Overlapping Activities of ELAV/Hu Family RNA Binding Proteins Specify the Extended Neuronal 3' UTR Landscape in Drosophila.

The tissue-specific deployment of highly extended neural 3' UTR isoforms, generated by alternative polyadenylation (APA), is a broad and conserved feature of metazoan genomes. However, the factors and mechanisms that control neural APA isoforms are not well understood. Here, we show that three ELAV/Hu RNA binding proteins (Elav, Rbp9, and Fne) have similar capacities to induce a lengthened 3' UTR landscape in an ectopic setting. These factors promote accumulation of chromatin-associated, 3' UTR-extended, nascent transcripts, through inhibition of proximal polyadenylation site (PAS) usage. Notably, Elav represses an unannotated splice isoform of fne, switching the normally cytoplasmic Fne toward the nucleus in elav mutants. We use genomic profiling to reveal strong and broad loss of neural APA in elav/fne double mutant CNS, the first genetic background to largely abrogate this distinct APA signature. Overall, we demonstrate how regulatory interplay and functionally overlapping activities of neural ELAV/Hu RBPs drives the neural APA landscape.

ELAV and FNE Determine Neuronal Transcript Signatures through EXon-Activated Rescue.

The production of alternative RNA variants contributes to the tissue-specific regulation of gene expression. In the animal nervous system, a systematic shift toward distal sites of transcription termination produces transcript signatures that are crucial for neuron development and function. Here, we report that, in Drosophila, the highly conserved protein ELAV globally regulates all sites of neuronal 3' end processing and directly binds to proximal polyadenylation sites of target mRNAs in vivo. We uncover an endogenous strategy of functional gene rescue that safeguards neuronal RNA signatures in an ELAV loss-of-function context. When not directly repressed by ELAV, the transcript encoding the ELAV paralog FNE acquires a mini-exon, generating a new protein able to translocate to the nucleus and rescue ELAV-mediated alternative polyadenylation and alternative splicing. We propose that exon-activated functional rescue is a more widespread mechanism that ensures robustness of processes regulated by a hierarchy, rather than redundancy, of effectors.

Short- and Long-Term Regulation of HuD: A Molecular Switch Mediated by Folic Acid?

The RNA-binding protein HuD has been shown to play a crucial role in gene regulation in the nervous system and is involved in various neurological and psychiatric diseases. In this study, through the creation of an interaction network on HuD and its potential targets, we identified a strong association between HuD and several diseases of the nervous system. Specifically, we focused on the relationship between HuD and the brain-derived neurotrophic factor (BDNF), whose protein is implicated in several neuronal diseases and is involved in the regulation of neuronal development, survival, and function. To better investigate this relationship and given that we previously demonstrated that folic acid (FA) is able to directly bind HuD itself, we performed in vitro experiments in neuron-like human SH-SY5Y cells in the presence of FA, also known to be a pivotal environmental factor influencing the nervous system development. Our findings show that FA exposure results in a significant increase in both HuD and BDNF transcripts and proteins after 2 and 4 h of treatment, respectively. Similar data were obtained after 2 h of FA incubation followed by 2 h of washout. This increase was no longer detected upon 24 h of FA exposure, probably due to a signaling shutdown mechanism. Indeed, we observed that following 24 h of FA exposure HuD is methylated. These findings indicate that FA regulates BDNF expression via HuD and suggest that FA can behave as an epigenetic modulator of HuD in the nervous system acting via short- and long-term mechanisms. Finally, the present results also highlight the potential of BDNF as a therapeutic target for specific neurological and psychiatric diseases.

The Labyrinthine Landscape of APP Processing: State of the Art and Possible Novel Soluble APP-Related Molecular Players in Traumatic Brain Injury and Neurodegeneration.

