Dual promoter architecture of NFE2L2 underlies isoform-specific translational control of NRF2.

Transcription factor NRF2 (encoded by the NFE2L2 gene) is a central signalling hub that coordinates antioxidant, detoxification, anti-inflammatory and metabolic programmes to maintain cellular homeostasis, with broad relevance to degenerative diseases, chronic inflammation and cancer. NFE2L2 is transcribed from two promoters: the canonical promoter (P1) and an alternative promoter (P2), generating transcripts that differ solely in their first exon. Exon 1 of P1-derived transcripts contains the AUG start codon, whereas the considerably longer exon 1' of P2-derived transcripts forms an extended 5' untranslated region (5'UTR). Consequently, the P1-derived NRF2 isoform 1 contains 16 additional N-terminal amino acids relative to the P2-derived isoform 2. Although transcripts from both promoters are expressed in human tissues, the existence of isoform 2 protein and the functional significance of alternative promoter usage have remained unresolved. Here, we identify an endogenous NRF2 protein species consistent with isoform 2 using an isoform-specific siRNA strategy. Isoform 2 is negatively regulated by KEAP1 and retains the ability to activate canonical NRF2 target genes. Despite similar stability of the two isoforms, isoform 2 accumulates at substantially lower basal levels, which is associated with reduced translational engagement of P2-derived transcripts. Polysome profiling revealed that P1-derived transcripts are predominantly associated with polysomes, whereas P2-derived transcripts are distributed between polysomal and subpolysomal fractions. The extended P2-specific 5'UTR contains evolutionarily conserved structural features that may contribute to translational regulation. Finally, the electrophilic NRF2 activator omaveloxolone (RTA-408) induced translation of transcripts from both promoters, demonstrating that NRF2 stress response is coordinated through the combined regulation of mRNA translation and protein stability.