Abstract
Nrf2 gene encodes a transcription factor that regulates the expression of a cluster of antioxidant and detoxification genes. Recent works from our laboratory indicate that oxidative stress causes rapid de novo synthesis of Nrf2 protein. We have found that 5' Untranslated Region (5'UTR) of Nrf2 allows the mRNA to undergo an Internal Ribosomal Entry Site (IRES) mediated protein translation. Using liquid chromatography tandem MS, we have discovered that La/SSB protein bound to Nrf2 5'UTR in response to oxidative stress. In vitro RNA binding and in vivo ribonucleoprotein immunoprecipitation showed H(2)O(2) dose and time dependent increases of La/SSB binding to Nrf2 5'UTR. La/SSB protein translocated from the nuclei to cytoplasm and distributed in the perinuclear space in cells treated with H(2)O(2). Isolation of ribosomal fractions indicated that oxidants caused an association of La/SSB with ribosomes. Physical interaction of La/SSB with representative proteins from the small or large subunits of ribosomes was found to increase in cells responding to H(2)O(2) treatment. Knocking down La/SSB gene with siRNA prevented Nrf2 protein elevation or Nrf2 5'UTR activation by oxidants. In contrast, overexpression of La/SSB gene was able to enhance Nrf2 5'UTR activation and Nrf2 protein increase. Our data suggest that oxidants cause nuclear export of La/SSB protein and subsequent association of La/SSB with Nrf2 5'UTR and ribosomes. These events contribute to de novo Nrf2 protein translation because of oxidative stress.
Highlights
Nuclear factor erythroid-2 related factor 2 (Nrf2)1 is a transcription factor that regulates the expression of a cluster of antioxidant and detoxification genes
Whereas stress causes an overall inhibition of protein synthesis, increasing evidence suggests that genes containing an Internal Ribosomal Entry Site (IRES) in the 5Ј Untranslated Region (5ЈUTR) of mRNA can bypass the mechanism of general protein synthesis, which is dependent on 7-methyl guanine cap at the 5Ј end of mRNA
H2O2 induced La/Sjogren Syndrome Antigen B (SSB) binding to Nrf2 5ЈUTR was confirmed by Far Western and RNA-protein complex immunoprecipitation approaches
Summary
Nuclear factor erythroid-2 related factor 2; UTR, untranslated region; IRES, Internal Ribosomal Entry Site; ARE, Antioxidant Response Element; ITAF, IRES transacting factor. Whereas stress causes an overall inhibition of protein synthesis, increasing evidence suggests that genes containing an Internal Ribosomal Entry Site (IRES) in the 5Ј Untranslated Region (5ЈUTR) of mRNA can bypass the mechanism of general protein synthesis, which is dependent on 7-methyl guanine cap at the 5Ј end of mRNA. A stable secondary structure containing “stems and loops” has been predicted via Zucker’s MFold algorithm (Xu et al, 2011 manuscript submitted) This suggests that Nrf2 5ЈUTR contains an IRES, allowing Nrf protein being translated under oxidative stress. A recent study using proteomic approaches found four proteins: G-rich RNA sequence binding factor 1 (GRSF-1), Y-box binding protein 1 (YB-1), PTB associated splicing factor (PSF), and its binding partner p54nrb, as ITAFs for regulating translation of myc family genes [10]. We report the finding of La autoantigen as an ITAF regulating Nrf protein translation under oxidative stress
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