Amyloid Precursor Protein (APP) and its cleavage processes have been widely investigated in the past, in particular in the context of Alzheimer's Disease (AD). Evidence of an increased expression of APP and its amyloidogenic-related cleavage enzymes, ß-secretase 1 (BACE1) and γ-secretase, at the hit axon terminals following Traumatic Brain Injury (TBI), firstly suggested a correlation between TBI and AD. Indeed, mild and severe TBI have been recognised as influential risk factors for different neurodegenerative diseases, including AD. In the present work, we describe the state of the art of APP proteolytic processing, underlining the different roles of its cleavage fragments in both physiological and pathological contexts. Considering the neuroprotective role of the soluble APP alpha (sAPPα) fragment, we hypothesised that sAPPα could modulate the expression of genes of interest for AD and TBI. Hence, we present preliminary experiments addressing sAPPα-mediated regulation of BACE1, Isthmin 2 (ISM2), Tetraspanin-3 (TSPAN3) and the Vascular Endothelial Growth Factor (VEGFA), each discussed from a biological and pharmacological point of view in AD and TBI. We finally propose a neuroprotective interaction network, in which the Receptor for Activated C Kinase 1 (RACK1) and the signalling cascade of PKCßII/nELAV/VEGF play hub roles, suggesting that vasculogenic-targeting therapies could be a feasible approach for vascular-related brain injuries typical of AD and TBI.

ELAV Proteins Bind and Stabilize C/EBP mRNA in the Induction of Long-Term Memory in Aplysia.

Long-term memory (LTM) formation is a critical survival process by which an animal retains information about prior experiences to guide future behavior. In the experimentally advantageous marine mollusk Aplysia, LTM for sensitization can be induced by the presentation of two aversive shocks to the animal's tail. Each of these training trials recruits distinct growth factor signaling systems that promote LTM formation. Specifically, whereas intact TrkB signaling during Trial 1 promotes an initial and transient increase of the immediate early gene apc/ebp mRNA, a prolonged increase in apc/ebp gene expression required for LTM formation requires the addition of TGFß signaling during Trial 2. Here we explored the molecular mechanisms by which Trial 2 achieves the essential prolonged gene expression of apc/ebp We find that this prolonged gene expression is not dependent on de novo transcription, but that apc/ebp mRNA synthesized by Trial 1 is post-transcriptionally stabilized by interacting with the RNA-binding protein ApELAV. This interaction is promoted by p38 MAPK activation initiated by TGFß. We further demonstrate that blocking the interaction of ApELAV with its target mRNA during Trial 2 blocks both the prolonged increase in apc/ebp gene expression and the behavioral induction of LTM. Collectively, our findings elucidate both when and how ELAV proteins are recruited for the stabilization of mRNA in LTM formation. Stabilization of a transiently expressed immediate early gene mRNA by a repeated training trial may therefore serve as a "filter" for learning, permitting only specific events to cause lasting transcriptional changes and behavioral LTM.SIGNIFICANCE STATEMENT: In the present paper, we significantly extend the general field of molecular processing in long-term memory (LTM) by describing a novel form of pretranslational processing required for LTM, which relies on the stabilization of a newly synthesized mRNA by a class of RNA binding proteins (ELAVs). There are now compelling data showing that important processing can occur after transcription of a gene, but before translation of the message into protein. Although the potential importance of ELAV proteins in LTM formation has previously been reported, the specific actions of ELAV proteins during LTM formation remained to be understood. Our new findings thus complement and extend this literature by demonstrating when and how this post-transcriptional gene regulation is mediated in the induction of LTM.

The lncRNA HMS recruits RNA-binding protein HuR to stabilize the 3'-UTR of HOXC10 mRNA.

Long noncoding RNAs (lncRNAs) have been reported to drive key cancer pathways but the functions of majority of lncRNAs are unknown making a case for comprehensive functional evaluation of lncRNAs. With an aim to identify lncRNAs dysregulated in human cancers, we analyzed the cancer patient database of lung adenocarcinoma (LUAD), which revealed an upregulated lncRNA, LINC02381 (renamed HOXC10mRNA stabilizing factor or HMS in this study), whose depletion results in proliferation defects and inhibition of colony formation of human cancer cells. In order to identify the binding targets of HMS, we screened for cis-genes and discovered that HOXC10, an oncogene, is downregulated in the absence of HMS. Depletion of HMS does not affect the HOXC10 promoter activity but inhibits the HOXC10 3'-UTR-linked luciferase reporter activity. Since lncRNAs have been known to associate with RNA-binding proteins (RBPs) to stabilize mRNA transcripts, we screened for different RBPs and discovered that HuR, an ELAV family protein, stabilizes HOXC10 mRNA. Using RNA pull-down and deletion mapping experiments, we show that HuR physically interacts with the cytosine-rich stretch of HMS and HOXC10 3'-UTR to stabilize HOXC10 mRNA. HOXC10 is overexpressed in many human cancers, and our discovery highlights that lncRNA HMS sustains the HOXC10 mRNA levels to maintain the invasive phenotypes of cancer cells.

