Fig 1.
Type-I IFN fails to activate the Akt-mTOR pathway in the majority of cell lines tested.
(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.
Fig 2.
The mTOR inhibitor Torin1 results in a global reduction of mRNA translation, including of ISG mRNAs during IFN treatment.
(A) WISH cells treated for 12 hrs with DMSO (-), 100nM rapamycin or 1μM Torin1 alone or in combination with IFN β (100pM) were subjected to western blot to assess phosphorylation of the indicated proteins. (B) Polysome profiles of WISH cells treated as in (A). (C) RNA abundance in nanograms (ng) as quantified by Nanodrop to determine yields of isolated RNA from cytoplasmic and polyribosome-associated (polysomal) fractions of WISH cells treated with DMSO (-), 100nM rapamycin, or 1μM Torin1 alone or in combination with IFN β (100pM) for 12 hrs. Shown are means +/- standard deviations (n = 7 biological replicates). * indicates p-values < 0.05. (D) Polysome profiles of WISH cells treated with Torin1 (1μM) for 0, 1.5, 4.5, or 12 hrs.
Fig 3.
Polysome-profiling to identify ISG mRNAs subject to mTOR-dependent translational control.
(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) NT5C3A mRNA variants 3 and 4 are transcriptionally induced by IFN. Schematic representation of NT5C3A 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 NT5C3A. 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 e and are shown as means and 95% confidence intervals. * indicates p-values < 0.05 and ** < 0.005.
Fig 4.
The ISGs TLR3, NT5C3A, and RNF19B encode mRNAs less efficiently translated during mTOR inhibition.
(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.
Fig 5.
Transcriptional start site determination using an oligo-capping approach.
(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.