Translation in amino-acid-poor environments is limited by tRNAGln charging

Abstract

An inadequate supply of amino acids leads to accumulation of uncharged tRNAs, which can bind and activate GCN2 kinase to reduce translation. Here, we show that glutamine-specific tRNAs selectively become uncharged when extracellular amino acid availability is compromised. In contrast, all other tRNAs retain charging of their cognate amino acids in a manner that is dependent upon intact lysosomal function. In addition to GCN2 activation and reduced total translation, the reduced charging of tRNAGln in amino-acid-deprived cells also leads to specific depletion of proteins containing polyglutamine tracts including core-binding factor α1, mediator subunit 12, transcriptional coactivator CBP and TATA-box binding protein. Treating amino-acid-deprived cells with exogenous glutamine or glutaminase inhibitors restores tRNAGln charging and the levels of polyglutamine-containing proteins. Together, these results demonstrate that the activation of GCN2 and the translation of polyglutamine-encoding transcripts serve as key sensors of glutamine availability in mammalian cells.

Fibroblast pyruvate carboxylase is required for collagen production in the tumour microenvironment

“…We next asked why collagen production in TGFβ-stimulated fibroblasts was impaired when the extracellular glutamine concentration was reduced. Based on a recent report suggesting that glutamine-tRNA is often uncharged when cells are cultured in low amino acid medium 20 , we used a qPCR-based method to analyze the charging state of tRNAs for several non-essential amino acids (NEAAs) and leucine-tRNA as control. We observed a profound uncharging of glutamine-tRNA in TGFβ- but not mock-treated NIH-3T3 cells cultured in low Gln ( Fig.…”

“…These data suggest that an inability to maintain glutamine tRNA charging could lead to activation of GCN2 and reduced translation in fibroblasts treated with TGFβ in a low glutamine environment, resulting in a reduction of ECM synthesis. Glutamine-tRNA charging has been reported to be restored in proliferative fibroblasts by inhibiting glutaminase with the allosteric inhibitor CB839 20 . However, CB839 treatment did not rescue GCN2 activation induced by TGFβ treatment in low Gln and only marginally increased collagen I protein levels ( Fig.…”

“…Charging status of the indicated tRNA isodecoders was measured as previously described 20 . In brief, Trizol-chloroform extracts were precipitated with 2.7x volumes of cold ethanol in presence of 30 µg GlycoBlue (ThermoFisher) overnight.…”

Protein synthesis, degradation, and energy metabolism in T cell immunity

“…In particular, the reduced availability of amino acids results in an accumulation of uncharged tRNAs, which can bind and activate the kinase GCN2. GCN2 phosphorylates eIF2a, preventing eIF2 from being recycled to the GTP-bound state required for translation initiation and thus inhibiting global protein synthesis [72,73]. Whether GCN2 plays a major regulatory role in controlling global protein synthesis in T cells and is a major signaling factor through which amino acid deprivation controls the T cell response are unclear.…”

Conditional covalent lethality driven by oncometabolite accumulation

“…Several recent studies have indicated the potential of tRNAs to serve as a critical capacitor of metabolic information. 40, 41 Evaluating whether oncometabolites can modulate endogenous tRNA modification levels - and what affect this has on cancer development - represents an important topic for future inquiry. Finally, we note with interest the parallels between the fumarate-dependent synthetic lethal mechanism invoked above and the ‘collateral vulnerability’ concept pioneered by Muller and coworkers.…”