mTOR-4EBP1/2-Independent Translational Regulation of mRNAs Encoding Ribosomal Proteins

Abstract

The PI3K-AKT-mTOR axis is a central signaling pathway that controls cell growth and proliferation, and exerts much of its phenotypic effects as a master regulator of RNA translation. Pharmacological inhibition of this pathway strongly and preferentially suppresses, in a 4EBP1/2-dependent manner, translation of mRNAs encoding ribosomal proteins and translation factors that comprise 5' terminal oligopyrimidine (TOP) motifs. Modulation of these transcripts consequently impacts ribosome biogenesis and cell-wide protein synthesis capacity. However, it is unclear whether mTOR-4EBP1/2 is the exclusive translational regulator of TOP-mRNA transcripts, and furthermore, systematic searches for novel translation modulators have been immensely challenging due to 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). To demonstrate its applicability, we utilized this method in a first-in-kind chemical screen for novel translation modulators, and identified numerous therapeutic compounds, with diverse nominal targets, that preferentially suppress TOP-mRNA translation. Surprisingly, we found that some of these compounds act in a manner that bypasses canonical regulation by mTOR-4EBP1/2, but instead elicit these same translational effects via GCN2 kinase. Furthermore, we identified metabolic perturbations that also modulate TOP-mRNA translation in an mTOR-independent manner. Together, we describe a novel translation assay that is directly applicable to large-scale RNA translation studies, and that enabled us to identify a non-canonical, mTOR-independent mode for translational regulation of ribosomal proteins and translation factors.