on stable complex isolation methods.

• What is the structural relationship between eIF3j and the ribosome?

Structural studies using directed hydroxyl radical probing and cryo-electron microscopy have demonstrated that eIF3j binds to the aminoacyl (A) site and mRNA entry channel of the 40S ribosomal subunit, positioning it directly in the ribosomal decoding center . This strategic location allows eIF3j to influence both mRNA and tRNA binding to the ribosome. Specifically, eIF3j has been shown to interact with eIF1A and can reduce the 40S subunit's affinity for mRNA .

For researchers studying this interaction, methods like hydroxyl radical footprinting or crosslinking combined with mass spectrometry are recommended approaches to map the precise contact points between eIF3j and the ribosome components.

• How does A. niger eIF3j interact with other components of the translation machinery?

A. niger eIF3j interacts with multiple components of the translation machinery, including:

• Other eIF3 subunits (particularly subunits a and b)

• The 40S ribosomal subunit

• Potentially with Rli1/ABCE1 (based on homology with yeast)

From studies in related organisms, we know that "eIF3j interacts with both the C terminus of Tif32/eIF3a and the N-terminal RNA recognition motif (RRM) domain of Prt1/eIF3b" . A conserved tryptophan residue in the eIF3j N-terminal acidic (NTA) motif is held in a "hydrophobic 'pocket' of the eIF3b RRM" .

The STRING database indicates high confidence (0.999 score) interactions between TIF32 (eIF3a) and other eIF3 components in Aspergillus species, suggesting a conserved interaction network .

Advanced Research Questions

• How can researchers differentiate between eIF3j's roles in initiation versus recycling?

To differentiate between eIF3j's roles in initiation versus recycling, researchers should employ a multi-faceted experimental approach:

Ribosome profiling analysis:

• Compare 3' UTR ribosome occupancy patterns between eIF3j deletion strains and known recycling factor mutants

• Analyze the correlation of 3' UTR:ORF ratios between different mutants (as done in studies showing "3' UTR:ORF ratios for hcr1Δ and rli1-d were correlated (Spearman's R² of 0.44)" )

Reporter assays:

• Design reporter constructs with varying intercistronic regions

• Analyze the size of translation products in eIF3j mutants compared to initiation and recycling factor mutants

AUG dependency tests:

• Examine ribosome density around AUG codons in 3' UTRs

• The finding that "no peak when ribosome P sites were aligned at AUGs" in hcr1Δ strains (similar to rli1-d strains) suggests recycling over initiation defects

Genetic interaction studies:

• Test if overexpression of known 40S recycling factors can suppress eIF3j deletion phenotypes

• Examine synthetic growth defects with mutations in initiation versus recycling factors

The data from these approaches should be collectively analyzed to determine which function predominates in your specific experimental context.

• What structural and functional domains of A. niger eIF3j are critical for its various roles?

Based on studies in related organisms, A. niger eIF3j likely contains several critical functional domains:

• N-terminal acidic (NTA) motif: Contains a conserved tryptophan residue crucial for interacting with the RRM domain of eIF3b

• Central region: Likely involved in 40S ribosomal subunit binding, particularly to the mRNA entry channel and A-site

• C-terminal region: May facilitate interaction with Rli1/ABCE1 for ribosome recycling function

To experimentally map these domains in A. niger eIF3j:

• Generate a series of truncation and point mutants

• Assess their ability to complement eIF3j deletion phenotypes

• Perform protein-protein interaction assays with suspected binding partners

• Analyze ribosome binding capabilities of each construct

• Test specific functions (initiation vs. recycling) with appropriate assays

The comparison between A. niger eIF3j and characterized homologs can guide the design of these experiments. For example, researchers found that "mutating the tryptophan and NTA motif of Hcr1/eIF3j (NTA1 mutant) or the corresponding pocket residues in Prt1/eIF3b eliminates Hcr1/eIF3j association with Prt1/eIF3b in vitro and in vivo" .

• How does eIF3j cooperate with other translation factors in A. niger?

Understanding eIF3j's cooperative relationships requires comprehensive interaction studies:

Physical interaction analysis:

• Perform co-immunoprecipitation with tagged eIF3j to identify interaction partners

• Use cross-linking followed by mass spectrometry to map interaction interfaces

• Similar studies in N. crassa revealed that "tagged Nc eIF3j was able to affinity purify a myriad of translation-related proteins including eIF3 subunits, ribosomal proteins and other translation initiation/elongation factors"

Genetic interaction networks:

• Test synthetic genetic interactions between eIF3j and other translation factors

• Analyze suppressor relationships (e.g., "the effect could be suppressed by overexpression of Rli1/ABCE1" )

Functional cooperation in translation:

• Analyze the effect of eIF3j mutations on the recruitment of other factors to ribosomes

• Test whether eIF3j is required for specific translation events (e.g., reinitiation, leaky scanning)

A key finding to build upon is that "affinity-tagged Rli1/ABCE1 has been shown to pull down components of the MFC (multi-initiation factor complex), presumably through an interaction with Hcr1/eIF3j" , suggesting eIF3j may bridge recycling and initiation processes.

• How can recombinant A. niger eIF3j be used as a tool to study translation dynamics?

Recombinant A. niger eIF3j can serve as a valuable research tool for studying translation dynamics:

As a probe for ribosome structural studies:

• Use labeled eIF3j to map conformational changes in ribosomes during translation

• Apply techniques like hydroxyl radical footprinting with eIF3j as the probe

For reconstitution experiments:

• Add recombinant eIF3j to in vitro translation systems lacking this component

• Assess how it modulates translation efficiency of different mRNA templates

As a competitor in translation reactions:

• Use excess eIF3j to disrupt normal translation patterns

• Analyze which translation steps are most sensitive to eIF3j competition

For structural biology applications:

• Use purified eIF3j in complex with binding partners for cryo-EM or X-ray crystallography

• Map the exact binding interfaces within translation complexes

This approach builds on observations that eIF3j "binds to the aminoacyl (A) site and mRNA entry channel of the 40S subunit" and "interacts with eIF1A and reduces 40S subunit affinity for mRNA" , suggesting its strategic use could reveal transition states in translation processes.