The ELAV family of RNA-binding proteins in synaptic plasticity and long-term memory.

The mechanisms of de novo gene expression and translation of specific gene transcripts have long been known to support long-lasting changes in synaptic plasticity and behavioral long-term memory. In recent years, it has become increasingly apparent that gene expression is heavily regulated not only on the level of transcription, but also through post-transcriptional gene regulation, which governs the subcellular localization, stability, and likelihood of translation of mRNAs. Specific families of RNA-binding proteins (RBPs) bind transcripts which contain AU-rich elements (AREs) within their 3' UTR and thereby govern their downstream fate. These post-transcriptional gene regulatory mechanisms are coordinated through the same cell signaling pathways that play critical roles in long-term memory formation. In this review, we discuss recent results that demonstrate the roles that these ARE-binding proteins play in LTM formation.

Protein Kinase C Activation as a Potential Therapeutic Strategy in Alzheimer's Disease: Is there a Role for Embryonic Lethal Abnormal Vision-like Proteins?

Alzheimer's disease (AD), the most common cause of dementia, is an irreversible and progressive neurodegenerative disorder. It affects predominantly brain areas that are critical for memory and learning and is characterized by two main pathological hallmarks: extracellular amyloid plaques and intracellular neurofibrillary tangles. Protein kinase C (PKC) has been classified as one of the cognitive kinases controlling memory and learning. By regulating several signalling pathways involved in amyloid and tau pathologies, it also plays an inhibitory role in AD pathophysiology. Among downstream targets of PKC are the embryonic lethal abnormal vision (ELAV)-like RNA-binding proteins that modulate the stability and the translation of specific target mRNAs involved in synaptic remodelling linked to cognitive processes. This MiniReview summarizes the current evidence on the role of PKC and ELAV-like proteins in learning and memory, highlighting how their derangement can contribute to AD pathophysiology. This last aspect emphasizes the potential of pharmacological activation of PKC as a promising therapeutic strategy for the treatment of AD.

Lymphotoxin α, a novel target of posttranscriptional gene regulation by HuR in HepG2 cells.

The role of the RNA-binding protein human antigen R (HuR) in hepatocarcinogenesis is still elusive. By employing short hairpin (sh)RNA-dependent knockdown approach, we demonstrate that lymphotoxin α (LTα) is a target of posttranscriptional gene regulation by HuR in hepatocellular carcinoma (HepG2) cells. Consequently, the increased mRNA decay upon HuR depletion significantly affects lymphotoxin expression at both, the mRNA and protein level. Biotin-pulldown assay showed that HuR specifically interacts with the 3'-untranslated region (3'-UTR) of the LTα mRNA. Furthermore, electrophoretic mobility shift assay (EMSA) implicates that the RNA-binding critically depends on the RNA recognition motif 2 (RRM2) and the hinge region of HuR.

The long intergenic noncoding RNA UFC1, a target of MicroRNA 34a, interacts with the mRNA stabilizing protein HuR to increase levels of ß-catenin in HCC cells.

BACKGROUND & AIMS: Altered activities of long noncoding RNAs (lncRNAs) have been associated with cancer development. We investigated the mechanisms by which the long intergenic noncoding RNA UFC1 (lincRNA-UFC1) promotes progression of hepatocellular carcinoma (HCC), using human tissues and cell lines. METHODS: We used microarrays to compare expression profiles of lncRNAs in HCC samples and adjacent nontumor tissues (controls) from 7 patients. HCC and nontumor tissues were collected from 2006 through 2012 from patients in Guangzhou, China. We used quantitative real-time polymerase chain reaction to measure levels of lincRNA-UFC1 in tissues from 49 patients, and in situ hybridization to measure levels in samples from 131 patients; clinical data were collected from patients for up to 5 years. The lincRNA-UFC1 was expressed transgenically, or knocked down with short hairpin RNAs, in BEL-7402, SK-Hep1, Huh7, and MHCC-97H HCC cell lines; luciferase reporter and RNA immunoprecipitation and pull-down assays were performed. We also analyzed growth of xenograft tumors from these cells in BALB/c nude mice. RESULTS: Levels of the lincRNA-UFC1 were increased in HCC tissues compared with controls, and associated with tumor size, Barcelona Clinic Liver Cancer stage, and patient outcomes. Transgenic expression of the lincRNA-UFC1 in HCC cells promoted their proliferation and cell-cycle progression and inhibited apoptosis, whereas short hairpin RNA knockdown of lincRNA-UFC1 had opposite effects. Xenograft tumors grown from cells overexpressing lincRNA-UFC1 had larger mean volumes and weights, and formed more rapidly, than tumors grown from control cells. Tumors grown from lincRNA-UFC1 knockdown were smaller than controls. The lincRNA-UFC1 interacted directly with the messenger RNA (mRNA) stabilizing protein HuR (encoded by ELAVL1) to increase levels of ß-catenin mRNA (encoded by CTNNB1) and protein. Levels of lincRNA-UFC1 correlated with those of ß-catenin in HCC tissues. In contrast, there was a negative correlation between levels of microRNA 34a and lincRNA-UFC1 in HCC tissues; microRNA 34a reduced the stability of lincRNA-UFC1. CONCLUSIONS: The lincRNA-UFC1, a target of microRNA 34a, promotes proliferation and reduces apoptosis in HCC cells to promote growth of xenograft tumors in mice. It interacts directly with the mRNA stabilizing protein HuR to regulate levels of ß-catenin in HCC cells.

