mTORC1 Balances Cellular Amino Acid Supply with Demand for Protein Synthesis through Post-transcriptional Control of ATF4  Yeonwoo Park, Andrea Reyna-Neyra, Lucas Philippe, Carson C. Thoreen  Cell Reports  Volume 19, Issue 6, Pages 1083-1090 (May 2017) DOI: 10.1016/j.celrep.2017.04.042 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 19, 1083-1090DOI: (10.1016/j.celrep.2017.04.042) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 mTOR Inhibition Depletes mRNAs with Functions in AA Transport and Metabolism (A) Global changes in mRNA levels following 24 hr of mTOR inhibition. HEK293T cells were treated with vehicle (DMSO) or 250 nM Torin 1 for 24 hr and analyzed by RNA-seq. (B) mTOR inhibition reduces mRNAs with functions in AA metabolism and transport. Gene set enrichment analysis (GSEA) was used to analyze changes in mRNA levels from (A). Vertical lines beneath the x axis denote the members of each gene set. (AA Metabolic process, MSIGDB M11769; AA/Oligopeptide SLC Transporters, MSIGDB 19056; tRNA Aminoacylation, MSIGDB M456). (C) Left panel: an mTOR-regulated gene module. CCLE expression data were clustered by Pearson correlation and shown as a heatmap (increases in red and decreases in blue). Middle panel: log2-fold changes in mRNA levels from (A) were summed in a 25-gene window and plotted at each position. Right panel: a zoomed-in view of the cluster indicated is shown. mRNAs repressed upon mTOR inhibition are highlighted in red. (D) ATF4-binding motif from the JASPAR database of transcription factor-binding sites, identified by motif analysis of promoters of genes in (C) (see the Supplemental Experimental Procedures). (E) Extracts from cells treated as in (A) were analyzed for the indicated proteins by immunoblotting. Cell Reports 2017 19, 1083-1090DOI: (10.1016/j.celrep.2017.04.042) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 mTOR Inhibition Causes Rapid ATF4-Dependent Reductions in AA-Related mRNAs (A) mTOR inhibition rapidly reduces ATF4 protein level. Extracts were prepared from HEK293T cells treated with vehicle (DMSO) or 250 nM Torin 1 for the indicated times and analyzed by immunoblotting. (B) mTOR inhibition depletes ATF4 target mRNAs. qPCR analysis of RNA isolated from cells treated with vehicle (DMSO) or 250 nM Torin 1 for the indicated durations is shown. RNA levels were normalized to that of GAPDH (n = 3, error bars are SEM). (C) ATF4−/− HEK293T cells generated using CRISPR/Cas9 lack ATF4 protein. Cell extracts were analyzed by immunoblotting for the indicated proteins. (D) mTOR control of AA-related mRNAs in ATF4+/+ and ATF4−/− HEK293T cells. Left panel: changes in mRNA levels in the indicated cell lines treated with 250 nM Torin 1 for 6 hr were analyzed by RNA-seq. Red, mRNAs that are mTOR-regulated in ATF4+/+ cells (q < 0.1); blue, mTOR-repressed mRNAs (q < 0.1) encoding proteins with AA-related functions (transport, metabolism, and tRNA aminoacylation from Figure 1B) (n = 2). Right panel: boxplot shows change in expression of mTOR-repressed mRNAs with AA-related functions in ATF4+/+ and ATF4−/− HEK293T cells. Boxplots are displayed with standard definitions: upper and lower hinges are 25th and 75th percentile, respectively. The bar represents the median. (E) Comparison of mRNA changes induced by Torin 1 and Tunicamycin (Tm). Left panel: changes in mRNA levels in HEK293T cells treated with either 250 nM Torin 1 or 1 μM tunicamycin for 6 hr were analyzed by RNA-seq. Blue, mTOR-regulated mRNAs (q < 0.1) with AA-related functions, as in (D); green, mRNAs induced by tunicamycin (q < 0.1) with ER stress-related functions (MSIGDB M10844) (n = 2). Right panel: boxplot shows change in expression of AA-related and ER-stress mRNAs in HEK293T cells treated with 250 nM Torin 1 