A unifying model for mTORC1-mediated regulation of mRNA translation

Abstract

The mTOR complex 1 (mTORC1) kinase nucleates a pathway that promotes cell growth and proliferation and is the target of rapamycin, a drug with many clinical uses. mTORC1 regulates messenger RNA translation, but the overall translational program is poorly defined and no unifying model exists to explain how mTORC1 differentially controls the translation of specific mRNAs. Here we use high-resolution transcriptome-scale ribosome profiling to monitor translation in mouse cells acutely treated with the mTOR inhibitor Torin 1, which, unlike rapamycin, fully inhibits mTORC1 (ref. 2). Our data reveal a surprisingly simple model of the mRNA features and mechanisms that confer mTORC1-dependent translation control. The subset of mRNAs that are specifically regulated by mTORC1 consists almost entirely of transcripts with established 5' terminal oligopyrimidine (TOP) motifs, or, like Hsp90ab1 and Ybx1, with previously unrecognized TOP or related TOP-like motifs that we identified. We find no evidence to support proposals that mTORC1 preferentially regulates mRNAs with increased 5' untranslated region length or complexity. mTORC1 phosphorylates a myriad of translational regulators, but how it controls TOP mRNA translation is unknown. Remarkably, loss of just the 4E-BP family of translational repressors, arguably the best characterized mTORC1 substrates, is sufficient to render TOP and TOP-like mRNA translation resistant to Torin 1. The 4E-BPs inhibit translation initiation by interfering with the interaction between the cap-binding protein eIF4E and eIF4G1. Loss of this interaction diminishes the capacity of eIF4E to bind TOP and TOP-like mRNAs much more than other mRNAs, explaining why mTOR inhibition selectively suppresses their translation. Our results clarify the translational program controlled by mTORC1 and identify 4E-BPs and eIF4G1 as its master effectors.

Figure 1. Profile of mTOR-regulated translation

(a) WT MEFs were treated with vehicle (DMSO), 250 nM rapamycin or Torin1, or starved for amino acids for 2 h and analyzed for protein levels. (b) WT MEFs were treated for 2 h with vehicle (DMSO), 250 nM rapamycin or Torin1, or 10 ug/ml cycloheximide, pulsed for 30 min with S-Cys/Met and S incorporation into protein quantified and normalized to the total protein. Data are mean +/− s.d. (n=3). (c) Polysome profiles of WT MEFs treated with DMSO or 250 nM Torin1 for 2 h. (d) Distributions of ribosome footprint (RF) frequency in vehicle- or Torin1-treated cells. RF libraries from cells treated as in (c) were used to determine RF frequencies (reads per million, RPM) for 4840 mRNAs. (e) β-actin mRNA abundance in fractions from (c) were quantified by qPCR, and calculated as a percentage of the total in all fractions. Data are means +/− s.e.m. (n=2). (f) Distribution of changes in translational efficiency from vehicle- or Torin1-treated cells. RF frequencies from (d) were normalized to transcript levels to calculate translational efficiencies. Ribosome densities (reads per kilobase per million, RPKM) from vehicle- and Torin1-treated cells are inset. mRNAs with suppressed (z-score < −1.5) or resistant (z-score > 1.5) translational efficiencies are indicated. (g) Torin1-dependent changes in translational efficiency for indicated mRNA classes. For histone mRNAs, results reflect changes in ribosome density only. Significance determined by two-tailed Mann-Whitney U test.