ELAVL1 regulates alternative splicing of eIF4E transporter to promote postnatal angiogenesis.

Posttranscriptional RNA regulation is important in determining the plasticity of cellular phenotypes. However, mechanisms of how RNA binding proteins (RBPs) influence cellular behavior are poorly understood. We show here that the RBP embryonic lethal abnormal vision like 1 (ELAVL1, also know as HuR) regulates the alternative splicing of eukaryotic translation initiation factor 4E nuclear import factor 1 (Eif4enif1), which encodes an eukaryotic translation initiation factor 4E transporter (4E-T) protein and suppresses the expression of capped mRNAs. In the absence of ELAVL1, skipping of exon 11 of Eif4enif1 forms the stable, short isoform, 4E-Ts. This alternative splicing event results in the formation of RNA processing bodies (PBs), enhanced turnover of angiogenic mRNAs, and suppressed sprouting behavior of vascular endothelial cells. Further, endothelial-specific Elavl1 knockout mice exhibited reduced revascularization after hind limb ischemia and tumor angiogenesis in oncogene-induced mammary cancer, resulting in attenuated blood flow and tumor growth, respectively. ELAVL1-regulated alternative splicing of Eif4enif1 leading to enhanced formation of PB and mRNA turnover constitutes a novel posttranscriptional mechanism critical for pathological angiogenesis.

Intestinal epithelial HuR modulates distinct pathways of proliferation and apoptosis and attenuates small intestinal and colonic tumor development.

HuR is a ubiquitous nucleocytoplasmic RNA-binding protein that exerts pleiotropic effects on cell growth and tumorigenesis. In this study, we explored the impact of conditional, tissue-specific genetic deletion of HuR on intestinal growth and tumorigenesis in mice. Mice lacking intestinal expression of HuR (Hur (IKO) mice) displayed reduced levels of cell proliferation in the small intestine and increased sensitivity to doxorubicin-induced acute intestinal injury, as evidenced by decreased villus height and a compensatory shift in proliferating cells. In the context of Apc(min/+) mice, a transgenic model of intestinal tumorigenesis, intestinal deletion of the HuR gene caused a three-fold decrease in tumor burden characterized by reduced proliferation, increased apoptosis, and decreased expression of transcripts encoding antiapoptotic HuR target RNAs. Similarly, Hur(IKO) mice subjected to an inflammatory colon carcinogenesis protocol [azoxymethane and dextran sodium sulfate (AOM-DSS) administration] exhibited a two-fold decrease in tumor burden. Hur(IKO) mice showed no change in ileal Asbt expression, fecal bile acid excretion, or enterohepatic pool size that might explain the phenotype. Moreover, none of the HuR targets identified in Apc(min/+)Hur(IKO) were altered in AOM-DSS-treated Hur(IKO) mice, the latter of which exhibited increased apoptosis of colonic epithelial cells, where elevation of a unique set of HuR-targeted proapoptotic factors was documented. Taken together, our results promote the concept of epithelial HuR as a contextual modifier of proapoptotic gene expression in intestinal cancers, acting independently of bile acid metabolism to promote cancer. In the small intestine, epithelial HuR promotes expression of prosurvival transcripts that support Wnt-dependent tumorigenesis, whereas in the large intestine epithelial HuR indirectly downregulates certain proapoptotic RNAs to attenuate colitis-associated cancer. Cancer Res; 74(18); 5322-35. ©2014 AACR.

HuR and post-transcriptional regulation in vascular aging.

HuR (ELAV11 (embryonic lethal, abnormal vision)-like 1), a ubiquitously expressed member of the ELAV-like RNA-binding protein family, has been shown to regulate the stability and translation of mRNAs that encode factors regulating cellular senescence, thereby impacting on aging. In this review, we discuss the current knowledge of HuR's role in vascular cell senescence and vascular aging.

