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.

Several subunits of the multifunctional eukaryotic translation initiation factor 3 (eIF3) contain well-defined domains. Among them is the conserved bipartite PCI domain, typically serving as the principal scaffold for multisubunit 26S proteasome lid, CSN and eIF3 complexes, which constitutes most of the C-terminal region of the c/NIP1 subunit. Interestingly, the c/NIP1-PCI domain is exceptional in that its deletion, despite being lethal, does not affect eIF3 integrity. Here, we show that a short C-terminal truncation and two clustered mutations directly disturbing the PCI domain produce lethal or slow growth phenotypes and significantly reduce amounts of 40S-bound eIF3 and eIF5 in vivo. The extreme C-terminus directly interacts with blades 1–3 of the small ribosomal protein RACK1/ASC1, which is a part of the 40S head, and, consistently, deletion of the ASC1 coding region likewise affects eIF3 association with ribosomes. The PCI domain per se shows strong but unspecific binding to RNA, for the first time implicating this typical protein–protein binding domain in mediating protein–RNA interactions also. Importantly, as our clustered mutations severely reduce RNA binding, we conclude that the c/NIP1 C-terminal region forms an important intermolecular bridge between eIF3 and the 40S head region by contacting RACK1/ASC1 and most probably 18S rRNA.

Background: AUG recognition is promoted by several initiation factors (eIFs).Results: eIF5 interacts with the extreme N terminus of eIF3c/Nip1 to promote pre-initiation complex assembly, and eIF1 binds the region that immediately follows.Conclusion: eIF1 binding to c/Nip1 is equally important for its 40 S ribosome recruitment and AUG selection.Significance: Understanding start codon selection that sets the reading frame for decoding is key in gene expression studies.