Posttranscriptional regulation of cyclin D1 by ARE-binding proteins AUF1 and HuR in cycling myoblasts

RNA binding proteins (RBPs) can regulate the stability and/or translatability of messengerRNAs (mRNAs) through interactions with their 3′-untranslated regions. However, individual mRNAs may be regulated simultaneously or successively by more than one RBP, as well as by Argonaute (AGO)-bound miRNAs; the coordination of these various influences on an individual mRNA is therefore complex and not well studied. In this report we examine the roles of two RBPs that bind to AU-rich elements (ARE) – AUF1 and HuR – in the stability and translation of cyclin D1 (Ccnd1) mRNA in rat myoblasts transiting the G phase of the cell cycle, and their interactions with miRNAs. Knockdown (KD) of AUF1 resulted in (1) transient upregulation of the mRNA level as well as an advancement of translation onset time (TOT) from 6 to 5 h post-serum addition, (2) loss of miRNA loading on AGO1 and AGO2 and (3) reduction in the level of AGO-1 and AGO-2 bound mRNA. In contrast, KD of HuR had no effect on the mRNA level, or on the AGO–mRNA complexes, but delayed TOT by 1 h independent of miRNA let-7. Thus the dynamics of RBP–mRNA binding and –RBP–AGO–miRNA interactions are coordinated to fine tune the expression of Ccnd1 in the G1 phase.     

Cooperative interplay of let-7 mimic and HuR with MYC RNA

Both RNA-binding proteins (RBP) and miRNA play important roles in the regulation of mRNA expression, often acting together to regulate a target mRNA. In some cases the RBP and miRNA have been reported to act competitively, but in other instances they function cooperatively. Here, we investigated HuR function as an enhancer of let-7-mediated translational repression of c-Myc despite the separation of their binding sites. Using an in vitro system, we determined that a let-7 mimic, consisting of single-stranded (ss)DNA complementary to the let-7 binding site, enhanced the affinity of HuR for a 122-nt MYC RNA encompassing both binding sites. This finding supports the biophysical principle of cooperative binding by an RBP and miRNA purely through interactions at distal mRNA binding sites.     

HuR protein attenuates miRNA-mediated repression by promoting miRISC dissociation from the target RNA

The microRNA (miRNA)-mediated repression of protein synthesis in mammalian cells is a reversible process. Target mRNAs with regulatory AU-rich elements (AREs) in their 3'-untranslated regions (3'-UTR) can be relieved of miRNA repression under cellular stress in a process involving the embryonic lethal and altered vision family ARE-binding protein HuR. The HuR-mediated derepression occurred even when AREs were positioned at a considerable distance from the miRNA sites raising questions about the mechanism of HuR action. Here, we show that the relief of miRNA-mediated repression involving HuR can be recapitulated in different in vitro systems in the absence of stress, indicating that HuR alone is sufficient to relieve the miRNA repression upon binding to RNA ARE. Using in vitro assays with purified miRISC and recombinant HuR and its mutants, we show that HuR, likely by its property to oligomerize along RNA, leads to the dissociation of miRISC from target RNA even when miRISC and HuR binding sites are positioned at a distance. Further, we demonstrate that HuR association with AREs can also inhibit miRNA-mediated deadenylation of mRNA in the Krebs-2 ascites extract, in a manner likewise depending on the potential of HuR to oligomerize.     

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.     