Krüppel -like factor 8 is a stress-responsive transcription factor that regulates expression of HuR.

BACKGROUND/AIMS: HuR is an RNA-binding protein that regulates the post-transcriptional life of thousands of cellular mRNAs and promotes cell survival. HuR is expressed as two mRNA transcripts that are differentially regulated by cell stress. The goal of this study is to define factors that promote transcription of the longer alternate form. METHODS: Effects of transcription factors on HuR expression were determined by inhibition or overexpression of these factors followed by competitive RT-PCR, gel mobility shift, and chromatin immunoprecipitation. Transcription factor expression patterns were identified through competitive RT-PCR and Western analysis. Stress responses were assayed in thapsigargin-treated proximal tubule cells and in ischemic rat kidney. RESULTS: A previously described NF-κB site and a newly identified Sp/KLF factor binding site were shown to be important for transcription of the long HuR mRNA. KLF8, but not Sp1, was shown to bind this site and increase HuR mRNA levels. Cellular stress in cultured or native proximal tubule cells resulted in a rapid decrease of KLF8 levels that paralleled those of the long HuR mRNA variant. CONCLUSIONS: These results demonstrate that KLF8 can participate in regulating expression of alternate forms of HuR mRNA along with NF-κB and other factors, depending on cellular contexts.

The RNA-binding protein ELAV regulates Hox RNA processing, expression and function within the Drosophila nervous system.

The regulated head-to-tail expression of Hox genes provides a coordinate system for the activation of specific programmes of cell differentiation according to axial level. Recent work indicates that Hox expression can be regulated via RNA processing but the underlying mechanisms and biological significance of this form of regulation remain poorly understood. Here we explore these issues within the developing Drosophila central nervous system (CNS). We show that the pan-neural RNA-binding protein (RBP) ELAV (Hu antigen) regulates the RNA processing patterns of the Hox gene Ultrabithorax (Ubx) within the embryonic CNS. Using a combination of biochemical, genetic and imaging approaches we demonstrate that ELAV binds to discrete elements within Ubx RNAs and that its genetic removal reduces Ubx protein expression in the CNS leading to the respecification of cellular subroutines under Ubx control, thus defining for the first time a specific cellular role of ELAV within the developing CNS. Artificial provision of ELAV in glial cells (a cell type that lacks ELAV) promotes Ubx expression, suggesting that ELAV-dependent regulation might contribute to cell type-specific Hox expression patterns within the CNS. Finally, we note that expression of abdominal A and Abdominal B is reduced in elav mutant embryos, whereas other Hox genes (Antennapedia) are not affected. Based on these results and the evolutionary conservation of ELAV and Hox genes we propose that the modulation of Hox RNA processing by ELAV serves to adapt the morphogenesis of the CNS to axial level by regulating Hox expression and consequently activating local programmes of neural differentiation.

Post-transcriptional gene regulation by HuR and microRNAs in angiogenesis.

PURPOSE OF REVIEW: This review summarizes recent findings in the area of post-transcriptional regulation of gene expression during angiogenesis, also known as new blood vessel formation. Specifically, we focus on gene regulation by HuR, an RNA-binding protein (RBP), and microRNAs (miRNAs) and their interplay, which ultimately influences cellular phenotypes of cells involved in angiogenesis. RECENT FINDINGS: Recently, RBPs and miRNAs have emerged as key regulators of angiogenesis. We and others have demonstrated that the RBP HuR (a.k.a. Elavl1) stabilizes vascular endothelial growth factor-A mRNA, a potent angiogenic factor in the settings of tumor development and inflammation. However, several miRNAs were shown to modulate gene expression during developmental (miR-126), physiological (miR-126, miR-92a), and pathological angiogenesis (miR-200b, miR-132). Moreover, the interplay of HuR and miRNAs in the regulation of genes involved in angiogenesis was described. In addition, recent work suggests a new role of circulating miRNAs as paracrine mediators in angiogenesis. SUMMARY: The elucidation of novel posttranscriptional gene regulatory mechanisms has expanded our understanding of angiogenesis in physiological and pathological conditions. We anticipate that this knowledge will ultimately lead to new insights for discovering novel therapeutic strategies to control pathological angiogenesis.

Sirtuin 1 stabilization by HuR represses TNF-α- and glucose-induced E-selectin release and endothelial cell adhesiveness in vitro: relevance to human metabolic syndrome.

