Chronic ethanol exposure increases the binding of HuR to the TNFalpha 3'-untranslated region in macrophages

Tumor necrosis factor alpha (TNFalpha) expression is a key mediator of ethanol-induced liver disease. Increased lipopolysaccharide (LPS)-stimulated TNFalpha expression in macrophages after chronic ethanol feeding is associated with a stabilization of TNFalpha mRNA (Kishore, R., McMullen, M. R., and Nagy, L. E. (2001) J. Biol. Chem. 276, 41930-41937). Here we show that the 3'-UTR of murine TNFalpha mRNA was sufficient to mediate increased LPS-stimulated expression of a luciferase reporter in RAW 264.7 macrophages after chronic ethanol exposure. Further, we show that HuR, a nuclear/cytoplasmic shuttling protein, which binds to TNFalpha mRNA, is required for increased expression of TNFalpha after chronic ethanol. In Kupffer cells, HuR was primarily localized to the nucleus and then translocated to the cytosol in response to LPS in both pair- and ethanol-fed rats. After chronic ethanol feeding, HuR quantity in the cytosol was greater, both at baseline and in response to LPS, compared with pair-fed controls. Using RNA gel shift assays, we found that LPS treatment increased HuR binding to the 65-nucleotide A + U-rich element of the TNFalpha 3'-UTR by 2-fold over baseline in Kupffer cells from pair-fed rats. After chronic ethanol feeding, HuR binding to the TNFalpha A + U-rich element was increased by more than 5-fold at baseline and in response to LPS, compared with pair-fed controls. Down-regulation of HuR expression by RNA interference prevented the chronic ethanol-induced increase in expression of luciferase reporters containing the TNFalpha 3'-UTR. Taken together, these data demonstrate that increased binding of HuR to the TNFalpha 3'-UTR contributes to increased LPS-stimulated TNFalpha expression in macrophages after chronic ethanol exposure.     

Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR

We purified the KH-type splicing regulatory protein (KSRP) as a protein interacting with the 3'-untranslated region (3'-UTR) of the human inducible nitric oxide (iNOS) mRNA. Immunodepletion of KSRP enhanced iNOS 3'-UTR RNA stability in in vitro-degradation assays. In DLD-1 cells overexpressing KSRP cytokine-induced iNOS expression was markedly reduced. In accordance, downregulation of KSRP expression increases iNOS expression by stabilizing iNOS mRNA. Co-immunoprecipitations showed interaction of KSRP with the exosome and tristetraprolin (TTP). To analyze the role of KSRP binding to the 3'-UTR we studied iNOS expression in DLD-1 cells overexpressing a non-binding mutant of KSRP. In these cells, iNOS expression was increased. Mapping of the binding site revealed KSRP interacting with the most 3'-located AU-rich element (ARE) of the human iNOS mRNA. This sequence is also the target for HuR, an iNOS mRNA stabilizing protein. We were able to demonstrate that KSRP and HuR compete for this binding site, and that intracellular binding to the iNOS mRNA was reduced for KSRP and enhanced for HuR after cytokine treatment. Finally, a complex interplay of KSRP with TTP and HuR seems to be essential for iNOS mRNA stabilization after cytokine stimulation.     

热激蛋白Hsp72促进热休克下HuR对p21CIP1的调控作用

RNA结合蛋白HuR可以结合并调控靶标mRNA稳定性与翻译,但影响HuR 结合活性的因素有待探讨。本研究从蛋白质-蛋白质相互作用角度对影响HuR 与RNA结合活性的因素做了探讨。结果发现,热激蛋白Hsp72在细胞浆与HuR相互作用并促进HuR与p21 (KIP1) 3′UTR（3′非翻译区）的结合; 热休克下Hsp72总蛋白质及细胞浆蛋白质水平上调、但HuR总蛋白质及细胞浆蛋白质水平不变|热休克下HuR与p21 3′UTR的相互作用加强、p21蛋白及mRNA水平上调。上述结果提示,Hsp72可通过与HuR相互作用促进后者与p21 mRNA的结合,进而加强热休克下HuR对p21的表达的促进作用。这些结果为进一步解析HuR的生物学作用机制提供了实验依据。     

Post-transcriptional regulation of RNase-L expression is mediated by the 3'-untranslated region of its mRNA