RNA-binding proteins La and HuR cooperatively modulate translation repression of PDCD4 mRNA.

Posttranscriptional regulation of gene expression plays a critical role in controlling the inflammatory response. An uncontrolled inflammatory response results in chronic inflammation, often leading to tumorigenesis. Programmed cell death 4 (PDCD4) is a proinflammatory tumor-suppressor gene which helps to prevent the transition from chronic inflammation to cancer. PDCD4 mRNA translation is regulated by an interplay between the oncogenic microRNA miR-21 and the RNA-binding protein (RBP) human antigen R (HuR) in response to lipopolysaccharide stimulation, but the role of other regulatory factors remains unknown. Here, we report that the RBP lupus antigen (La) interacts with the 3'-untranslated region of PDCD4 mRNA and prevents miR-21-mediated translation repression. While lipopolysaccharide causes nuclear-cytoplasmic translocation of HuR, it enhances cellular La expression. Remarkably, La and HuR were found to bind cooperatively to the PDCD4 mRNA and mitigate miR-21-mediated translation repression. The cooperative action of La and HuR reduced cell proliferation and enhanced apoptosis, reversing the pro-oncogenic function of miR-21. Together, these observations demonstrate a cooperative interplay between two RBPs, triggered differentially by the same stimulus, which exerts a synergistic effect on PDCD4 expression and thereby helps maintain a balance between inflammation and tumorigenesis.

Cellular Protein HuR Regulates the Switching of Genomic RNA Templates for Differential Functions during the Coxsackievirus B3 Life Cycle.

Coxsackievirus B3 (CVB3) is an enterovirus belonging to the family Picornaviridae. Its 5' untranslated region (UTR) contains a cloverleaf structure followed by an internal ribosome entry site (IRES). The cloverleaf forms an RNA-protein complex known to regulate virus replication, translation, and stability of the genome, and the IRES regulates virus RNA translation. For positive-strand RNA-containing viruses, such as members of the flaviviruses or enteroviruses, the genomic RNA is used for translation, replication, and encapsidation. Only a few regulatory mechanisms which govern the accessibility of genomic RNA templates for translation or replication have been reported. Here, we report the role of human antigen R (HuR) in regulating the fate of CVB3 positive-strand RNA into the replication cycle or translation cycle. We have observed that synthesis of HuR is induced during CVB3 infection, and it suppresses viral replication by displacing PCBP-2 (a positive regulator of virus replication) at the cloverleaf RNA. Silencing of HuR increases viral RNA replication and consequently reduces viral RNA translation in a replication-dependent manner. Furthermore, we have shown that HuR level is upregulated upon CVB3 infection. Moreover, HuR limits virus replication and can coordinate the availability of genomic RNA templates for translation, replication, or encapsidation. Our study highlights the fact that the relative abundance of translation factors and replication factors in the cell decides the outcome of viral infection. IMPORTANCE A positive-strand RNA virus must balance the availability of its genomic template for different viral processes at different stages of its life cycle. A few host proteins are shown to be important to help the virus in switching the usage of a template between these processes. These proteins inhibit translation either by displacing a stimulator of translation or by binding to an alternative site. Both mechanisms lead to ribosome clearance and availability of the genomic strand for replication. We have shown that HuR also helps in maintaining this balance by inhibiting replication and subsequently promoting translation and packaging.

Targeting the RNA-Binding Protein HuR as Potential Thera-Peutic Approach for Neurological Disorders: Focus on Amyo-Trophic Lateral Sclerosis (ALS), Spinal Muscle Atrophy (SMA) and Multiple Sclerosis.

The importance of precise co- and post-transcriptional processing of RNA in the regulation of gene expression has become increasingly clear. RNA-binding proteins (RBPs) are a class of proteins that bind single- or double-chain RNA, with different affinities and selectivity, thus regulating the various functions of RNA and the fate of the cells themselves. ELAV (embryonic lethal/abnormal visual system)/Hu proteins represent an important family of RBPs and play a key role in the fate of newly transcribed mRNA. ELAV proteins bind AU-rich element (ARE)-containing transcripts, which are usually present on the mRNA of proteins such as cytokines, growth factors, and other proteins involved in neuronal differentiation and maintenance. In this review, we focused on a member of ELAV/Hu proteins, HuR, and its role in the development of neurodegenerative disorders, with a particular focus on demyelinating diseases.

