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.
Highlights
To be utilized in protein synthesis, amino acids must be first covalently attached, or charged, onto corresponding tRNA isoacceptors by amino-acid-specific tRNA synthetases
In agreement with previous reports, depriving immortalized mouse embryonic fibroblasts (MEFs) of all amino acids resulted in an initial loss of mTORC1 activity, as measured by the phosphorylation of its substrate, S6 kinase 1 (S6K1), yet a prolonged period of amino acid deprivation resulted in a near-complete reactivation of mTORC1 (Figure 1A)
Even after 6 hr without extracellular amino acids, there remained ongoing translation in amino-acid-deprived cells in comparison to cells treated with the translation elongation inhibitor cycloheximide, demonstrating that amino-acid-deprived cells can continue to engage in adaptive translation even while bulk translation continues to decline
Summary
To be utilized in protein synthesis, amino acids must be first covalently attached, or charged, onto corresponding tRNA isoacceptors by amino-acid-specific tRNA synthetases. Eukaryotic cells possess sensors for monitoring both free and tRNA-charged amino acid reserves (reviewed in Gonzalez and Hall, 2017) The former is carried out by a set of sensors that sample individual amino acids - namely, leucine, arginine, and S-adenosylmethionine (a derivative of methionine), and relay the amino acid sufficiency information to the mTORC1 complex (Chantranupong et al, 2016; Gu et al, 2017; Wolfson et al, 2016), whereas the latter is mediated by the GCN2 kinase, which becomes activated via autophosphorylation in response to uncharged tRNA binding (Dong et al, 2000). We use a tRNA charging profiling method (CHARGEseq) to survey the pools of charged tRNAs in amino-acid-deprived cells and investigate how amino acid deficit-associated patterns of tRNA charging affect protein synthesis
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