mTOR Controls ATF4 Translation and mRNA Stability (A) mTOR reduces ATF4 mRNA levels. RNA was isolated from cells treated with vehicle (DMSO) or 250 nM Torin 1 for the indicated times and analyzed by qPCR. RNA levels were normalized to GAPDH (n = 3, error bars are SD). (B) mTOR activity has little effect on ATF4 transcription. HEK293T cells were treated with vehicle or 250 nM Torin 1 for 4 hr and then pulsed for 15 and 30 min with 100 μM 4sU. RNA was reacted with MTS-biotin, isolated by streptavidin-affinity purification, and analyzed by qPCR. Synthesis rates were determined by comparing 4sU labeling at 15 and 30 min and compared to changes in steady-state mRNA levels (n = 3, error bars are SD). (C) mTOR inhibition decreases the half-life of ATF4 mRNA. ATF4 −/− HEK293T cells simultaneously expressing doxycycline-repressible constructs encoding ATF4 and GFP were pre-treated with vehicle or 250 nM Torin 1 for 30 min, and then 1 μg/mL doxycycline. mRNA was collected at 0 and 6 hr post-doxycycline addition and analyzed by qPCR. mRNA levels were normalized to GAPDH (n = 3, error bars are SD, but are too small to be visible). (D) ATF4 protein stability is unaffected by mTOR inhibition. Extracts were prepared from HEK293T cells pre-treated with 100 μg/mL cycloheximide for 5 min and then with vehicle (DMSO) or 250 nM Torin 1 for the indicated times, and they were analyzed for the indicated proteins by immunoblotting (left panel) and quantified by normalizing levels of ATF4 to EIF3B (right panel) (n = 3, error bars are SD). (E) mTOR inhibition preferentially decreases translation of the ATF4-coding ORF. Top panel: ribosome profiling data from HEK293T cells treated for 24 hr with vehicle (DMSO) or 250 nM Torin 1 are shown. Bar heights are reads per million (RPM) for each position in the spliced ATF4 transcript, and they are the combined values of two replicate libraries. Bottom panel: organization of ORFs in the ATF4 mRNA is shown. (F) mTOR-regulated change in the translation efficiency of ATF4 ORFs. Translation efficiencies of ATF4 uORF3 and main ORF (mORF) were calculated by normalizing ribosome-protected fragment (RPF) reads from (E) from non-overlapping segments of uORF3 or mORF to RNA levels in DMSO- and Torin 1-treated conditions (n = 2, error bars are SD, significance calculated by t test). (G) Top panel: reporter design. 5′ UTRs are from wild-type human ATF4 (WT), ATF4 with start codon of uORF3 mutated to TAC (DuORF3), or ACTB. Bottom panel: cells were treated with 10 μM TMP to stabilize YFP concurrently with vehicle (DMSO) or 250 nM Torin 1, and they were monitored for fluorescence at the indicated times (n = 9, error bars are SEM). (H) uORF3 is required for mTOR control of full-length ATF4. ATF4 −/− HEK293T cells stably expressing dox-inducible WT or DuORF3 ATF4 were treated with 1.0 μg/mL (WT) or 0.5 μg/mL (ΔuORF3) doxycycline for 40 hr, and then with vehicle (DMSO) or 250 nM Torin 1 for 1 hr. Cell extracts were prepared and analyzed by immunoblotting for the indicated proteins. (I) Gcn2 is required for mTOR control of eIF2α phosphorylation, but not ATF4 translation. Extracts were prepared from Gcn2 +/+ or Gcn2 −/− MEFs treated with vehicle (DMSO) or 250 nM Torin 1 for 4 hr, and they were analyzed by immunoblotting for the indicated proteins.