AU-rich element-mediated mRNA decay via the butyrate response factor 1 controls cellular levels of polyadenylated replication-dependent histone mRNAs.

Replication-dependent histone (RDH) mRNAs have a nonpolyadenylated 3'-UTR that ends in a highly conserved stem-loop structure. Nonetheless, a subset of RDH mRNAs has a poly(A) tail under physiological conditions. The biological meaning of poly(A)-containing (+) RDH mRNAs and details of their biosynthesis remain elusive. Here, using HeLa cells and Western blotting, qRT-PCR, and biotinylated RNA pulldown assays, we show that poly(A)+ RDH mRNAs are post-transcriptionally regulated via adenylate- and uridylate-rich element-mediated mRNA decay (AMD). We observed that the rapid degradation of poly(A)+ RDH mRNA is driven by butyrate response factor 1 (BRF1; also known as ZFP36 ring finger protein-like 1) under normal conditions. Conversely, cellular stresses such as UV C irradiation promoted BRF1 degradation, increased the association of Hu antigen R (HuR; also known as ELAV-like RNA-binding protein 1) with the 3'-UTR of poly(A)+ RDH mRNAs, and eventually stabilized the poly(A)+ RDH mRNAs. Collectively, our results provide evidence that AMD surveils poly(A)+ RDH mRNAs via BRF1-mediated degradation under physiological conditions.

Molecular and cytological analysis of widely-used Gal4 driver lines for Drosophila neurobiology.

BACKGROUND: The Drosophila central nervous system (CNS) is a convenient model system for the study of the molecular mechanisms of conserved neurobiological processes. The manipulation of gene activity in specific cell types and subtypes of the Drosophila CNS is frequently achieved by employing the binary Gal4/UAS system. However, many Gal4 driver lines available from the Bloomington Drosophila Stock Center (BDSC) and commonly used in Drosophila neurobiology are still not well characterized. Among these are three lines with Gal4 driven by the elav promoter (BDSC #8760, #8765, and #458), one line with Gal4 driven by the repo promoter (BDSC #7415), and the 69B-Gal4 line (BDSC #1774). For most of these lines, the exact insertion sites of the transgenes and the detailed expression patterns of Gal4 are not known. This study is aimed at filling these gaps. RESULTS: We have mapped the genomic location of the Gal4-bearing P-elements carried by the BDSC lines #8760, #8765, #458, #7415, and #1774. In addition, for each of these lines, we have analyzed the Gal4-driven GFP expression pattern in the third instar larval CNS and eye-antennal imaginal discs. Localizations of the endogenous Elav and Repo proteins were used as markers of neuronal and glial cells, respectively. CONCLUSIONS: We provide a mini-atlas of the spatial activity of Gal4 drivers that are widely used for the expression of UAS-target genes in the Drosophila CNS. The data will be helpful for planning experiments with these drivers and for the correct interpretation of the results.

Regulation of Kv11.1 potassium channel C-terminal isoform expression by the RNA-binding proteins HuR and HuD.

The potassium voltage-gated channel subfamily H member 2 (KCNH2) gene encodes the Kv11.1 potassium channel, which conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative polyadenylation and forms a functional, full-length Kv11.1a isoform if exon 15 is polyadenylated or a nonfunctional, C-terminally truncated Kv11.1a-USO isoform if intron 9 is polyadenylated. The molecular mechanisms that regulate Kv11.1 isoform expression are poorly understood. In this study, using HEK293 cells and reporter gene expression, pulldown assays, and RNase protection assays, we identified the RNA-binding proteins Hu antigen R (HuR) and Hu antigen D (HuD) as regulators of Kv11.1 isoform expression. We show that HuR and HuD inhibit activity at the intron 9 polyadenylation site. When co-expressed with the KCNH2 gene, HuR and HuD increased levels of the Kv11.1a isoform and decreased the Kv11.1a-USO isoform in the RNase protection assays and immunoblot analyses. In patch clamp experiments, HuR and HuD significantly increased the Kv11.1 current. siRNA-mediated knockdown of HuR protein decreased levels of the Kv11.1a isoform and increased those of the Kv11.1a-USO isoform. Our findings suggest that the relative expression levels of Kv11.1 C-terminal isoforms are regulated by the RNA-binding HuR and HuD proteins.

Neural specificity of the RNA-binding protein Elav is achieved by post-transcriptional repression in non-neural tissues.

