The ISGs <i>TLR3</i>, <i>NT5C3A</i>, and <i>RNF19B</i> encode mRNAs less efficiently translated during mTOR inhibition.

<p>(A) Polysomal-to-cytoplasmic mRNA ratios from three biological replicates of WISH cells treated with DMSO or Torin1 (1μM) in combination with IFN β (100pM) for 12 hrs were calculated (Student’s t-test, n = 3) for the genes indicated and are shown as means and 95% confidence intervals. (B) Polysome profiles of WISH cells treated as in A. mRNA abundance was assessed by qPCR for the indicated genes in each fraction. Abs254, absorbance of light at 254nm.</p

Transcriptional start site determination using an oligo-capping approach.

<p>(A) Ethidium bromide stained agarose gels show for each indicated genes the PCR products obtained using an oligo-capped forward primer and nested gene-specific reverse primers to amplify 5’ capped and polyadenylated mRNA from WISH cells treated with DMSO (-), 100nM rapamycin, or 1μM Torin1 alone or in combination with IFN β (100pM) for 12 hrs. (B-E), Major identified transcriptional start sites are shown for each gene as the sequence immediately following that of the oligo-capped primer. Representative Sanger sequencing dendograms (boxes) are shown displaying the reverse complement of the associated sequence. RC, reverse complement.</p

Type-I IFN fails to activate the Akt-mTOR pathway in the majority of cell lines tested.

<p>(A) IFN α2b and IFN β, added for various times to WISH cells cultured in 10% serum, fail to significantly alter 4E-BP1 phosphorylation at Ser65. (B) Western blot analyses of the activation of the Akt-mTOR and ERK pathways in WISH cells subjected to serum starvation for 24 hrs prior to IFN or serum stimulation. (C) Western blot analyses of the activation of the Akt-mTOR and ERK pathways in NB4 cells subjected to serum starvation for 24 hrs prior to IFN or serum stimulation. (D) Western blot analyses comparing activation of the Akt-mTOR and ERK pathways in Daudi, NB4, Jurkat, Molt-4, and U266 cells subjected to serum starvation for 4 hrs prior to IFN β stimulation.</p

Polysome-profiling to identify ISG mRNAs subject to mTOR-dependent translational control.

<p>(A) RNA isolated from WISH cells treated with IFN β (100pM) in combination with DMSO or 1μM Torin1 for 12 hrs was subjected to comparative genome-wide mRNA expression profiling, and genes showing differential translation were identified using anota. Genes are plotted according to changes (Δ) in cytoplasmic and polysomal mRNA levels upon the addition of Torin1 to IFN β-treated cells. Genes showing repressed (blue) and enhanced (orange) mRNA translation in response to Torin1 are indicated. (B) Correlation between Torin1 (square)- and rapamycin (triangle)-induced mRNA translation changes (Δ) in IFN β-treated cells. Genes identified in either comparison that show increased (yellow) or decreased (blue) translation are indicated by condition where they were identified (squares or triangles). (C) A lack of correlation is observed when Torin1-induced mRNA translation changes (y-axis) and IFN β-induced cytoplasmic mRNA changes (x-axis) are plotted for all assessed genes. (D) <i>NT5C3A</i> mRNA variants 3 and 4 are transcriptionally induced by IFN. Schematic representation of <i>NT5C3A</i> mRNA variant-specific PCR primers used for qPCR. Arrows represent forward and reverse primers. mRNA abundance in nanograms (ng) was assessed by quantitative polymerase chain reaction (qPCR) for the indicated genes, including mRNA variants 1–4 of <i>NT5C3A</i>. Shown are means +/- standard deviations (n = 3). Theoretical transcriptional start sites (arrows) based on NCBI reference sequences are shown (box). (E) Cytoplasmic and polysomal mRNA abundance (ng) was assessed by qPCR for the indicated genes. Shown are means +/- standard deviations (n = 3). * indicates p-values < 0.05 and ** < 0.005. (F) Polysomal-to-cytoplasmic mRNA ratios were calculated (Student’s t-test) for select genes in <b>e</b> and are shown as means and 95% confidence intervals. * indicates p-values < 0.05 and ** < 0.005.</p

eIF4E induction stimulates the translation of a subset of mRNAs.

<p>A) Western blotting of extracts from 3T3-tTA versus 3T3-tTA-eIF4E cells after eIF4E induction (0 to 24 hr) and from NIH 3T3 cells and MEFs that constitutively express HA-eIF4E or vector alone. eIF4E induction was determined by using anti-HA and anti-eIF4E antibodies. Fold increase at the 24 hr time point was determined using NIH Image. B) Western blotting experiments were performed as described in (A). These experiments were repeated three times using three different sets of whole-cell extracts.</p

eIF4E and TOP mRNA translation.

