Fig 2: eIF6 translational activity controls the expression of lipogenic transcription factors.(a) Global initiation is reduced on eIF6 inhibition and insulin administration, as shown by polysome/80S ratio. Data are represented as mean±s.d. Statistical P values were calculated by two-tailed t-test (n=4. ***P value =0.001). (b) Outline of the analysis and quantitative results: a subset of mRNA is differentially depleted (green) from eIF6 shRNA polysomes. The normalized ratio was obtained by the quantile method. Correction taking in account the reduction of polysomal area shows that more genes are affected by eIF6 deficiency. (c) Gene Ontology analysis shows that metabolism predominates in eIF6-affected genes, from the normalized ratio pool (P value =10-12). (d) Structural 5'-UTR analysis of genes affected by eIF6 deficiency. Polysomes from eIF6-depleted cells show reduction of G/C rich 5' mRNAs (green box) (e) Transcription factors involved in lipogenesis are depleted from polysomes upon eIF6 shRNA. Structural features of their 5' UTR are included. (f) C/EBPd protein expression is modulated by eIF6. eIF6 increase, by lentiviral administration, induces C/EBPd. eIF6 depletion, by lentiviral-mediated shRNA, reduces C/EBPd. Representative experiments done in triplicates on AML12 hepatocytes and reconstituted mesenchymal stem cells (g) Lipogenic transcription factor C/EBPß (LIP isoform) is reduced in eIF6-depleted cells. Left, mRNA structure of C/EBPß generating three isoforms by differential translation (arrows: translation start sites). Blue small box with blue arrows is the C/EBP uORF. Right panel show a blot with LAP, LIP and C/EBPß isoforms: LIP isoform is specifically downregulated. (Representative experiment done in triplicates). (h) ATF4 reporter translation depends from eIF6. Reporter assay of reinitiation with luciferase (blue box) cloned downstream of natural ATF4 uORF. eIF6 upregulation increases ATF4 translational reporter activity, eIF6 downregulation decreases it. Data are normalized on translation of a cap-dependent firefly control. The large black arrow represents the stop codon of the second uORF. Small arrows are start codons. Blue box is the reporter. Statistical P values were calculated by two-tailed t-test, as above (*P value =0.05; **P=0.01; ***P=0.001).