A dual fluorescent reporter for the investigation of methionine mistranslation in live cells

Abstract

In mammalian cells under oxidative stress, the methionyl-tRNA synthetase (MetRS) misacylates noncognate tRNAs at frequencies as high as 10% distributed among up to 28 tRNA species. Instead of being detrimental for the cell, misincorporation of methionine residues in the proteome reduces the risk of oxidative damage to proteins, which aids the oxidative stress response. tRNA microarrays have been essential for the detection of the full pattern of misacylated tRNAs, but have limited capacity to investigate the misacylation and mistranslation mechanisms in live cells. Here we develop a dual-fluorescence reporter to specifically measure methionine misincorporation at glutamic acid codons GAA and GAG via tRNA Glu mismethionylation in human cells. Our method relies on mutating a specific Met codon in the active site of the fluorescent protein mCherry to a Glu codon that renders mCherry nonfluorescent when translation follows the genetic code. Mistranslation utilizing mismethionylated tRNA Glu restores fluorescence in proportion to the amount of misacylated tRNA Glu . This cellular approach works well for both transient transfection and established stable HEK293 lines. It is rapid, straightforward, and well suited for high-throughput activity analysis under a wide range of physiological conditions. As a proof of concept, we apply this method to characterize the effect of human tRNA Glu isodecoders on mistranslation and discuss the implications of our findings.

Determination of tRNA aminoacylation levels by high-throughput sequencing

“…For example, different tRNA isodecoders of tRNA Ser (UAG) show different stop codon suppression efficiency ( 20 ). Additionally, tRNA Glu isodecoders seem to have different mistranslation efficiency ( 21 ). A tRNA Arg (UCU) isodecoder is specifically expressed in central nervous system, and its expression is needed to alleviate ribosome pausing ( 22 ).…”

Determining the fidelity of tRNA aminoacylation via microarrays

“…This information greatly reduces the difficulty of searching for such mistranslational events in proteins through methods such as mass spectrometry and fluorescence detection [28]. Furthermore, since mistranslation can significantly alter the function and activity optima of a protein [8, 9, 35], elucidating specific mistranslational processes can help determine how specific amino acid substitutions may affect protein function as well as determine how these mistranslation might affect cellular physiology.…”

“…The general protocol for in vivo pulse-labeling involves an optional short amino acid starvation period, concentration of cells, pulse-labeling with radiolabeled amino acid, and subsequent charged tRNA isolation. Protocols for in vivo pulse labeling and total charged tRNA isolation of different organisms can be found in the literature and are easily adapted to new organisms [8, 10, 13, 28]. Since in vivo pulse labeling methods vary based on the organism and this review aims to describe on the array method itself, we will focus on the standard assessment of the fidelity of recombinant synthetases in vitro [8, 10, 12].…”

Pathways to disease from natural variations in human cytoplasmic tRNAs

“…As reviewed elsewhere (83), a recurrent finding is that stress conditions reduce aminoacylation fidelity (84). For example, upon oxidative stress, methionyl-tRNA synthetase increases its mis-aminoacylation of noncognate tRNAs up to 10-fold in bacteria and mammalian cells (82,85,86); the additional methionine in proteins is thought to protect the proteome from oxidative damage.…”