<p>A) DNA segments encompassing the promoters and 5′ UTRs of L32, L32 mut (non-TOP), S16, S16 mut (non-TOP), L30 and β-actin were subcloned upstream of the firefly luciferase gene. 3T3-tTA-eIF4E cells were transfected with the various firefly luciferase reporters and a renilla luciferase reporter, which was used for transfection efficiency, and were cultured for 32 hr. Tetracycline containing medium was then replaced by a tetracycline free medium for 16 hr; control (non-induced) cells were cultured in parallel with tetracycline. Firefly luciferase activity (FLU) was measured and normalized against renilla luciferase activity (RLU). B) Luciferase activity of the reporters was measured in the parental cell line 3T3-tTA as described in (A). C) TOP sequences of L32, S16 and L30 are depicted. The arrows indicate the transcriptional start site. The nucleotide changes between L32 and S16 and L32 and L30 are underlined. D) Mutated reporters were generated by exchanging the TOP sequences of L32, S16 and L30. Luciferase assays were performed as described in (A). E) Luciferase activity of the reporters was measured in the parental cell line 3T3-tTA as described in (A). Assays were carried out in triplicate. Luciferase activities represent an average obtained from three independent experiments.</p

eIF4E induction causes an increase in the recruitment of a subset of mRNAs to polysomes.

<p>A) Total and polysomal (24 fractions) RNA from induced (−tet for 5 hr) and uninduced (+tet for 5 hr) 3T3-tTA-eIF4E cells was reverse transcribed into cDNA. Primers for BI-1, survivin, MIF, L23, L34, L9, S17 and actin were used to assess mRNA levels. Amplified PCR bands from the polysomal fractions were quantified using NIH Image, and absolute values were plotted. B) The effect of eIF4E induction on L34 mRNA distribution was assessed by northern blotting. Polysomal RNA was isolated from induced (−tet for 5 hr) and uninduced (+tet for 5 hr) 3T3-tTA-eIF4E cells and fractionated into 12 fractions (for purpose of detection). The RNA was loaded on an agarose denaturing gel and transferred to a nitrocellulose membrane. Membranes were probed with radiolabeled murine L34 and actin probes. Bands were quantified using NIH Image, and absolute values were plotted.</p

siRNA mediated knockdown of eIF4E in NIH 3T3 cells.

<p>NIH 3T3 cells were transiently transfected with an siRNA against murine eIF4E or with a control siRNA, 4E-T-inv (scrambled sequence of human 4E-T), for 48 hr. Cells were lysed, and protein extracts were subjected to SDS-PAGE, followed by western blot analysis. The RNAi-mediated knockdown was repeated three times.</p

eIF4E induction protects cells against ER-mediated apoptosis.

<p>A) 3T3 cells that stably express HA-eIF4E were treated with 5 µM ionomycin for different periods, fixed and stained with propidium iodide. The percentage of apoptosis was quantified by flow cytometry (triplicates were pooled to generate the s.d.) B) 3T3-tTA and 3T3-tTA-eIF4E cells were seeded at 75% confluency and cultured for 8 hr. Tetracycline containing medium was then replaced by a tetracycline free medium for 16 hr to induce eIF4E expression. Uninduced cells were cultured for the same period without removal of tetracycline. Cells were treated with ionomycin and processed as described in (A). C) 3T3-tTA and 3T3-tTA-eIF4E cells were seeded and cultured with or without tetracycline as described in (B). Cells were then cultured for 24 hr in complete medium±5 µM ionomycin. Protein extracts were resolved by SDS-PAGE and transferred to membranes, which were immunoblotted with anti–caspase 3 and anti–caspase 12. eIF4E expression was also examined by immunoblot; elevated eIF4E levels were only detected in 3T3-tTA-eIF4E induced cells (−tet).</p

Induction of eIF4E in NIH 3T3 fibroblast cells and microarray analysis.

<p>A) eIF4E overexpression was induced by culturing 3T3-tTA-eIF4E cells in a tetracycline free medium. Immunoblots for eIF4E and β-actin were performed. B) A characteristic fractionation profile of 3T3-tTA and 3T3-tTA-eIF4E cells is depicted. Absorbance at 254 nm was monitored. C) Fractions from 3T3-tTA and 3T3-tTA-eIF4E (induced) cells were analyzed on a denaturing agarose gel to visualize the 18S and 28S rRNAs. D) The experimental design used for microarray analysis is shown.</p