Chronic inflammation and hyperglycaemia, typical features of metabolic diseases, trigger endothelial damage and release of E-selectin, a marker of endothelial activation. In the present study, we investigated molecular pathways involved in the regulation of endothelial cell activation induced by tumour necrosis factor-α (TNF-α) and high glucose. In cultured human umbilical vein endothelial cells (HUVECs), we studied the role of HuR, an ELAV (embryonic lethal, abnormal vision, Drosophila) family RNA-binding protein, and Sirtuin 1 (SIRT1) on E-selectin release and cell adhesion at different glucose concentrations. HuR expression and binding to SIRT1 were also analysed ex vivo in peripheral blood mononuclear cells (PBMCs) of subjects with and without the metabolic syndrome (MS), by immunoprecipitation (IP) of the ribonucleoprotein (RNP) complex. We found that SIRT1 overexpression prevented TNF-α- and high-glucose-dependent nuclear factor-κB (NF-κB)-p65 acetylation, E-selectin promoter activity, E-selectin release and adhesion of THP-1 cells to HUVECs. The same was mimicked by HuR overexpression, which binds and stabilizes SIRT1 mRNA. Importantly, in PBMCs of individuals with MS compared with those without, SIRT1 expression was lower, and the ability of HuR to bind SIRT1 mRNA was significantly reduced, while plasma E-selectin was increased. We conclude that post-transcriptional stabilization of SIRT1 by HuR represses inflammation- and hyperglycaemia-induced E-selectin release and endothelial cell activation. Therefore, increasing SIRT1 expression represents a strategy to counter the accelerated vascular disease in metabolic disorders.

The role of HuR in the post-transcriptional regulation of interleukin-3 in T cells.

Human Interleukin-3 (IL-3) is a lymphokine member of a class of transiently expressed mRNAs harboring Adenosine/Uridine-Rich Elements (ARE) in their 3' untranslated regions (3'-UTRs). The regulatory effects of AREs are often mediated by specific ARE-binding proteins (ARE-BPs). In this report, we show that the human IL-3 3'-UTR plays a post-transcriptional regulation role in two human transformed cell lines. More specifically, we demonstrate that the hIL-3 3'-UTR represses the translation of a luciferase reporter both in HeLa and Jurkat T-cells. These results also revealed that the hIL-3 3'-UTR-mediated translational repression is exerted by an 83 nt region comprised mainly by AREs and some non-ARE sequences. Moreover, electrophoretic mobility shift assays (EMSAs) and UV-crosslinking analysis show that this hIL-3 ARE-rich region recruits five specific protein complexes, including the ARE-BPs HuR and TIA-1. HuR binding to this ARE-rich region appears to be spatially modulated during T-cell activation. Together, these results suggest that HuR recognizes the ARE-rich region and plays a role in the IL-3 3'-UTR-mediated post-transcriptional control in T-cells.

HuR posttranscriptionally regulates WEE1: implications for the DNA damage response in pancreatic cancer cells.

HuR (ELAV1), an RNA-binding protein abundant in cancer cells, primarily resides in the nucleus, but under specific stress (e.g., gemcitabine), HuR translocates to the cytoplasm in which it tightly modulates the expression of mRNA survival cargo. Here, we demonstrate for the first time that stressing pancreatic ductal adenocarcinoma (PDA) cells by treatment with DNA-damaging anticancer agents (mitomycin C, oxaliplatin, cisplatin, carboplatin, and a PARP inhibitor) results in HuR's translocation from the nucleus to the cytoplasm. Importantly, silencing HuR in PDA cells sensitized the cells to these agents, whereas overexpressing HuR caused resistance. HuR's role in the efficacy of DNA-damaging agents in PDA cells was, in part, attributed to the acute upregulation of WEE1 by HuR. WEE1, a mitotic inhibitor kinase, regulates the DNA damage repair pathway, and therapeutic inhibition of WEE1 in combination with chemotherapy is currently in early phase trials for the treatment of cancer. We validate WEE1 as a HuR target in vitro and in vivo by demonstrating (i) direct binding of HuR to WEE1's mRNA (a discrete 56-bp region residing in the 3' untranslated region) and (ii) HuR siRNA silencing and overexpression directly affects the protein levels of WEE1, especially after DNA damage. HuR's positive regulation of WEE1 increases γ-H2AX levels, induces Cdk1 phosphorylation, and promotes cell-cycle arrest at the G2-M transition. We describe a novel mechanism that PDA cells use to protect against DNA damage in which HuR posttranscriptionally regulates the expression and downstream function of WEE1 upon exposure to DNA-damaging agents.

HuR and other turnover- and translation-regulatory RNA-binding proteins: implications for the kidney.

