Recent reports have begun unraveling the details of various roles of individual eukaryotic translation initiation factor 3 (eIF3) subunits in translation initiation. Here we describe functional characterization of two essential Saccharomyces cerevisiae eIF3 subunits, g/Tif35 and i/Tif34, previously suggested to be dispensable for formation of the 48S preinitiation complexes (PICs) in vitro. A triple-Ala substitution of conserved residues in the RRM of g/Tif35 (g/tif35-KLF) or a single-point mutation in the WD40 repeat 6 of i/Tif34 (i/tif34-Q258R) produces severe growth defects and decreases the rate of translation initiation in vivo without affecting the integrity of eIF3 and formation of the 43S PICs in vivo. Both mutations also diminish induction of GCN4 expression, which occurs upon starvation via reinitiation. Whereas g/tif35-KLF impedes resumption of scanning for downstream reinitiation by 40S ribosomes terminating at upstream open reading frame 1 (uORF1) in the GCN4 mRNA leader, i/tif34-Q258R prevents full GCN4 derepression by impairing the rate of scanning of posttermination 40S ribosomes moving downstream from uORF1. In addition, g/tif35-KLF reduces processivity of scanning through stable secondary structures, and g/Tif35 specifically interacts with Rps3 and Rps20 located near the ribosomal mRNA entry channel. Together these results implicate g/Tif35 and i/Tif34 in stimulation of linear scanning and, specifically in the case of g/Tif35, also in proper regulation of the GCN4 reinitiation mechanism.

Laboratory of Regulation of Gene Expression Institute of Microbiology AVCR v v i Videnska 1083 Prague Czech Republic

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Adapted formaldehyde gradient cross-linking protocol implicates human eIF3d and eIF3c, k and l subunits in the 43S and 48S pre-initiation complex assembly, respectively

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

An aggregation-prone mutant of eIF3a forms reversible assemblies escaping spatial control in exponentially growing yeast cells

Please do not recycle! Translation reinitiation in microbes and higher eukaryotes

Embraced by eIF3: structural and functional insights into the roles of eIF3 across the translation cycle

ABCE1: A special factor that orchestrates translation at the crossroad between recycling and initiation

Human eIF3b and eIF3a serve as the nucleation core for the assembly of eIF3 into two interconnected modules: the yeast-like core and the octamer

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

In-depth analysis of cis-determinants that either promote or inhibit reinitiation on GCN4 mRNA after translation of its four short uORFs

Translation initiation factor eIF3 promotes programmed stop codon readthrough

Fail-safe mechanism of GCN4 translational control--uORF2 promotes reinitiation by analogous mechanism to uORF1 and thus secures its key role in GCN4 expression

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

Translation initiation factors eIF3 and HCR1 control translation termination and stop codon read-through in yeast cells

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)

The eIF3c/NIP1 PCI domain interacts with RNA and RACK1/ASC1 and promotes assembly of translation preinitiation complexes

Structural analysis of an eIF3 subcomplex reveals conserved interactions required for a stable and proper translation pre-initiation complex assembly

Translation reinitiation relies on the interaction between eIF3a/TIF32 and progressively folded cis-acting mRNA elements preceding short uORFs