Interactions of eukaryotic translation initiation factor 3 (eIF3) subunit NIP1/c with eIF1 and eIF5 promote...

Area of Science:

• Molecular Biology
• Protein-protein interactions
• Translation regulation

Background:

• The N-terminal domain (NTD) of NIP1/eIF3c is essential for forming the multifactor complex (MFC).
• MFC formation involves interactions with eIF1, eIF5, and the eIF2 ternary complex (TC).
• Understanding the physiological importance of these interactions is key to deciphering translation initiation regulation.

Purpose of the Study:

• To investigate the physiological importance of NIP1-NTD interactions.
• To identify specific segments within the NIP1-NTD critical for its function.
• To elucidate the role of NIP1-NTD in start codon selection and preinitiation complex assembly.

Main Methods:

• Site-directed mutagenesis of 16 segments within the NIP1-NTD.
• Assessing the impact of mutations on eIF1 and eIF5 binding.
• Evaluating effects on UUG start codon utilization (Sui(-) phenotype).
• Analyzing genetic interactions with mutations in eIF1 and eIF5.
• Investigating effects on GCN4 translation and TC recruitment.

Main Results:

• Mutations in multiple NIP1-NTD segments impaired eIF1 or eIF5 binding.
• A C-terminal NTD mutation caused increased UUG start codon usage and lethality with hyperactive eIF5-G31R.
• This mutation's effects were suppressed by eIF1 overexpression.
• N-terminal NTD mutations suppressed Sui(-) phenotypes caused by eIF1 and eIF5 mutations.
• Two NTD mutations derepressed GCN4 translation, indicating MFC formation stimulates TC recruitment.

Conclusions:

• The NIP1-NTD coordinates eIF1 and eIF5 interactions to inhibit GTP hydrolysis at non-AUG codons.
• The NIP1-NTD is essential for efficient preinitiation complex assembly.
• The NIP1-NTD regulates AUG start codon selection in vivo.
• MFC formation, mediated by the NIP1-NTD, is critical for TC recruitment to 40S ribosomes.