title: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Ana Cristina
authors.initials: AC
authors.lastname: Gomes
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Anna J
authors.initials: AJ
authors.lastname: Kordala
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Rita
authors.initials: R
authors.lastname: Strack
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Renaud
authors.initials: R
authors.lastname: Geslain
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Kamila
authors.initials: K
authors.lastname: Delaney
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Wesley C
authors.initials: WC
authors.lastname: Clark
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Keenan
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, USA Institute of Biophysical Dynamics, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Tao
authors.initials: T
authors.lastname: Pan
mesh_terms: D001483:Base Sequence; D005456:Fluorescent Dyes; D006801:Humans; D008715:Methionine / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012352:RNA, Transfer, Glu / Q000737:chemistry / Q000235:genetics
chemical_list: D005456:Fluorescent Dyes; D012352:RNA, Transfer, Glu; D008715:Methionine
keywords: FACS; fluorescent protein; mistranslation; reporter; tRNA isodecoder