Identification of RNA binding proteins associated with the 5'UTR of Nrf2 during oxidative stress

ABSTRACT: Nrf2 transcript factor plays an important role in cellular defense against oxidative stress due to its control for the expression of antioxidant and detoxification genes. We have found that Nrf2 undergoes de novo protein translation when mammalian cells encounter oxidative stress. Here, we report the discovery of DHX9 as a binding protein for Nrf2 5’UTR. H2O2 treatment causes dose- or time- dependent increases of DHX9 binding to Nrf2 5’UTR, in parallel with elevation of Nrf2 protein. Inhibiting DHX9 expression with siRNA or its activity with YK-4-279 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 total ribosomal collections in cells treated with H2O2, most significantly with 100 uM H2O2, and at 60 mins. 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 eIF3 The binding of DHX9 to Nrf2 5’UTR probe was enhanced by H2O2 treated cells or by deducting the length of Nrf2 5’UTR, suggesting a negative element normally prohibiting DHX9 binding to 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 pre-initiation complex for translation initiation.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Daniel Wurm  

LAB HEAD: Qin Chen

PROVIDER: PXD055838 | Pride | 2025-06-23

REPOSITORIES: Pride