The posttranscriptional regulation of gene expression occurs through cis RNA regulatory elements by the action of trans factors, which are represented by noncoding RNAs (especially microRNAs) and turnover- and translation-regulatory (TTR) RNA-binding proteins (RBPs). These multifactorial proteins are a group of heterogeneous RBPs primarily implicated in controlling the decay and translation rates of target mRNAs. TTR-RBPs usually shuttle between cellular compartments (the nucleus and cytoplasm) in response to various stimuli and undergo posttranslational modifications such as phosphorylation or methylation to ensure their proper subcellular localization and function. TTR-RBPs are emerging as key regulators of a wide variety of genes influencing kidney physiology and pathology. This review summarizes the current knowledge of TTR-RBPs that influence renal metabolism. We will discuss the role of TTR-RBPs as regulators of kidney ischemia, fibrosis and matrix remodeling, angiogenesis, membrane transport, immunity, vascular tone, hypertension, and acid-base balance as well as anemia, bone mineral disease, and vascular calcification.

Elavl1a regulates zebrafish erythropoiesis via posttranscriptional control of gata1.

The RNA-binding protein Elavl1 (also known as HuR) regulates gene expression at the posttranscriptional level. Early embryonic lethality of the mouse knockout challenges investigation into hematopoietic functions for Elavl1. We identified 2 zebrafish elavl1 genes, designated elavl1a (the predominant isoform during embryogenesis) and elavl1b. Knockdown of Elavl1a using specific morpholinos resulted in a striking loss of primitive embryonic erythropoiesis. Transcript levels for early hematopoietic regulatory genes including lmo2 and scl are unaltered, but levels of gata1 transcripts, encoding a key erythroid transcription factor, are significantly reduced in elavl1a morphants. Other mesoderm markers are mostly unchanged by depletion of Elav1a. The 3'-untranslated region (UTR) of gata1 contains putative Elavl1a-binding sites that support robust expression levels when fused to a transfected luciferase reporter gene, and Elavl1a binds the gata1 3'-UTR sequences in a manner dependent on these sites. Moreover, expression of a transgenic reporter specifically in developing embryonic erythroid cells is enhanced by addition of the gata1 3'UTR with intact Elavl1-binding sites. Injection of gata1 messenger RNA partially rescues the erythropoiesis defect caused by Elavl1 knockdown. Our study reveals a posttranscriptional regulatory mechanism by which RNA-binding protein Elavl1a regulates embryonic erythropoiesis by maintaining appropriate levels of gata1 expression.

A feedforward regulatory loop between HuR and the long noncoding RNA linc-MD1 controls early phases of myogenesis.

The muscle-specific long noncoding RNA linc-MD1 was shown to be expressed during early phases of muscle differentiation and to trigger the switch to later stages by acting as a sponge for miR-133 and miR-135. Notably, linc-MD1 is also the host transcript of miR-133b, and their biogenesis is mutually exclusive. Here, we describe that this alternative synthesis is controlled by the HuR protein, which favors linc-MD1 accumulation through its ability to bind linc-MD1 and repress Drosha cleavage. We show that HuR is under the repressive control of miR-133 and that the sponging activity of linc-MD1 consolidates HuR expression in a feedforward positive loop. Finally, we show that HuR also acts in the cytoplasm, reinforcing linc-MD1 sponge activity by cooperating for miRNA recruitment. An increase in miR-133 synthesis, mainly from the two unrelated miR-133a coding genomic loci, is likely to trigger the exit from this circuitry and progression to later differentiation stages.

HuR maintains a replicative life span by repressing the ARF tumor suppressor.

p19(ARF) plays an essential role in the senescence of mouse cells, and its expression is lost by methylation or deletion of the ARF locus; otherwise, p53 is inactivated to bypass senescence. ARF expression is tightly regulated, but little is known about its posttranscriptional regulation. Here, we show that an RNA-binding protein, HuR (human antigen R), represses ARF mRNA translation, thereby maintaining the replicative life span of mouse embryonic fibroblasts (MEFs). Loss of HuR results in premature senescence, with concomitant increases in p19(ARF) but not p16(Ink4a) levels, and this senescence is not observed in ARF-null MEFs that retain an intact Ink4a locus. HuR depletion does not alter ARF transcription or stability but enhances ribosome association with ARF mRNA. Under these conditions, ARF mRNA accumulates in nucleoli, where it associates with nucleolin. Furthermore, adipose-specific deletion of the HuR gene results in increased p19(ARF) expression in aged animals, which is accompanied by decreased insulin sensitivity. Together, our findings demonstrate that p19(ARF) is also regulated at the translational level, and this translational regulation restrains the cellular life span and tissue functions in vivo.