The Ribosome Associated Gene Transcripts During Oxidative Stress

Abstract ID 91811 Poster Board 543 Redox homeostasis is compromised when cells are exposed to elevated levels of Reactive Oxygen Species (ROS). Excessive ROS alter cell fate by inducing damage to macromolecules, while low to mild levels of ROS trigger a cascade of signaling events contributing to cell survival. Stress in general causes a global inhibition of protein synthesis. Increasing evidence supports that selective protein translation occurs and benefits cellular repair and adaptation. Since the mRNA species in the process of being translated are bound with ribosomes, to investigate which proteins are translated during oxidative stress, we sequenced poly (A) RNAs (RNA-Seq) associated with ribosomes from Hela cells treated with a low dose of H2O2 (100 microM) for 1 hour. A total of 21 transcripts showed statistically significant changes ranging from 1.7 to 40 fold. Two genes were verified for expression at total mRNA, polysomal RNA and protein level: ATF3 and sMafF. ATF3 mRNA showed a 2.9 fold increase, whereas sMafF showed a 2.1 fold increase in association with ribosomes by H2O2 treatment per RNA-Seq analyses. The observed increases were confirmed using qRT-PCR, which revealed 50 to 300 microM H2O2 dose-dependent increase. Despite the increases of both mRNA species in the ribosomes, ATF3 exhibited an elevation of mRNA in total cellular RNA and an increase of protein expression, whereas sMafF showed no change in total mRNA or the level of its protein within the observed time frame of its increased mRNA in the ribosomes. Therefore, ATF3 appears to undergo transcription coupled translation, while the functional significance of sMafF transcript accumulation in ribosomes remains to be determined. ATF3, an activating transcription factor belonging to CREB family, regulates transcription of genes involved in cell proliferation and apoptosis in response to cellular stress. sMafF is a basic region leucine zipper transcription factor lacking a transactivation domain but forms a heterodimer with antioxidant transcription factor Nrf2 or BACH1 for transcriptional regulation. Our data highlights the selective accumulation of mRNA transcripts in the ribosomes and the complexity of protein translation mechanisms under oxidative stress. NIH R01 GM125212, R01 GM126165, Holsclaw Endowment and the University of Arizona College of Pharmacy start-up fund