Eukaryotic initiation factor 6 is rate-limiting in translation, growth and transformation

Abstract

Cell growth and proliferation require coordinated ribosomal biogenesis and translation. Eukaryotic Initiation Factors (eIF) control translation at the rate-limiting step of initiation 1,2 . So far, only two eIFs connect extracellular stimuli to global translation rates 3 ; eIF4E acts in the eIF4F complex and regulates binding of capped mRNA to 40S subunits, downstream of growth factors 4 ; eIF2 controls loading of the ternary complex on the 40S subunit and is inhibited upon stress stimuli [5][6] . No eIFs have been found to link extracellular stimuli to the activity of the large 60S ribosomal subunit. eIF6 binds 60S ribosomes precluding ribosome joining in vitro [7][8][9] . However studies in yeasts showed that eIF6 is required for ribosome biogenesis rather than translation [10][11][12][13] . We show that mammalian eIF6 is required for efficient initiation of translation, in vivo. eIF6 null embryos are lethal at preimplantation. Heterozygous mice have 50% reduction of eIF6 levels in all tissues, and show reduced mass of hepatic and adipose tissues due to a lower number of cells and to impaired G1/S cell cycle progression. eIF6 +/− cells retain sufficient nucleolar eIF6 and normal ribosome biogenesis. The liver of eIF6 +/− mice displays an increase of 80S in polysomal profiles, indicating a defect in initiation of translation. Consistently, isolated hepatocytes have impaired insulin-stimulated translation. Heterozygous mouse embryonic fibroblasts (MEFs) recapitulate the organism phenotype and have normal ribosome biogenesis, reduced insulin-stimulated translation, and delayed G1/S phase progression. Furthermore, eIF6 +/− cells resist to oncogene-induced transformation. Thus, eIF6 is the first eIF associated with the large 60S subunit that regulates translation in response to extracellular signals.The eIF6 gene was deleted by homologous recombination using embryonic stem (ES) cell technology ( Supplementary Fig. 1a). The portion of the gene containing the first two exons and the first two introns was substituted by a cassette containing the neomycin resistance gene. The presence of the neomycin resistance cassette did not affect expression of wt eIF6 and of adjacent genes ( Supplementary Fig. 2). Germline transmission was achieved and intercrossingCorrespondence and requests for materials should be addressed to stefano.biffo@hsr.it. * These authors contributed equally Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. Table 1). The lethality of eIF6 −/− embryos is consistent with the early expression of the protein in the blastocysts ( Supplementary Fig. 1d).Heterozygous eIF6 +/− mice were viable and indistinguishable from wt counterparts up to 30 days after birth. At three months of age, heterozygous mice, independently from gender and genetic background, weighted less than their wt littermates (Fig. 1a). The head-anus length of eIF6 +/− and wt mice was identical, suggesting that the reduction of body mass in eIF6 +/− mice could be due to smaller size of specific...

Suboptimal T-cell receptor signaling compromises protein translation, ribosome biogenesis, and proliferation of mouse CD8 T cells

“…For example, differences in the abundance of proteins, such as eIF6, between WT and Lck −/− T cells may have influenced the range of mRNA species that are translated. The antiassociation factor eIF6 prevents premature binding of 40S and 60S ribosome subunits (38) and has been shown to affect G1/S cell cycle transition (39,40) and the expression of metabolic enzymes controlling fatty acid synthesis and glycolysis (41). A G1/S block was observed in Lck −/− cells at 24 h after stimulation, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that fatty acid metabolism was highly enriched in the translationally repressed gene subset in Lck −/− cells.…”

Translational control by 80S formation and 60S availability: The central role of eIF6, a rate limiting factor in cell cycle progression and tumorigenesis

“…eIF6 heterozygote MEFs (mouse embryonic fibroblasts) have resistance to MYC or HRAS-mediated oncogenic transformation and impairment in G 1 /S cell cycle progression. 19 In addition, eIF6 inactivation delays EμMyc-induced model, PKC stimulation leads to the phosphorylation of eIF6 through interaction with PKC/RACK1 dimer, reducing the affinity of eIF6 for 60S and promoting its release.…”

“…7 Interestingly, in mammalian cells, a minor pool of eIF6 is sufficient for normal ribosomal biogenesis; in fact, 80% depletion of eIF6 by siRNA still generates normal rRNA synthesis. 19 Total depletion of eIF6 is lethal. 19 These data are consistent with an evolutionarily conserved role for a pool of eIF6 in the generation of 60S subunits.…”

“…19 Total depletion of eIF6 is lethal. 19 These data are consistent with an evolutionarily conserved role for a pool of eIF6 in the generation of 60S subunits. date, none of these steps has been linked to extracellular signaling, suggesting that this process is made of strict mechanistic operations.…”

Translation factors and ribosomal proteins control tumor onset and progression: how?

“…eIF6 is necessary for ribosome biogenesis of 60S subunits 88,89 and for high levels of translation. 90 Thus, eIF6 translation activity is dispensable for translation in vitro, but is required for maximal stimulation of mitogens in vivo. eIF6 involvement in tumorigenesis is described below.…”