Regulation of HuR by DNA Damage Response Kinases

As many DNA-damaging conditions repress transcription, posttranscriptional processes critically influence gene expression during the genotoxic stress response. The RNA-binding protein HuR robustly influences gene expression following DNA damage. HuR function is controlled in two principal ways: (1) by mobilizing HuR from the nucleus to the cytoplasm, where it modulates the stability and translation of target mRNAs and (2) by altering its association with target mRNAs. Here, we review evidence that two main effectors of ataxia-telangiectasia-mutated/ATM- and Rad3-related (ATM/ATR), the checkpoint kinases Chk1 and Chk2, jointly influence HuR function. Chk1 affects HuR localization by phosphorylating (hence inactivating) Cdk1, a kinase that phosphorylates HuR and thereby blocks HuR's cytoplasmic export. Chk2 modulates HuR binding to target mRNAs by phosphorylating HuR's RNA-recognition motifs (RRM1 and RRM2). We discuss how HuR phosphorylation by kinases including Chk1/Cdk1 and Chk2 impacts upon gene expression patterns, cell proliferation, and survival following genotoxic injury

The RNA-binding protein HuR contributes to neuroinflammation by promoting C-C chemokine receptor 6 (CCR6) expression on Th17 cells.

In both multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), the C-C chemokine receptor 6 (CCR6) is critical for pathogenic T helper 17 (Th17) cell migration to the central nervous system (CNS). Whereas many cytokines and their receptors are potently regulated via post-transcriptional mechanisms in response to various stimuli, how CCR6 expression is post-transcriptionally regulated in Th17 cells is unknown. Here, using RNA-binding protein HuR conditional knock-out (KO) and wild-type (WT) mice, we present evidence that HuR post-transcriptionally regulates CCR6 expression by binding to and stabilizing Ccr6 mRNA and by promoting CCR6 translation. We also found that HuR down-regulates several microRNA expressions, which could target the 3\u27-UTR of Ccr6 mRNA for decay. Accordingly, knock-out of HuR reduced CCR6 expression on Th17 cells and impaired their migration to CNS compared with the response of WT Th17 cells and thereby ameliorated EAE. Together, these findings highlight how HuR contributes to Th17 cell-mediated autoimmune neuroinflammation and support the notion that targeting HuR might be a potential therapeutic intervention for managing autoimmune disorders of the CNS

Post-Transcriptional Control of the Hypoxic Response by RNA-Binding Proteins and MicroRNAs

Mammalian gene expression patterns change profoundly in response to low oxygen levels. These changes in gene expression programs are strongly influenced by post-transcriptional mechanisms mediated by mRNA-binding factors: RNA-binding proteins (RBPs) and microRNAs (miRNAs). Here, we review the RBPs and miRNAs that modulate mRNA turnover and translation in response to hypoxic challenge. RBPs such as HuR (human antigen R), PTB (polypyrimidine tract-binding protein), heterogeneous nuclear ribonucleoproteins (hnRNPs), tristetraprolin, nucleolin, iron-response element-binding proteins (IRPs), and cytoplasmic polyadenylation-element-binding proteins (CPEBs), selectively bind to numerous hypoxia-regulated transcripts and play a major role in establishing hypoxic gene expression patterns. MiRNAs including miR-210, miR-373, and miR-21 associate with hypoxia-regulated transcripts and further modulate the levels of the encoded proteins to implement the hypoxic gene expression profile. We discuss the potent regulation of hypoxic gene expression by RBPs and miRNAs and their integrated actions in the cellular hypoxic response

The Transcription Factor NRF2 Has Epigenetic Regulatory Functions Modulating HDACs, DNMTs, and miRNA Biogenesis

The epigenetic regulation of gene expression is a complex and tightly regulated process that defines cellular identity and is associated with health and disease processes. Oxidative stress is capable of inducing epigenetic modifications. The transcription factor NRF2 (nuclear factor erythroid-derived 2-like 2) is a master regulator of cellular homeostasis, regulating genes bearing antioxidant response elements (AREs) in their promoters. Here, we report the identification of ARE sequences in the promoter regions of genes encoding several epigenetic regulatory factors, such as histone deacetylases (HDACs), DNA methyltransferases (DNMTs), and proteins involved in microRNA biogenesis. In this research, we study this possibility by integrating bioinformatic, genetic, pharmacological, and molecular approaches. We found ARE sequences in the promoter regions of genes encoding several HDACs, DNMTs, and proteins involved in miRNA biogenesis. We confirmed that NRF2 regulates the production of these genes by studying NRF2-deficient cells and cells treated with dimethyl fumarate (DMF), an inducer of the NRF2 signaling pathway. In addition, we found that NRF2 could be involved in the target RNA-dependent microRNA degradation (TDMD) of miR-155-5p through its interaction with Nfe2l2 mRNA. Our data indicate that NRF2 has an epigenetic regulatory function, complementing its traditional function and expanding the regulatory dimensions that should be considered when developing NRF2-centered therapeutic strategiesThis work was supported MINECO (SAF2016-76520-R to I.L-B., PID2019-105600RB-I00 to I.L-B) and ISCiii CIBERNED (CB06/05/0089 to I.L.-B.