or 1 μM tunicamycin for 6 hr (n = 2). Boxplots are displayed with standard definitions: upper and lower hinges are 25th and 75th percentile, respectively. The bar represents the median. (F) mTOR regulation of ATF4-target transcripts in ATF4−/− cells. RNA was isolated from ATF4+/+ and ATF4−/− HEK293T cells treated with vehicle (DMSO) or 250 nM Torin 1 for 4 hr and analyzed by qPCR. Values for each mRNA are normalized to GAPDH and are relative to levels in untreated wild-type cells (n = 3, error bars are SD). (G) Loss of ATF4 reduces AA uptake. Uptake of 14C-labeled AAs was measured in ATF4+/+, ATF4−/−, or ATF4−/− HEK293T cells expressing ectopic ATF4 by scintillation counting and normalized to DNA content. n = 3, error bars are SD, significance by t test. (H) mTOR inhibition reduces AA uptake. ATF4+/+ and ATF4−/− HEK293T cells were treated with vehicle (DMSO) or 250 nM Torin 1 for 24 hr. AA uptake was assessed as in (G). n = 3, error bars are SD, significance by t test. Cell Reports 2017 19, 1083-1090DOI: (10.1016/j.celrep.2017.04.042) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 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 (ΔuORF3), 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 ΔuORF3 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. Cell Reports 2017 19, 1083-1090DOI: (10.1016/j.celrep.2017.04.042) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 mTOR Controls ATF4 Translation through 4E-BPs (A) 4E-BPs are required for mTOR control of ATF4. 4E-BP1/2 wild-type (WT), double knockout (DKO), or DKO MEFs stably expressing 4ebp1 (DKO + 4ebp1) were treated with vehicle (DMSO) or 250 nM Torin 1 for 4 hr. Cell extracts were analyzed by immunoblotting for the indicated proteins. (B) mTOR control of ATF4 mRNA in MEFs is independent of 4E-BPs. RNA was isolated from wild-type or 4E-BP-deficient cells treated as in (A) and analyzed by qPCR for levels of ATF4 mRNA, normalized to RPS20 mRNA (n = 3, error bars are SD). (C) 4E-BP1 is sufficient to deplete ATF4 protein in HEK293T cells. HEK293T cells stably expressing a doxycycline-inducible 4× Ala 4E-BP1 mutant were treated with dox for 48 hr. Cell extracts were analyzed by immunoblotting for the indicated proteins. (D) 4E-BP1 reduces ATF4 mRNAs in HEK293T cells. RNA was isolated from cells treated as in (C) and analyzed by qPCR for ATF4 mRNA levels, normalizing it to GAPDH (n = 3, error bars are SD). (E) Overexpression of EIF4E increases ATF4 protein levels. HEK293T cells stably transduced with dox-inducible EIF4E were treated with 0.1 or 1.0 μg/mL doxycycline for 24 hr, and they were analyzed by immunoblotting for the indicated proteins. (F) 4E-BPs are required for mTOR control of ATF4 target mRNAs in MEFs. RNA was isolated from 4E-BP1/2 WT or DKO MEFs treated with vehicle (DMSO) or 250 nM Torin 1 and analyzed by qPCR for the indicated mRNAs. All mRNAs were normalized to Gapdh (n = 3, error bars are SD). (G) Growth factor and AA starvation have opposite effects on ATF4 protein levels. Left panel: WT MEFs were incubated in media with or without AAs + 10% dialyzed FBS for 4 hr. Cell extracts were analyzed by immunoblotting for the indicated proteins. Right panel: WT MEFs were incubated in serum-free media for 24 hr and then stimulated with 500 nM insulin for 4 hr. Cell extracts were analyzed by immunoblotting for the indicated proteins. (H) Depletion of ATF4 by serum deprivation is mTORC1 dependent. Cell extracts were prepared from Tsc2−/− MEFs serum-starved for 24 hr, and then stimulated with 500 nM insulin ± 250 nM Torin 1 and analyzed by immunoblotting for the indicated proteins. (I) Model for cellular control of ATF4. Cell Reports 2017 19, 1083-1090DOI: (10.1016/j.celrep.2017.04.042) Copyright © 2017 The Author(s) Terms and Conditions