Dephosphorylation of HuR Protein during Alphavirus Infection Is Associated with HuR Relocalization to the Cytoplasm

We have demonstrated previously that the cellular HuR protein binds U-rich elements in the 3′ untranslated region (UTR) of Sindbis virus RNA and relocalizes from the nucleus to the cytoplasm upon Sindbis virus infection in 293T cells. In this study, we show that two alphaviruses, Ross River virus and Chikungunya virus, lack the conserved high-affinity U-rich HuR binding element in their 3′ UTRs but still maintain the ability to interact with HuR with nanomolar affinities through alternative binding elements. The relocalization of HuR protein occurs during Sindbis infection of multiple mammalian cell types as well as during infections with three other alphaviruses. Interestingly, the relocalization of HuR is not a general cellular reaction to viral infection, as HuR protein remained largely nuclear during infections with dengue and measles virus. Relocalization of HuR in a Sindbis infection required viral gene expression, was independent of the presence of a high-affinity U-rich HuR binding site in the 3′ UTR of the virus, and was associated with an alteration in the phosphorylation state of HuR. Sindbis virus-induced HuR relocalization was mechanistically distinct from the movement of HuR observed during a cellular stress response, as there was no accumulation of caspase-mediated HuR cleavage products. Collectively, these data indicate that virus-induced HuR relocalization to the cytoplasm is specific to alphavirus infections and is associated with distinct posttranslational modifications of this RNA-binding protein.     

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.     

A 40-bp RNA element that mediates stabilization of vascular endothelial growth factor mRNA by HuR

VEGF is a critical mediator of hypoxia-induced angiogenesis in numerous physiological and pathophysiological conditions. The hypoxic induction of VEGF is due in large part to an increase in the stability of its mRNA. We recently demonstrated that the stabilization of VEGF mRNA by hypoxia is dependent upon the RNA-binding protein HuR. This report describes the identification of a 40-bp functional HuR binding site in the VEGF mRNA 3'-untranslated region. This element can confer HuR-mediated stabilization of a heterologous gene in vitro and in vivo. Furthermore, the element is sufficient to confer an increase in the hypoxic induction of a heterologous gene. Deletion of the HuR binding site within this 40-bp element as mapped by RNase T1 and lead footprinting uncouples a stabilizing sequence from a destabilizing sequence, thus providing a novel RNA-protein regulatory model that might be exploited to manipulate VEGF expression and hypoxia-induced angiogenesis.     

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.     

HuR Is Necessary for Mammary Epithelial Cell Proliferation and Polarity at Least in Part via ΔNp63

HuR, a RNA binding protein, is known to function as a tumor maintenance gene in breast cancer and associated with tumor growth and poor prognosis. However, the cellular function of this protein remains largely unknown in normal mammary epithelial cells. Here, we showed that in immortalized MCF10A mammary epithelial cells, HuR knockdown inhibits cell proliferation and enhances premature senescence. We also showed that in three-dimensional culture, MCF10A cells with HuR knockdown form abnormal acini with filled lumen and an aberrant expression pattern of the extracellular matrix protein laminin V. In addition, we showed that HuR knockdown increases ΔNp63, but decreases wild-type p53, expression in MCF10A cells. Moreover, we showed that ΔNp63 knockdown partially rescues the proliferative defect induced by HuR knockdown in MCF10A cells. Consistent with this, we identified two U-rich elements in the 3′-untranslated region of p63 mRNA, to which HuR specifically binds. Finally, we showed that HuR knockdown enhances ΔNp63 mRNA translation but has no effect on p63 mRNA turnover. Together, our data suggest that HuR maintains cell proliferation and polarity of mammary epithelial cells at least in part via ΔNp63.     

Tissue distribution of AU-rich mRNA-binding proteins involved in regulation of mRNA decay

Short lived cytokine and proto-oncogene mRNAs are destabilized by an A+U-rich element (ARE) in the 3'-untranslated region. Several regulatory proteins bind to AREs in cytokine and proto-oncogene mRNAs, participate in inhibiting or promoting their rapid degradation of ARE mRNAs, and influence cytokine expression and cellular transformation in experimental models. The tissue distribution and cellular localization of the different AU-rich binding proteins (AUBPs), however, have not been uniformly characterized in the mouse, a model for ARE mRNA decay. We therefore carried out immunoblot and immunohistochemical analyses of the different AUBPs using the same mouse tissues. We show that HuR protein, a major AUBP that stabilizes the ARE mRNAs, is most strongly expressed in the thymus, spleen (predominantly in lymphocytic cells), intestine, and testes. AUF1 protein, a negative regulator of ARE mRNA stability, displayed strong expression in thymus and spleen cells within lymphocytic cells, moderate expression in the epithelial linings of lungs, gonadal tissues, and nuclei of most neurons in the brain, and little expression in the other tissues. Tristetraprolin, a negative regulator of ARE mRNA stability, displayed a largely non-overlapping tissue distribution with AUF1 and was predominantly expressed in the liver and testis. KH-type splicing regulatory protein, a presumptive negative regulator of ARE mRNA stability, was distributed widely in murine organs. These results indicate that HuR and AUF1, which functionally oppose each other, have generally similar distributions, suggesting that the balance between HuR and AUF1 is likely important in control of short lived mRNA degradation, lymphocyte development, and/or cytokine production, and possibly in certain aspects of neurological function.     

Purification and characterization of beta-adrenergic receptor mRNA-binding proteins

Beta-adrenergic receptors (beta-ARs), like other G-protein-coupled receptors, can undergo post-transciptional regulation at the level of mRNA stability. In particular, the human beta(1)- and beta(2)-ARs and the hamster beta(2)-AR mRNA undergo beta-agonist-mediated destabilization. By UV cross-linking, we have previously described an approximately M(r) 36,000 mRNA-binding protein, betaARB, that binds to A/C+U-rich nucleotide regions within 3'-untranslated regions. Further, we have demonstrated previously that betaARB is immunologically distinct from AUF1/heterogeneous nuclear ribonucleoprotein (hnRNP) D, another mRNA-binding protein associated with destabilization of A+U-rich mRNAs (Pende, A., Tremmel, K. D., DeMaria, C. T., Blaxall, B. C., Minobe, W., Sherman, J. A., Bisognano, J., Bristow, M. R., Brewer, G., and Port, J. D. (1996) J. Biol. Chem. 271, 8493-8501). In this report, we describe the peptide composition of betaARB. Mass spectrometric analysis of an approximately M(r) 36,000 band isolated from ribosomal salt wash proteins revealed the presence of two mRNA-binding proteins, hnRNP A1, and the elav-like protein, HuR, both of which are known to bind to A+U-rich nucleotide regions. By immunoprecipitation, HuR appears to be the biologically dominant RNA binding component of betaARB. Although hnRNP A1 and HuR can both be immunoprecipitated from ribosomal salt wash proteins, the composition of betaARB (HuR alone versus HuR and hnRNP A1) appears to be dependent on the mRNA probe used. The exact role of HuR and hnRNP A1 in the regulation of beta-AR mRNA stability remains to be determined.     

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.