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Translation errors mediated by oxidized and methylated mRNA
Author(s) -
Tanaka Mikiei,
Tanaka Hikari,
Chock P. Boon
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.09553
Subject(s) - luciferase , messenger rna , translation (biology) , chemistry , reticulocyte , microbiology and biotechnology , biochemistry , protein biosynthesis , biology , gene , transfection
Post translational modification of biomacromolecules play an important role in their normal physiological functions. However, under pathological conditions, extracellular toxic compounds are known to induce various covalent modification of the biomacromolecules to exert their toxic effects. It is known that oxidative damage and alkylation can cause mutation or strand break of genomic DNA leading to tumorigenesis. However, the consequences of these modifications to mRNA has not been well investigated. The aim of this study is to assess the protein synthesis activity mediated by the oxidized and methylated mRNA. Here, the synthesized protein was monitored by the luciferase activity of the in vitro synthesized mRNA encoding FLAG‐tagged luciferase gene. The synthesized mRNA was subjected to iron‐catalyzed oxidation (Fe‐ox) or monomethylation by methylmethane sulfonate (MMS). The data reveal that the modified mRNAs induces a decrease in luciferase activity both in the reticulocyte lysate and wheat germ lysate in in vitro translation system. Conversely, mRNA was synthesized in the presence of modified nucleotide, to generate mRNA that incorporated with modified base, N1‐mA, N6‐mA, N7‐mG, or 8‐oxo‐G. Our data reveal that N6‐mA and 8‐oxo‐G mRNA exhibit a reduced luciferase activity, however the reduction was modest. To ensure that the activity of the full‐‐length protein was not compromised by amino acid substitution caused by mistranslation, LC/MS method was used to analyze these products. Interestingly, the proteins generated with N1‐mA mRNA and N7‐mG mRNA exhibited full luciferase activity. In addition, Western blotting data showed an increase in the generation of fragmented peptides in the Fe‐ox and MMS modified mRNAs, and in the 8‐oxo‐G or N1‐m6A mRNA,suggesting that these modified nucleotides tend to induce translation errors.