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. 2019 Dec;33(12):14636-14652.
doi: 10.1096/fj.201901930R. Epub 2019 Nov 1.

Post-transcriptional regulation of Nrf2-mRNA by the mRNA-binding proteins HuR and AUF1

Jesse R Poganik  1   2 , Marcus J C Long  2 , Michael T Disare  2 , Xuyu Liu  1 , Sung-Hee Chang  3 , Timothy Hla  4   5 , Yimon Aye  1
Affiliations

Post-transcriptional regulation of Nrf2-mRNA by the mRNA-binding proteins HuR and AUF1

Jesse R Poganik et al. FASEB J. 2019 Dec.

Abstract

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.

Keywords: antioxidant response; mRNA-maturation; mRNA-stabilization; mRNA-trafficking; zebrafish.

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Conflict of interest statement

The authors thank Dr. Jen Grenier [Cornell RNA Sequencing Core (RSC) Cornell University] for assistance with RNA sequencing. The authors also thank Alexandra Van Hall-Beauvais and Gene Hu (Cornell University) for assistance with initial studies of HuR. The following organizations have supported this study: American Heart Association predoctoral fellowship (17PRE33670395 to J.R.P.); Swiss National Science Foundation (SNSF); Swiss Federal Institute of Technology Lausanne (EPFL); Novartis Medical-Biological Research Foundation; National Centre of Competence in Research Chemical Biology (NCCR) Chemical Biology; and U.S. National Institutes of Health (NIH) Director’s New Innovator (Office of the Director, 1DP2GM114850 to Y.A.). The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
RNA-seq expression profiling indicates context-specific HuR regulation in global and Nrf2-specific transcriptional activities. A, B) Differential expression from RNA-seq analyses in shHuR or shControl HEK293T cells following HNE (A, 25 μM, 18 h) or H2O2 (B, 225 μM, 18 h) stimulation relative to respective nonstimulated cells. Genes SDE are denoted with dark, opaque points; Nrf2-driven genes SDE in at least 1 comparison are labeled. Dashed lines to gene names indicate that the gene was not SDE in that comparison. See also Supplemental Figs. S1A, S2, and S3 and Supplemental Tables S2 and S3. C) Differential expression from RNA-seq analysis in nonstimulated shHuR cells relative to shControl cells. Genes SDE are denoted with blue points. Nrf2-driven genes found to be SDE are labeled. See also Supplemental Tables S3–S5. D) Nrf2 activity reporter system in which firefly luciferase is driven by Nrf2 and a constitutive Renilla luciferase is coexpressed as an internal normalization control. E) Relative Nrf2 activity in unstimulated shHuR and shControl cells (mean ± sem, n = 24); shControl set to 1.0. F) Relative Nrf2 activity was measured as in F, but ectopic HuR was expressed in non-HNE-stimulated shHuR cells (mean ± sem, n = 7 for shHuR+HuR and n = 8 for other conditions) and shHuR + Empty vector was set to 1.0. See also Supplemental Fig. S4. G) Relative Nrf2 activity up-regulation in HNE-stimulated shHuR (25 μM, 18 h) and shControl (mean ± sem, n = 16) lines was calculated by measuring (Nrf2 activity + HNE/Nrf2 activity +DMSO); shControl was set to 1.0. See also Supplemental Fig. S4. H) Relative Nrf2 activity up-regulation was measured as in G, but ectopic HuR was expressed in HNE-stimulated (25 μM, 18 h) shHuR cells (mean ± sem, n = 8); shHuR + Empty vector was set to 1.0. See also Supplemental Fig. S4. I) Nrf2 activity in H2O2-stimulated (225 μM, 18 h) shHuR and shControl (mean ± sem, n = 7 for shHuR and 8 for shControl) lines was calculated by measuring (Nrf2 activity with H2O2/Nrf2 activity with water); shControl was set to 1.0. See also Supplemental Fig. S4. All P values were calculated with Student’s t test. For (A, B), data were derived from 2 independent biologic replicates per treatment condition. Skewness was calculated with Prism. FPKM, fragments per kilobase of transcript per million mapped reads; ns, not significant.