Drosophila Elav is the founding member of the conserved family of Hu RNA-binding proteins (RBPs), which play crucial and diverse roles in post-transcriptional regulation. Elav has long served as the canonical neuronal marker. Surprisingly, although Elav has a well-characterized neural cis-regulatory module, we find endogenous Elav is also ubiquitously transcribed and post-transcriptionally repressed in non-neural settings. Mutant clones of multiple miRNA pathway components derepress ubiquitous Elav protein. Our re-annotation of the elav transcription unit shows not only that it generates extended 3' UTR isoforms, but also that its universal 3' UTR isoform is much longer than previously believed. This longer common 3' UTR includes multiple conserved, high-affinity sites for the miR-279/996 family. Of several miRNA mutants tested, endogenous Elav and a transgenic elav 3' UTR sensor are derepressed in mutant clones of mir-279/996 We also observe cross-repression of Elav by Mei-P26, another RBP derepressed in non-neural miRNA pathway clones. Ubiquitous Elav has regulatory capacity, since derepressed Elav can stabilize an Elav-responsive sensor. Repression of Elav in non-neural territories is crucial as misexpression here has profoundly adverse consequences. Altogether, we define unexpected post-transcriptional mechanisms that direct appropriate cell type-specific expression of a conserved neural RBP.

Neurogenesis in sea urchin embryos and the diversity of deuterostome neurogenic mechanisms.

A single origin to the diverse mechanisms of metazoan neurogenesis is suggested by the involvement of common signaling components and similar classes of transcription factors. However, in many forms we lack details of where neurons arise, patterns of cell division, and specific differentiation pathway components. The sea urchin larval nervous system is composed of an apical organ, which develops from neuroepithelium and functions as a central nervous system, and peripheral neurons, which differentiate in the ciliary band and project axons to the apical organ. To reveal developmental mechanisms of neurogenesis in this basal deuterostome, we developed antibodies to SoxC, SoxB2, ELAV and Brn1/2/4 and used neurons that develop at specific locations to establish a timeline for neurogenesis. Neural progenitors express, in turn, SoxB2, SoxC, and Brn1/2/4, before projecting neurites and expressing ELAV and SynB. Using pulse-chase labeling of cells with a thymidine analog to identify cells in S-phase, we establish that neurons identified by location are in their last mitotic cycle at the time of hatching, and S-phase is coincident with expression of SoxC. The number of cells expressing SoxC and differentiating as neurons is reduced in embryos injected with antisense morpholino oligonucleotides to SoxC, SoxB2 or Six3. Injection of RNA encoding SoxC into eggs does not enhance neurogenesis. In addition, inhibition of FGF receptors (SU5402) or a morpholino to FGFR1 reduces expression of SoxC. These data indicate that there are common features of neurogenesis in deuterostomes, and that sea urchins employ developmental mechanisms that are distinct from other ambulacraria.

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.

Regeneration of the acorn worm pygochord with the implication for its convergent evolution with the notochord.

The origin of the notochord is a central issue in chordate evolution. This study examined the development of the acorn worm pygochord, a putative homologue of the notochord. Because the pygochord differentiates only after metamorphosis, the developmental was followed process by inducing regeneration after artificial amputation in Ptychodera flava. It was found that although the regeneration of the posterior part of the body did not proceed via formation of an obvious regeneration bud, pygochord regeneration was observed within a few weeks, possibly via trans-differentiation of endoderm cells. The expression of the fibrillary collagen gene (Fcol) and elav in the pygochord during regeneration was detected. This indicates that pygochord cells are not part of gut epithelial cells, but that they differentiated as a distinct cell type. Our gene expression analyses do not provide supporting evidence for the homology between the pygochord and notochord, but rather favored the convergent evolution between them.

RNA-Binding Proteins HuB, HuC, and HuD are Distinctly Regulated in Dorsal Root Ganglia Neurons from STZ-Sensitive Compared to STZ-Resistant Diabetic Mice.

