DExH-Box Helicase 9 Participates in De Novo Nrf2 Protein Translation Under Oxidative Stress

Nrf2 transcript factor plays an important role in cellular defense against oxidative stress due to its control for expression of antioxidant and detoxification genes. We have found that Nrf2 gene undergoes de novo protein translation when mammalian cells encounter oxidative stress. Here, we report the discovery of DExH-box helicase-9 (DHX9), also known as RNA helicase A, as a binding protein for Nrf2 mRNA at 5'UTR. DHX9 binding to Nrf2 5'UTR increased with increasing doses (50-300 μM) of H₂O₂ or treatment time (10-120 min). This incease was in parallel with elevation of Nrf2 protein. Inhibiting DHX9 expression with siRNA or its activity with YK-4-279 inhibitor blocked H₂O₂ from inducing Nrf2 mRNA recruitment to the ribosomes or Nrf2 protein elevation. As a nuclear protein, DHX9 was found to increase its abundance in the cytosol with oxidative stress. An increase of DHX9 was detected in the ribosomes from cells treated with H₂O₂, most significantly with 100 μM H₂O₂, and at 60 min. Ribosomal fractionation revealed an increase of DHX9 protein at 43/48S and 80S fractions in H₂O₂-treated cells. H₂O₂ treatment caused an RNA-dependent increase of DHX9 interaction with eIF3η. The binding of DHX9 to Nrf2 5'UTR was enhanced by H₂O₂-treated cells or by deducting the length of Nrf2 5'UTR. RNase digestion enhanced DHX9 association with the ribosomes. Our data have revealed a novel mechanism of de novo Nrf2 protein translation under oxidative stress involving DHX9 binding to Nrf2 5'UTR, perhaps via removal of a negative RNA element, to recruit 43S preinitiation complex for translation initiation.