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.

Laboratory of Regulation of Gene Expression Institute of Microbiology AVCR v v i Prague the Czech Republic

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A trans locus causes a ribosomopathy in hypertrophic hearts that affects mRNA translation in a protein length-dependent fashion

uS3/Rps3 controls fidelity of translation termination and programmed stop codon readthrough in co-operation with eIF3

Binding of eIF3 in complex with eIF5 and eIF1 to the 40S ribosomal subunit is accompanied by dramatic structural changes

Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex

Translation initiation factor eIF3 promotes programmed stop codon readthrough

Functional and biochemical characterization of human eukaryotic translation initiation factor 3 in living cells

Structural integrity of the PCI domain of eIF3a/TIF32 is required for mRNA recruitment to the 43S pre-initiation complexes

Functional characterization of the role of the N-terminal domain of the c/Nip1 subunit of eukaryotic initiation factor 3 (eIF3) in AUG recognition

Small ribosomal protein RPS0 stimulates translation initiation by mediating 40S-binding of eIF3 via its direct contact with the eIF3a/TIF32 subunit

'Ribozoomin'--translation initiation from the perspective of the ribosome-bound eukaryotic initiation factors (eIFs)