The nuclear factor erythroid 2‐related factor 2 (Nrf2) signaling axis is a target of covalent drugs and bioactive native electrophiles. However, much of our understanding of Nrf2 regulation has been focused at the protein level. Here we report a post‐transcriptional modality to directly regulate Nrf2‐mRNA. Our initial studies focused on the effects of the key mRNA‐binding protein (mRBP) HuR on global transcriptomic changes incurred upon oxidant or electrophile stimulation. These RNA‐sequencing data and subsequent mechanistic analyses led us to discover a novel role of HuR in regulating Nrf2 activity, and in the process, we further identified the related mRBP AUF1 as an additional novel Nrf2 regulator. Both mRBPs regulate Nrf2 activity by direct interaction with the Nrf2 transcript. Our data showed that HuR enhances Nrf2‐mRNA maturation and promotes its nuclear export, whereas AUF1 stabilizes Nrf2‐mRNA. Both mRBPs target the 3'‐UTR of Nrf2‐mRNA. Using a Nrf2 activity‐reporter zebrafish strain, we document that this post‐transcriptional control of Nrf2 activity is conserved at the whole‐vertebrate level.—Poganik, J. R., Long, M. J. C., Disare, M. T., Liu, X., Chang, S.‐H., Hla, T., Aye, Y. Post‐transcriptional regulation of Nrf2‐mRNA by the mRNA‐binding proteins HuR and AUF1. FASEB J. 33, 14636‐14652 (2019). www.fasebj.org

The anophelins are small protein thrombin inhibitors that are produced in the salivary glands of the Anopheles mosquito to fulfill a vital role in blood feeding. A bioinformatic analysis of anophelin sequences revealed the presence of conserved tyrosine residues in an acidic environment that were predicted to be post-translationally sulfated in vivo. To test this prediction, insect cell expression of two anophelin proteins, from Anopheles albimanus and Anopheles gambiae, was performed, followed by analysis by mass spectrometry, which showed heterogeneous sulfation at the predicted sites. Homogeneously sulfated variants of the two proteins were subsequently generated by chemical synthesis via a one-pot ligation–desulfurization strategy. Tyrosine sulfation of the anophelins was shown to significantly enhance the thrombin inhibitory activity, with a doubly sulfated variant of the anophelin from A. albimanus exhibiting a 100-fold increase in potency compared with the unmodified homologue. Sulfated anophelins were also shown to exhibit potent in vivo anticoagulant and antithrombotic activity.