RNase-L mediates critical cellular functions including antiviral, pro-apoptotic, and tumor suppressive activities; accordingly, its expression must be tightly regulated. Little is known about the control of RNASEL expression; therefore, we examined the potential regulatory role of a conserved 3'-untranslated region (3'-UTR) in its mRNA. The 3'-UTR mediated a potent decrease in the stability of RNase-L mRNA, and of a chimeric beta-globin-3'-UTR reporter mRNA. AU-rich elements (AREs) are cis-acting regulatory regions that modulate mRNA stability. Eight AREs were identified in the RNase-L 3'-UTR, and deletion analysis identified positive and negative regulatory regions associated with distinct AREs. In particular, AREs 7 and 8 served a strong positive regulatory function. HuR is an ARE-binding protein that stabilizes ARE-containing mRNAs, and a predicted HuR binding site was identified in the region comprising AREs 7 and 8. Co-transfection of HuR and RNase-L enhanced RNase-L expression and mRNA stability in a manner that was dependent on this 3'-UTR region. Immunoprecipitation demonstrated that RNase-L mRNA associates with a HuR containing complex in intact cells. Activation of endogenous HuR by cell stress, or during myoblast differentiation, increased RNase-L expression, suggesting that RNase-L mRNA is a physiologic target for HuR. HuR-dependent regulation of RNase-L enhanced its antiviral activity demonstrating the functional significance of this regulation. These findings identify a novel mechanism of RNase-L regulation mediated by its 3'-UTR.     

Sequence requirements for RNA binding by HuR and AUF1

The stability of RNAs bearing AU-rich elements in their 3'-UTRs, and thus the level of expression of their protein products, is regulated by interactions with cytoplasmic RNA-binding proteins. Binding by HuR generally leads to mRNA stabilization and increased protein production, whereas binding by AUF1 isoforms generally lead to rapid degradation of the mRNA and reduced protein production. The exact nature of the interplay between these and other RNA-binding proteins remains unclear, although recent studies have shown close interactions between them and even suggested competition between the two for binding to their cognate recognition sequences. Other recent reports have suggested that the sequences recognized by the two proteins are different. We therefore performed a detailed in vitro analysis of the binding site(s) for HuR and AUF1 present in androgen receptor mRNA to define their exact target sequences, and show that the same sequence is contacted by both proteins. Furthermore, we analysed a proposed HuR target within the 3'-UTR of MTA1 mRNA, and show that the contacted bases lie outside of the postulated motif and are a better match to a classical ARE than the postulated motif. The defining features of these HuR binding sites are their U-richness and single strandedness.     

The ELAV RNA-stability factor HuR binds the 5'-untranslated region of the human IGF-IR transcript and differentially represses cap-dependent and IRES-mediated translation

The type I insulin-like growth factor receptor (IGF-IR) is an integral component in the control of cell proliferation, differentiation and apoptosis. The IGF-IR mRNA contains an extraordinarily long (1038 nt) 5'-untranslated region (5'-UTR), and we have characterized a diverse series of proteins interacting with this RNA sequence which may provide for intricate regulation of IGF-IR gene expression at the translational level. Here, we report the purification and identification of one of these IGF-IR 5'-UTR-binding proteins as HuR, using a novel RNA crosslinking/RNase elution strategy. Because HuR has been predominantly characterized as a 3'-UTR-binding protein, enhancing mRNA stability and generally increasing gene expression, we sought to determine whether HuR might serve a different function in the context of its binding the IGF-IR 5'-UTR. We found that HuR consistently repressed translation initiation through the IGF-IR 5'-UTR. The inhibition of translation by HuR was concentration dependent, and could be reversed in trans by addition of a fragment of the IGF-IR 5'-UTR containing the HuR binding sites as a specific competitor, or abrogated by deletion of the third RNA recognition motif of HuR. We determined that HuR repressed translation initiation through the IGF-IR 5'-UTR in cells as well, and that siRNA knockdown of HuR markedly increased IGF-IR protein levels. Interestingly, we also found that HuR potently inhibited IGF-IR translation mediated through internal ribosome entry. Kinetic assays were performed to investigate the mechanism of translation repression by HuR and the dynamic interplay between HuR and the translation apparatus. We found that HuR, occupying a cap-distal position, significantly delayed translation initiation mediated by cap-dependent scanning, but was eventually displaced from its binding site, directly or indirectly, as a consequence of ribosomal scanning. However, HuR perpetually blocked the activity of the IGF-IR IRES, apparently arresting the IRES-associated translation pre-initiation complex in an inactive state. This function of HuR as a 5'-UTR-binding protein and dual-purpose translation repressor may be critical for the precise regulation of IGF-IR expression essential to normal cellular homeostasis.     

