RNA-binding protein HuR autoregulates its expression by promoting alternative polyadenylation site usage

RNA-binding protein HuR modulates the stability and translational efficiency of messenger RNAs (mRNAs) encoding essential components of the cellular proliferation, growth and survival pathways. Consistent with these functions, HuR levels are often elevated in cancer cells and reduced in senescent and quiescent cells. However, the molecular mechanisms that control HuR expression are poorly understood. Here we show that HuR protein autoregulates its abundance through a negative feedback loop that involves interaction of the nuclear HuR protein with a GU-rich element (GRE) overlapping with the HuR major polyadenylation signal (PAS2). An increase in the cellular HuR protein levels stimulates the expression of long HuR mRNA species containing an AU-rich element (ARE) that destabilizes the mRNAs and thus reduces the protein production output. The PAS2 read-through occurs due to a reduced recruitment of the CstF-64 subunit of the pre-mRNA cleavage stimulation factor in the presence of the GRE-bound HuR. We propose that this mechanism maintains HuR homeostasis in proliferating cells. Since only the nuclear HuR is expected to contribute to the auto-regulation, our model may explain the longstanding observation that the increase in the total HuR expression in cancer cells often correlates with the accumulation of its substantial fraction in the cytoplasm.     

Activity‐dependent expression of ELAV/Hu RBPs and neuronal mRNAs in seizure and cocaine brain

Growing evidence indicates that both seizure (glutamate) and cocaine (dopamine) treatment modulate synaptic plasticity within the mesolimbic region of the CNS. Activation of glutamatergic neurons depends on the localized translation of neuronal mRNA products involved in modulating synaptic plasticity. In this study, we demonstrate the dendritic localization of HuR and HuD RNA‐binding proteins (RBPs) and their association with neuronal mRNAs following these two paradigms of seizure and cocaine treatment. Both the ubiquitously expressed HuR and neuronal HuD RBPs were detected in different regions as well as within dendrites of the brain and in dissociated neurons. Quantitative analysis revealed an increase in HuR, HuD and p‐glycogen synthase kinase 3β (GSK3β) protein levels as well as neuronal mRNAs encoding Homer, CaMKIIα, vascular early response gene, GAP‐43, neuritin, and neuroligin protein products following either seizure or cocaine treatment. Inhibition of the Akt/GSK3β signaling pathway by acute or chronic LiCl treatment revealed changes in HuR, HuD, pGSK3β, p‐Akt, and β‐catenin protein levels. In addition, a genetically engineered hyperdopaminergic mouse model (dopamine transporter knockout) revealed decreased expression of HuR protein levels, but no significant change was observed in HuD or fragile‐X mental retardation protein RBPs. Finally, our data suggest that HuR and HuD RBPs potentially interact directly with neuronal mRNAs important for differentiation and synaptic plasticity.     

Scaffolding protein Grb2-associated binder 1 sustains epidermal growth factor-induced mitogenic and survival signaling by multiple positive feedback loops

Grb2-associated binder 1 (GAB1) is a scaffold protein involved in numerous interactions that propagate signaling by growth factor and cytokine receptors. Here we explore in silico and validate in vivo the role of GAB1 in the control of mitogenic (Ras/MAPK) and survival (phosphatidylinositol 3-kinase (PI3K)/Akt) signaling stimulated by epidermal growth factor (EGF). We built a comprehensive mechanistic model that allows for reliable predictions of temporal patterns of cellular responses to EGF under diverse perturbations, including different EGF doses, GAB1 suppression, expression of mutant proteins, and pharmacological inhibitors. We show that the temporal dynamics of GAB1 tyrosine phosphorylation is significantly controlled by positive GAB1-PI3K feedback and negative MAPK-GAB1 feedback. Our experimental and computational results demonstrate that the essential function of GAB1 is to enhance PI3K/Akt activation and extend the duration of Ras/MAPK signaling. By amplifying positive interactions between survival and mitogenic pathways, GAB1 plays the critical role in cell proliferation and tumorigenesis.     

Grb10 and active Raf-1 kinase promote Bad-dependent cell survival

The proapoptotic protein Bad is a key player in cell survival decisions, and is regulated post-translationally by several signaling networks. We expressed Bad in mouse embryonic fibroblasts to sensitize them to apoptosis, and tested cell lines derived from knock-out mice to establish the significance of the interaction between the adaptor protein Grb10 and the Raf-1 protein kinase in anti-apoptotic signaling pathways targeting Bad. When compared with wild-type cells, both Grb10 and Raf-1-deficient cells exhibit greatly enhanced sensitivity to apoptosis in response to Bad expression. Structure-function analysis demonstrates that, in this cellular model, the SH2, proline-rich, and pleckstrin homology domains of Grb10, as well as its Akt phosphorylation site and consequent binding by 14-3-3, are all necessary for its anti-apoptotic functions. As for Raf-1, its kinase activity, its ability to be phosphorylated by Src on Tyr-340/341 and the binding of its Ras-associated domain to the Grb10 SH2 domain are all necessary to promote cell survival. Silencing the expression of either Grb10 or Raf-1 by small interfering RNAs as well as mutagenesis of specific serine residues on Bad, coupled with signaling inhibitor studies, all indicate that Raf-1 and Grb10 are required for the ability of both the phosphatidylinositol 3-kinase/Akt and MAP kinase pathways to modulate the phosphorylation and inactivation of Bad. Because total Raf-1, ERK, and Akt kinase activities are not impaired in the absence of Grb10, we propose that this adapter protein creates a subpopulation of Raf-1 with specific anti-apoptotic activity.     

Phosphorylation of HuR by Chk2 regulates SIRT1 expression

The RNA binding protein HuR regulates the stability of many target mRNAs. Here, we report that HuR associated with the 3' untranslated region of the mRNA encoding the longevity and stress-response protein SIRT1, stabilized the SIRT1 mRNA, and increased SIRT1 expression levels. Unexpectedly, oxidative stress triggered the dissociation of the [HuR-SIRT1 mRNA] complex, in turn promoting SIRT1 mRNA decay, reducing SIRT1 abundance, and lowering cell survival. The cell cycle checkpoint kinase Chk2 was activated by H(2)O(2), interacted with HuR, and was predicted to phosphorylate HuR at residues S88, S100, and T118. Mutation of these residues revealed a complex pattern of HuR binding, with S100 appearing to be important for [HuR-SIRT1 mRNA] dissociation after H(2)O(2). Our findings demonstrate that HuR regulates SIRT1 expression, underscore functional links between the two stress-response proteins, and implicate Chk2 in these processes.     

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.     

Posttranscriptional Orchestration of an Anti-Apoptotic Program by HuR

The RNA-binding protein HuR can stabilize and/or regulate the translation of target mRNAs, thereby affecting the cellular responses to immune, proliferative, and damaging agents. Here, we discuss emerging evidence that HuR elicits a broad anti-apoptotic function through its influence on the expression of multiple target mRNAs. HuR was previously shown to bind to the mRNA encoding the apoptosome inhibitor prothymosin α (ProTα) and enhanced its translation and cytoplasmic abundance. More recently, HuR was shown to increase the stability of a target mRNA encoding the pro-survival deacetylase SIRT1. The discovery that HuR likewise binds to and promotes the expression of mRNAs encoding Bcl-2 and Mcl-1, two major anti-apoptotic effectors, strongly supports HuR’s role as a key upstream coordinator of a constitutive pro-survival program.