Targeted profiling of RNA translation reveals mTOR-4EBP1/2-independent translation regulation of mRNAs encoding ribosomal proteins

Abstract

The PI3K-Akt-mTOR signaling pathway is a master regulator of RNA translation. Pharmacological inhibition of this pathway preferentially and coordinately suppresses, in a 4EBP1/2-dependent manner, translation of mRNAs encoding ribosomal proteins. However, it is unclear whether mechanistic target of rapamycin (mTOR)-4EBP1/2 is the exclusive translation regulator of this group of genes, and furthermore, systematic searches for novel translation modulators have been immensely challenging because of difficulties in scaling existing RNA translation profiling assays. Here, we developed a rapid and highly scalable approach for gene-specific quantitation of RNA translation, termed Targeted Profiling of RNA Translation (TPRT). We applied this technique in a chemical screen for translation modulators, and identified numerous preclinical and clinical therapeutic compounds, with diverse nominal targets, that preferentially suppress translation of ribosomal proteins. Surprisingly, some of these compounds act in a manner that bypasses canonical regulation by mTOR-4EBP1/2. Instead, these compounds exert their translation effects in a manner that is dependent on GCN2-eIF2α, a central signaling axis within the integrated stress response. Furthermore, we were also able to identify metabolic perturbations that also suppress ribosomal protein translation in an mTOR-independent manner. Together, we describe a translation assay that is directly applicable to large-scale RNA translation studies, and that enabled us to identify a noncanonical, mTOR-independent mode for translation regulation of ribosomal proteins.

Keywords: GCN2-eIF2α; mTOR; ribosomal proteins; translation control; translation profiling.

Fig. 1.

Design and validation of the TPRT protocol. (A) Overview of TPRT, compared with ribosome profiling. (B) Changes in translation of RPs, other TOP- and TOP-like mRNAs, and non-RP internal controls, under Torin-1 treatment (100 nM, 1 h) in MDA-MB-468 cells, as measured by TPRT and ribosome profiling. Translation changes are relative to normalization factor of non-RP internal controls, and DMSO vehicle control, as described in SI Appendix, Materials and Methods. For TPRT, fold changes are derived as means over four biological replicates, each with four technical replicates; error bars denote SEs. For ribosomal profiling, fold changes derived as means of 2 biological replicates. (Inset) Direct comparison between TPRT and ribosome profiling mean fold changes in translation.

Fig. 2.

Chemical screen for RP translation inhibitors. Change in translation of RPS27, under acute treatment by compound library (1 μM, 1 h) in HMEC-CT2 cells. Translation changes, measured by TPRT, are relative to normalization factor of non-RP internal controls (PPIA, ACTB), and DMSO vehicle control; normalization and z-value thresholds are described in SI Appendix, Materials and Methods (SI Appendix, Fig. S3 for supporting data). (Inset) Effects of 10 RP translation inhibitors, with diverse nominal targets, on mTOR signaling as measured 4EBP1 phosphorylation shifts. Compounds that demonstrate no substantial change in 4EBP1/2 mobility shift are highlighted in red.

Fig. 3.

Validation of Dabrafenib (Dbr) and MK1775 (MK) as mTOR-4EBP1/2-independent translation inhibitors. (A) mTOR signaling under acute drug treatment (1 μM, 1 h) in HMEC-CT2 parental cells. mTOR activity is measured by phosphorylation shifts in total 4EBP1 and 4EBP2 blots, and band intensities in phosphosite-specific p-4EBP1 blots. (B–D) Changes in RP translation under acute drug treatment (1 μM, 1 h) in HMEC-CT2 subjected to CRISPR-Cas9 targeting GFP or 4EBP1/2 (SI Appendix, Fig. S4 G–J for supporting data). Translation changes, measured by TPRT, are relative to normalization factor of non-RP internal controls, and DMSO vehicle control, as described in SI Appendix, Materials and Methods. Fold changes are derived as means over three biological replicates, each with three technical replicates; error bars denote SEs.

Fig. 4.

Dependency of Dabrafenib (Dbr) and MK1775 (MK) translation effects on GCN2 and eIF2α. (A) TREEspot visualization of likelihood score (LS) for targets modulated by Dabrafenib and MK1775, and not by AZ628 (SI Appendix, Fig. S5 for target modulation by individual compounds, SI Appendix, Table S5 for LS values, and SI Appendix, Materials and Methods for LS definition). (B–G) Changes in RP translation under acute drug treatment (1 μM, 1 h) in HMEC-CT2 subjected to negative control siRNA, or siRNA targeting GCN2 (B–D) or eIF2α (E–G) (SI Appendix, Fig. S6 A–H for supporting data). Translation changes, measured by TPRT, are relative to normalization factor of non-RP internal controls, and DMSO vehicle control, as described in SI Appendix, Materials and Methods. Fold changes are derived as a mean of four (B–D) or three (E–G) biological replicates, each with three technical replicates; error bars denote SEs.

Fig. 5.

mTOR signaling and RP translation under limitation of glucose (–Gluc) or cysteine/cystine (–C/C). (A) mTOR signaling under Torin-1 treatment (1 μM, 30 min), or acute metabolic perturbations (30 min), in Src-transformed MCF10A cells. mTOR activity is measured by phosphorylation shifts in total 4EBP1 and 4EBP2 blots, and band intensities in phosphosite-specific p-4EBP1 blots. (B) Changes in transcriptome-wide translation efficiency under acute metabolic perturbations (30 min) in Src-transformed MCF10A cells. TE changes, measured by ribosome profiling, are filtered for genes with at least 100 reads per million reads in both total and ribosome footprint mRNA samples, and were computed as described in SI Appendix, Materials and Methods; RPs are highlighted in red.

Fig. 6.

Dependency of glucose (–Gluc) or cysteine/cystine (–C/C) limitation-mediated translation effects on GCN2 and eIF2α. Changes in RP translation under acute Torin-1 treatment (1 μM, 30 min), or acute metabolic perturbations (30 min) in Src-transformed MCF10A cells subjected to negative control siRNA or siRNA targeting (A–C) GCN2 or (D–F) eIF2α (SI Appendix, Fig. S8 A–H for supporting data). Translation changes, measured by TPRT, are relative to normalization factor of non-RP internal controls, and DMSO vehicle control, as described in SI Appendix, Materials and Methods. Fold changes derived as a mean of four (A–C) or three (D–F) biological replicates, each with three technical replicates; error bars denote SEs.

Fig. 7.

Schematic of chemical and metabolic perturbations identified as RP translation modulators.