Figure 2
Figure 2
Depletion of HuR or AUF1 in cells and larval zebrafish suppresses Nrf2 activity. A, B) Nrf2 activity in HEK293T cells depleted of AUF1 and HuR, respectively [mean ± sem of n = 12 (AUF1) and n = 8 (HuR) independent replicates per condition]. See also Supplemental Fig. S1B, C. C) Nrf2 activity upon simultaneous knockdown of HuR and AUF1 (mean ± sem of n = 4 independent replicates). See also Supplemental Fig. S7A. D, E) Nrf2 activity in Tg(gstp1:GFP) zebrafish upon knockdown of zHur and zAuf1. Larvae are stained with a red fluorescent antibody because green background fluorescence at this developmental stage prevents accurate quantitation of the GFP reporter signal. Inset: quantitation (mean ± sem) of mean fluorescence intensity measured using the Measure tool of ImageJ [sample sizes analyzed: D: n = 26 (Control MO), 44 (ATG-MO); E: n = 38 (Control MO), 12 (ATG-MO), 32 (SPL-MO)]. Each point represents a single fish. P values were calculated with Student’s t test. Scale bars, 500 μm. See also Supplemental Figs. S1D, E and S8. ATG-MO, translation initiation blocking morpholino oligonucleotide; SPL-MO, splice blocking morpholino oligonucleotide.
Figure 3
Figure 3
HuR and AUF1 bind directly to the 3′–UTR of Nrf2-mRNA in cells. A) RIP was carried out by expressing flag-tagged HuR or AUF1 in HEK293T cells and subjecting lysates to Flag IP. B) Nrf2-mRNA coeluting with enriched proteins was detected with real-time qPCR (mean ± sem of n = 4 independent replicates). C) 3′–UTR reporter system consisting of Nrf2-mRNA fused to a firefly luciferase transcript and normalized to Renilla luciferase. D, E) Knockdown of HuR and AUF1, respectively, reduces the 3′–UTR reporter activity in HEK293T cells (mean ± sem of n = 8 independent replicates for each bar). F) Knockdown of both proteins leads to a further suppression of the 3′–UTR reporter activity (mean ± sem of n = 4 independent replicates). See also Supplemental Fig. S7B. All P values were calculated with Student’s t test. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ZNF200, zinc finger protein 200.
Figure 4
Figure 4
HuR knockdown suppresses Nrf2-mRNA splicing and nuclear export of Nrf2-mRNA. A) Integrative Genomics Viewer (71) view of splicing tracks for Nrf2 (NFE2L2) from RNA-seq analysis of shHuR and shControl HEK293T cells. The area of the blue tracks corresponds to the levels of intronic RNA detected. Shown is 1 representative replicate per cell line. Inset at right: areas of splicing tracks integrated with ImageJ (mean ± sem, n = 4 introns). See also Supplemental Fig. S11. B) Reporters to read out the effect of introns were constructed by fusing a portion of Nrf2-mRNA (with or without the intron) upstream of a firefly luciferase reporter. As with the 3′–UTR reporter (Fig. 3C), effects on this construct can be assayed by measuring luciferase activity in the lysates of cells expressing these reporters. C) Reporter levels upon HuR knockdown in nonstimulated HEK293T cells for both the intron-less reporter (left) and the intron reporter (right) (mean ± sem of n ≥ 7 per set). Inset at right: Comparison of reporter activity in shHuR cells (i.e., blue bars from the main plot in C upon introduction of introns. D) Real-time qPCR was used to measure the ratio of endogenous Nrf2-mRNA in nuclear and cytosolic extracts of HEK293T cells depleted of HuR [mean ± sem of n = 8 for siHuR (1, 2) and n = 7 for siControl]. E) Model of post-transcriptional regulation of Nrf2-mRNA by HuR and AUF1. HuR regulates Nrf2-mRNA maturation and nuclear export, and AUF1 stabilizes Nrf2-mRNA. Shown in blue text/boxes is the experimental evidence supporting each facet of this regulatory program. All P values were calculated with Student’s t test.
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