Impact of alanyl-tRNA synthetase editing deficiency in yeast

Abstract

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that provide the ribosome with aminoacyl-tRNA substrates for protein synthesis. Mutations in aaRSs lead to various neurological disorders in humans. Many aaRSs utilize editing to prevent error propagation during translation. Editing defects in alanyl-tRNA synthetase (AlaRS) cause neurodegeneration and cardioproteinopathy in mice and are associated with microcephaly in human patients. The cellular impact of AlaRS editing deficiency in eukaryotes remains unclear. Here we use yeast as a model organism to systematically investigate the physiological role of AlaRS editing. Our RNA sequencing and quantitative proteomics results reveal that AlaRS editing defects surprisingly activate the general amino acid control pathway and attenuate the heatshock response. We have confirmed these results with reporter and growth assays. In addition, AlaRS editing defects downregulate carbon metabolism and attenuate protein synthesis. Supplying yeast cells with extra carbon source partially rescues the heat sensitivity caused by AlaRS editing deficiency. These findings are in stark contrast with the cellular effects caused by editing deficiency in other aaRSs. Our study therefore highlights the idiosyncratic role of AlaRS editing compared with other aaRSs and provides a model for the physiological impact caused by the lack of AlaRS editing.

Gain of C-Ala enables AlaRS to target the L-shaped tRNAAla

“…Unfortunately, no transformants carrying the CeAlaRS m -(C-Ala) fusion were available for the rescue assays. As the editing activity of ScAlaRS is crucial for the survival of yeast ( 35 ), perhaps fusion of C-Ala to the C-terminus of CeAlaRS m disturbed the enzyme’s editing activity, leading to mistranslation and cellular toxicity.…”

Serine mistranslation induces the integrated stress response without accumulation of uncharged tRNAs

“…Western blot was essentially performed as described (26). Yeast cultures were grown overnight, and total proteins were extracted using TCA precipitation.…”

“…Both AlaRS and ThrRS mischarge Ser onto tRNAs and require editing to remove the mischarged tRNAs. In our previous work, we show that an AlaRS editing-site mutation (C719A) leads to increased Ser mistranslation and activation of the ISR (25), but the underlying mechanism is unclear. In this work, we show that Ser misincorporation at Ala and Thr codons induces phosphorylation of eIF2α and GCN4 expression.…”

“…It has been shown that mutations in alanyl-tRNA synthetase (AlaRS) leading to an editing defect cause damage to neurons and cardiomyocytes (24). Mutations in the editing domain of yeast AlaRS and threonyl-tRNA synthetase (ThrRS) cause sensitivity to heat stress (25,26). However, translational infidelity may benefit bacteria under certain stress conditions (27)(28)(29), partially due to the activation of stress responses by moderate mistranslation.…”

Translational Fidelity during Bacterial Stresses and Host Interactions

“…This is in line with studies showing that the ThrRS C182A mutation results in little growth defect [ 13 , 48 ]. In contrast, mutating the editing site Cys (C666 in E. coli and C719 in Saccharomyces cerevisiae ) of AlaRS inhibits growth at elevated temperatures [ 48 , 59 ]. The striking difference between ThrRS and AlaRS editing defects is presumably due to the nature of translational errors.…”