Protein kinase C alpha-dependent phosphorylation of the mRNA-stabilizing factor HuR: implications for posttranscriptional regulation of cyclooxygenase-2

In this study, we investigated the molecular mechanisms underlying the ATP analogue adenosine-5'-O-(3-thio)triphosphate-induced nucleocytoplasmic shuttling of the mRNA stabilizing factor HuR in human (h) mesangial cells (MC). Using synthetic protein kinase C (PKC) inhibitors and small interfering RNA approaches, we demonstrated that knockdown of PKC alpha efficiently blocked the ATP-dependent nuclear HuR export to the cytoplasm. The functional importance of PKC alpha in HuR shuttling is highlighted by the high cytosolic HuR content detected in hMC stably overexpressing PKC alpha compared with mock-transfected cells. The ATP-induced recruitment of HuR to the cytoplasm is preceded by a direct interaction of PKC alpha with nuclear HuR and accompanied by increased Ser phosphorylation as demonstrated by coimmunoprecipitation experiments. Mapping of putative PKC target sites identified serines 158 and 221 as being indispensable for HuR phosphorylation by PKC alpha. RNA pull-down assay and RNA electrophoretic mobility shift assay demonstrated that the HuR shuttling by ATP is accompanied by an increased HuR binding to cyclooxygenase (COX)-2 mRNA. Physiologically, the ATP-dependent increase in RNA binding is linked with an augmentation in COX-2 mRNA stability and subsequent increase in prostaglandin E(2) synthesis. Regulation of HuR via PKC alpha-dependent phosphorylation emphasizes the importance of posttranslational modification for stimulus-dependent HuR shuttling.     

Interplay between microRNAs and RNA-binding proteins determines developmental processes

MicroRNAs (miRNAs) are genes involved in normal development and cancer. They inhibit gene expression by associating with 3'-Untranslated regions (3' UTRs) of messenger RNAs (mRNAs), and are thought to regulate a large proportion of protein coding genes. However, it is becoming apparent that miRNA activity is not necessarily always determined by its expression in the cell. MiRNA activity can be affected by RNA-binding proteins (RBPs). For example, the RNA-binding protein HuR associates with the 3'UTR of the CAT1 mRNA after stress, counteracting the effect of miR-122. Second, we found that the expression of an RNA-binding protein called Dead end (Dnd1) prohibits the function of several miRNAs by blocking the accessibility of target mRNAs. Dnd1 function is essential for proper development of primordial germ cells (PGCs) in zebrafish and mammals, indicating a crucial role for RBP/miRNA interplay on 3'UTRs of mRNAs in developmental decisions. In this perspective we discuss the interplay between RBPs and miRNAs in the context of germ cells and review current observations implicating RBPs in miRNA function.     

Role of AUF1 and HuR in the pH-responsive stabilization of phosphoenolpyruvate carboxykinase mRNA in LLC-PK₁-F⁺ cells

Onset of metabolic acidosis leads to a rapid and pronounced increase in expression of phosphoenolpyruvate carboxykinase (PEPCK) in rat renal proximal convoluted tubules. This adaptive response is modeled by treating a clonal line of porcine LLC-PK(1)-F(+) cells with an acidic medium (pH 6.9, 9 mM HCO(3)(-)). Measurement of the half-lives of PEPCK mRNA in cells treated with normal (pH 7.4, 26 mM HCO(3)(-)) and acidic medium established that the observed increase is due in part to stabilization of the PEPCK mRNA. The pH-responsive stabilization was reproduced in a Tet-responsive chimeric reporter mRNA containing the 3'-UTR of PEPCK mRNA. This response was lost by mutation of a highly conserved AU sequence that binds AUF1 and is the primary element that mediates the rapid turnover of PEPCK mRNA. However, siRNA knockdown of AUF1 had little effect on the basal levels and the pH-responsive increases in PEPCK mRNA and protein. Electrophoretic mobility shift assays established that purified recombinant HuR, another AU element binding protein, also binds with high affinity and specificity to multiple sites within the final 92-nucleotides of the 3'-UTR of the PEPCK mRNA, including the highly conserved AU-rich element. siRNA knockdown of HuR caused pronounced decreases in basal expression and the pH-responsive increases in PEPCK mRNA and protein. Therefore, basal expression and the pH-responsive stabilization of PEPCK mRNA in LLC-PK(1)-F(+) cells, and possibly in the renal proximal tubule, may require the remodeling of HuR and AUF1 binding to the elements that mediate the rapid turnover of PEPCK mRNA.     

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.