The RNA-binding protein HuR stabilizes cytosolic phospholipase A2α mRNA under interleukin-1β treatment in non-small cell lung cancer A549 Cells

The activation of cytosolic phospholipase A(2)α (cPLA(2)α) plays an important role in initiating the inflammatory response. The regulation of cPLA(2)α mRNA turnover has been proposed to control cPLA(2)α gene expression under cytokine and growth factor stimulation. However, the detailed mechanism is still unknown. In this report, we have demonstrated that the cPLA(2)α mRNA stability was increased under IL-1β treatment in A549 cells. By using EMSAs, HuR was identified as binding with the cPLA(2)α mRNA 3'-UTR, and the binding region was located at nucleotides 2716-2807, a fragment containing AUUUA flanked by U-rich sequences. IL-1β treatment enhanced the association of cPLA(2)α mRNA with cytosolic HuR. The reduction of HuR expression by RNA interference technology inhibited IL-1β-induced cPLA(2)α mRNA and protein expression. Furthermore, blocking the p38 MAPK signaling pathway with SB203580 abolished the effect of IL-1β-induced cPLA(2)α gene expression. Phosphorylation at residue Thr-118 of HuR is crucial in regulating the interaction between HuR and its target mRNAs. Mutation of HuR Thr-118 reduced the association between HuR and cPLA(2)α mRNA under IL-1β treatment. This inhibitory effect was also observed in binding with COX-2 mRNA. This result indicated that p38 MAPK-mediated Thr-118 phosphorylation may play a key role in regulating the interaction of HuR with its target mRNAs in inflammation.     

Distinct roles of heterogeneous nuclear ribonuclear protein K and microRNA-16 in cyclooxygenase-2 RNA stability induced by S100b, a ligand of the receptor for advanced glycation end products

Advanced glycation end products play major roles in diabetic complications. They act via their receptor RAGE to induce inflammatory genes such as cyclooxygenase-2 (COX-2). We examined the molecular mechanisms by which the RAGE ligand, S100b, induces COX-2 in monocytes. S100b significantly increased COX-2 mRNA accumulation in THP-1 monocytes at 2 h via mRNA stability. This was further confirmed by showing that S100b increased stability of luciferase-COX-2 3'-UTR mRNA. Chromatin immunoprecipitation and RNA immunoprecipitation revealed that S100b decreased occupancy of the DNA/RNA-binding protein, heterogeneous nuclear ribonuclear protein K (hnRNPK), at the COX-2 promoter but simultaneously increased its binding to the COX-2 3'-UTR. S100b treatment promoted the translocation of nuclear hnRNPK to cytoplasm, whereas a cytoplasmic translocation-deficient hnRNPK mutant inhibited S100b-induced COX-2 mRNA stability. Small interfering RNA-mediated specific knockdown of hnRNPK blocked S100b-induced COX-2 mRNA stability, whereas on the other hand, overexpression of hnRNPK increased S100b-induced COX-2 mRNA stability. S100b promoted the release of entrapped COX-2 mRNA from cytoplasmic processing bodies, sites of mRNA degradation. Furthermore, S100b significantly down-regulated the expression of a key microRNA, miR-16, which can destabilize COX-2 mRNA by binding to its 3'-UTR. MiR-16 inhibitor oligonucleotides increased, whereas, conversely, miR-16 mimic oligonucleotides decreased COX-2 mRNA stability in monocytes, further supporting the inhibitory effects of miR-16. Interestingly, hnRNPK knockdown increased miR-16 binding to COX-2 3'-UTR, indicating a cross-talk between them. These new results demonstrate that diabetic stimuli can efficiently stabilize inflammatory genes via opposing actions of key RNA-binding proteins and miRs.