Identification of a Signature Motif in Target mRNAs of RNA-binding Protein AUF1

The ubiquitous RNA-binding protein AUF1 promotes the degradation of some target mRNAs, but increases the stability and translation of other targets. Here, we isolated AUF1-associated mRNAs by immunoprecipitation of (AUF1–RNA) ribonucleoprotein (RNP) complexes from HeLa cells, identified them using microarrays, and used them to elucidate a signature motif shared among AUF1 target transcripts. The predicted AUF1 motif (29–39 nucleotides) contained 79% As and Us, consistent with the AU-rich sequences of reported AUF1 targets. Importantly, 10 out of 15 previously reported AUF1 target mRNAs contained the AUF1 motif. The predicted interactions between AUF1 and target mRNAs were recapitulated in vitro using biotinylated RNAs. Interestingly, further validation of predicted AUF1 target transcripts revealed that AUF1 associates with both the pre-mRNA and the mature mRNA forms. The consequences of AUF1 binding to 10 predicted target mRNAs were tested by silencing AUF1, which elevated the steady-state levels of only four mRNAs, and by overexpressing AUF1, which also lowered the levels of only four mRNAs. In total, we have identified a signature motif in AUF1 target mRNAs, have found that AUF1 also associates with the corresponding pre-mRNAs, and have discovered that altering AUF1 levels alone only modifies the levels of subsets of target mRNAs

NSUN2‐Mediated m5C Methylation and METTL3/METTL14‐Mediated m6A Methylation Cooperatively Enhance p21 Translation

N6‐methyladenosine (m6A) and m5C methylation are two major types of RNA methylation, but the impact of joint modifications on the same mRNA is unknown. Here, we show that in p21 3′UTR, NSUN2 catalyzes m5C modification and METTL3/METTL14 catalyzes m6A modification. Interestingly, methylation at m6A by METTL3/METTL14 facilitates the methylation of m5C by NSUN2, and vice versa. NSUN2‐mediated m5C and METTL3/METTL14‐mediated m6A methylation synergistically enhance p21 expression at the translational level, leading to elevated expression of p21 in oxidative stress‐induced cellular senescence. Our findings on p21 mRNA methylation and expression reveal that joint m6A and m5C modification of the same RNA may influence each other, coordinately affecting protein expression patterns. J. Cell. Biochem. 118: 2587–2598, 2017. © 2017 Wiley Periodicals, Inc.In p21 3’UTR,NSUN2 catalyzes m5C modification and METTL3/METTL14 catalyzes m6A modification. Methylation at m6A by METTL3/METTL14 facilitates the methylation of m5C by NSUN2, and vice versa. NSUN2‐mediated m5C and METTL3/METTL14‐mediated m6A methylation synergistically enhance p21 expression at the translational level, leading to elevated expression of p21 in oxidative stress‐induced cellular senescence.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/137760/1/jcb25957.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137760/2/jcb25957_am.pd

En masse nascent transcription analysis to elucidate regulatory transcription factors

Despite exhaustively informing about steady-state mRNA abundance, DNA microarrays have been used with limited success to identify regulatory transcription factors (TFs). The main limitation of this approach is that altered mRNA stability also strongly governs the patterns of expressed genes. Here, we used nuclear run-on assays and microarrays to systematically interrogate changes in nascent transcription in cells treated with the topoisomerase inhibitor camptothecin (CPT). Analysis of the promoters of coordinately transcribed genes after CPT treatment suggested the involvement of TFs c-Myb and Rfx1. The predicted CPT-dependent associations were subsequently confirmed by chromatin immunoprecipitation assays. Importantly, after RNAi-mediated knockdown of each TF, the CPT-elicited induction of c-Myb- and/or Rfx1-regulated mRNAs was diminished and the overall cellular response was impaired. The strategies described here permit the successful identification of the TFs responsible for implementing adaptive gene expression programs in response to cellular stimulation