Figure 2. Translation of TOP and TOP-like mRNAs is hyper-sensitive to mTOR inhibition

(a) Torin1-induced changes in translational efficiencies of 65 known TOP mRNAs in WT MEFs (outlined bars) compared to changes in all 4840 mRNAs (solid bars). Significance determined by the Mann-Whitney U test. (b) The pyrimidine content of the 10 nt surrounding the TSS for 3025 mRNAs where the TSS could confidently identified, excluding 65 known TOP mRNAs (expected frequency = 0.518). Boxplots indicate the TSS pyrimidine content for mRNAs binned according to Torin1-dependent change in translational efficiency. Significance determined by binomial test. (c) Numbers of indicated mRNA classes. (d) TSS annotations for selected TOP and TOP-like mRNAs. Primary and secondary TSS locations from dbTSS (purple) are indicated, as are annotations from Refseq (gray), Ensemble (blue), and UCSC (green). (e) Polysome analyses of selected TOP (eEF2, Rps20), unrecognized TOP (Hsp90ab1) and TOP-like (Vim, Ybx1) mRNAs. Data are means +/− s.e.m. (n=2).

Figure 3. mTOR regulates general protein synthesis and TOP mRNA translation through the 4E-BPs

(a) WT and 4EBP1/2 double-knockout (DKO) MEFs were treated with DMSO, 250 nM rapamycin or Torin1 for 2 h, lysates subjected to mGTP pull-downs, and analyzed for levels of indicated proteins. (b) WT and DKO MEFs expressing FLAG-GFP or FLAG-eIF4E were treated as in (a), and immunoprecipitates analyzed for indicated proteins. (c) DKO MEFs were treated for 2 h with vehicle (DMSO), 250 nM rapamycin or Torin1, or 10 μg/ml cycloheximide were analyzed as in Figure 1b. Data are mean +/− s.d. (n=3). (d) Polysome profiles of DKO MEFs treated with DMSO or Torin1 for 2 h (e) Torin1-dependent changes in translational efficiency in DKO (gray bars) and WT MEFs (blue bars). (f) Torin1-dependent translational suppression of 65 TOP mRNAs in WT and DKO MEFs. Significance determined by Mann-Whitney U test. (g) Polysome analyses of selected non-TOP (β-actin), known TOP (eEF2, Rps20), unrecognized TOP (Hsp90ab1) and TOP-like (Vim, Ybx1) mRNAs in DKO cells. Data are means +/− s.e.m. (n=2).

Figure 4. Destabilization of the eIF4E/eIF4G1 interaction dissociates TOP mRNAs from eIF4E and inhibits their translation

(a) WT and DKO MEFs were treated for 2 h with DMSO or 250 nM Torin1 and eIF3b immunoprecipitates analyzed for indicated proteins. (b) FLAG-eIF4E was immunoprecipitated from WT MEFs treated with DMSO or 250 nM Torin1 for 2 h. RNA was extracted, and abundance of TOP and TOP-like (TOP/L) (Eef2, Rps20, Hsp90ab1, Pabpc1, Ybx1, Vim) and non-TOP (Actb, Mrpl22, Ccnd1, Slc2a1, Gabarapl1, Myc) mRNAs quantified by QPCR. Changes in eIF4E binding of mRNAs were plotted against changes in translational efficiency from Fig. 1f. eIF4E binding data are means +/− s.e.m. (n=4). (c) Levels of indicated proteins in cells expressing indicated shRNAs. (d) Cells expressing indicated shRNAs were pulsed for 30 min with S-labeled Cys/Met and analyzed as in Figure 1b. Data are mean +/− s.d. (n=3). Significance determined by t-test. (e) Polysome profiles for WT or DKO cells expressing indicated shRNAs. (f) RNA isolated from gradients in (e) was analyzed by qPCR for the indicated mRNAs as in Fig. 1e. Data are means +/− s.e.m. (n=2). (g) Abundance of indicated transcripts from RNA-seq analysis. Data are means +/− s.e.m. (n=3). (h) Lysates from cells expressing shGFP or eIF4G2-specific shRNAs were analyzed by immunoblotting. (i) Fractions from shEIF4G2-2 gradients in (e) were analyzed as in (f). (j) mTORC1 regulates the selective translation of TOP and TOP-like mRNAs through the 4EBP-dependent control of eIF4G1-mediated initiation.