Despite the recent progress in our understanding of the numerous functions of individual subunits of eukaryotic translation initiation factor 3 (eIF3), there is still only little known on the molecular level. Using NMR spectroscopy, we determined the first solution structure of an interaction between eIF3 subunits. We revealed that a conserved tryptophan residue in the human eIF3j N-terminal acidic domain (NTA) is held in the helix α1 – loop L5 hydrophobic pocket of the human eIF3b-RRM. Mutating the corresponding “pocket” residues in its yeast orthologue reduces cellular growth rate, eliminates eIF3j/HCR1 association with eIF3b/PRT1 in vitro and in vivo, affects 40S-occupancy of eIF3, and produces a leaky scanning defect indicative of a deregulation of the AUG selection process. Unexpectedly, we found that the N-terminal half (NTD) of eIF3j/HCR1 containing the NTA motif is indispensable and sufficient for wild-type growth of yeast cells. Furthermore, we demonstrate that deletion of either j/HCR1 or its NTD only, or mutating the key tryptophan residues results in the severe leaky scanning phenotype partially suppressible by overexpressed eIF1A, which is thought to stabilize properly formed pre-initiation complexes at the correct start codon. These findings indicate that eIF3j/HCR1 remains associated with the scanning pre-initiation complexes and does not dissociate from the small ribosomal subunit upon mRNA recruitment as previously believed. Finally, we provide further support for earlier mapping of the ribosomal binding site for human eIF3j by identifying specific interactions of eIF3j/HCR1 with small ribosomal proteins RPS2 and RPS23 located in the vicinity of the mRNA entry channel. Taken together we propose that eIF3j/HCR1 closely co-operates with eIF3b/PRT1-RRM and eIF1A on the ribosome to ensure proper formation of the scanning-arrested conformation required for stringent AUG recognition.

Mammalian eIF3 is a 700-kDa multiprotein complex essential for initiation of protein synthesis in eukaryotic cells. It consists of 13 subunits (eIF3a to -m), among which eIF3b serves as a major scaffolding protein. Here we report the solution structure of the N-terminal RNA recognition motif of human eIF3b (eIF3b-RRM) determined by NMR spectroscopy. The structure reveals a noncanonical RRM with a negatively charged surface in the ␤-sheet area contradictory with potential RNA binding activity. Instead, eIF3j, which is required for stable 40 S ribosome binding of the eIF3 complex, specifically binds to the rear ␣-helices of the eIF3b-RRM, opposite to its ␤-sheet surface. Moreover, we identify that an N-terminal 69-amino acid peptide of eIF3j is sufficient for binding to eIF3b-RRM and that this interaction is essential for eIF3b-RRM recruitment to the 40 S ribosomal subunit. Our results provide the first structure of an important subdomain of a core eIF3 subunit and detailed insights into protein-protein interactions between two eIF3 subunits required for stable eIF3 recruitment to the 40 S subunit.

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