Tanaka, M., Chock, P.B., and Stadtman, E.R.
Notes: Oxidative damage has been associated with a range of age-related neurological conditions. In this study, the effect of mRNA oxidation was investigated. A direct correlation was observed between the extent of oxidation and the frequency of translation errors. The authors excised the firefly luciferase (luc2) gene from the pGL4.14 Vector, attached a FLAG tag to the 5´ terminus and a Myc tag to the 3´ terminus, and subcloned the gene into a pGEM-4Z Vector that had been modified to append a poly(A) sequence. The construct was transfected into HEK293 cells, which were then cultured in the presence of an oxidizing agent. The occurrence of truncated protein fragments and short peptides increased in the presence of the oxidizing agent in a concentration-dependent manner. The effects of oxidation of mRNA were also investigated in in vitro translation experiments using mRNA treated with an iron-ascorbate mixture and hydrogen peroxide. Translation in vitro was performed using rabbit reticulocyte lysate supplemented with protease inhibitors. The translation products were detected using anti-FLAG and anti-c-Myc antibodies. (3630)