The neuron-specific Elav-like Hu RNA-binding proteins were described to play an important role in neuronal differentiation and plasticity by ensuring the post-transcriptional control of RNAs encoding for various proteins. Although Elav-like Hu proteins alterations were reported in diabetes or neuropathy, little is known about the regulation of neuron-specific Elav-like Hu RNA-binding proteins in sensory neurons of dorsal root ganglia (DRG) due to the diabetic condition. The goal of our study was to analyze the gene and protein expression of HuB, HuC, and HuD in DRG sensory neurons in diabetes. The diabetic condition was induced in CD-1 adult male mice with single-intraperitoneal injection of streptozotocin (STZ, 150 mg/kg), and 8-weeks (advanced diabetes) after induction was quantified the Elav-like proteins expression. Based on the glycemia values, we identified two types of responses to STZ, and mice were classified in STZ-resistant (diabetic resistant, glycemia < 260 mg/dL) and STZ-sensitive (diabetic, glycemia > 260 mg/dL). Body weight measurements indicated that 8-weeks after STZ-induction of diabetes, control mice have a higher increase in body weight compared to the diabetic and diabetic resistant mice. Moreover, after 8-weeks, diabetic mice (19.52 ± 3.52 s) have longer paw withdrawal latencies in the hot-plate test than diabetic resistant (11.36 ± 1.92 s) and control (11.03 ± 1.97 s) mice, that correlates with the installation of warm hypoalgesia due to the diabetic condition. Further on, we evidenced the decrease of Elav-like gene expression in DRG neurons of diabetic mice (Elavl2, 0.68 ± 0.05 fold; Elavl3, 0.65 ± 0.01 fold; Elavl4, 0.53 ± 0.07 fold) and diabetic resistant mice (Ealvl2, 0.56 ± 0.07 fold; Elavl3, 0.32 ± 0.09 fold) compared to control mice. Interestingly, Elav-like genes have a more accentuated downregulation in diabetic resistant than in diabetic mice, although hypoalgesia was evidenced only in diabetic mice. The Elav-like gene expression changes do not always correlate with the Hu protein expression changes. To detail, HuB is upregulated and HuD is downregulated in diabetic mice, while HuB, HuC, and HuD are downregulated in diabetic resistant mice compared to control mice. To resume, we demonstrated HuD downregulation and HuB upregulation in DRG sensory neurons induced by diabetes, which might be correlated with altered post-transcriptional control of RNAs involved in the regulation of thermal hypoalgesia condition caused by the advanced diabetic neuropathy.

Post-transcriptional regulation of ERBB2 by miR26a/b and HuR confers resistance to tamoxifen in estrogen receptor-positive breast cancer cells.

Tamoxifen-resistant (TAMR) estrogen receptor-positive (ER+) breast cancer is characterized by elevated Erb-B2 receptor tyrosine kinase 2 (ERBB2) expression. However, the underlying mechanisms responsible for the increased ERBB2 expression in the TAMR cells remain poorly understood. Herein, we reported that the ERBB2 expression is regulated at the post-transcriptional level by miR26a/b and the RNA-binding protein human antigen R (HuR), both of which associate with the 3'-UTR of the ERBB2 transcripts. We demonstrated that miR26a/b inhibits the translation of ERBB2 mRNA, whereas HuR enhances the stability of the ERBB2 mRNA. In TAMR ER+ breast cancer cells with elevated ERBB2 expression, we observed a decrease in the level of miR26a/b and an increase in the level of HuR. The forced expression of miR26a/b or the depletion of HuR decreased ERBB2 expression in the TAMR cells, resulting in the reversal of tamoxifen resistance. In contrast, the inactivation of miR26a/b or forced expression of HuR decreased tamoxifen responsiveness of the parental ER+ breast cancer cells. We further showed that the increase in HuR expression in the TAMR ER+ breast cancer cells is attributable to an increase in the HuR mRNA isoform with shortened 3'-UTR, which exhibits increased translational activity. This shortening of the HuR mRNA 3'-UTR via alternative polyadenylation (APA) was observed to be dependent on cleavage stimulation factor subunit 2 (CSTF2/CstF-64), which is up-regulated in the TAMR breast cancer cells. Taken together, we have characterized a model in which the interplay between miR26a/b and HuR post-transcriptionally up-regulates ERBB2 expression in TAMR ER+ breast cancer cells.

Hu antigen R (HuR) multimerization contributes to glioma disease progression.

Among primary brain cancers, gliomas are the most deadly and most refractory to current treatment modalities. Previous reports overwhelmingly support the role of the RNA-binding protein Hu antigen R (HuR) as a positive regulator of glioma disease progression. HuR expression is consistently elevated in tumor tissues, and a cytoplasmic localization appears essential for HuR-dependent oncogenic transformation. Here, we report HuR aggregation (multimerization) in glioma and the analysis of this tumor-specific HuR protein multimerization in clinical brain tumor samples. Using a split luciferase assay, a bioluminescence resonance energy transfer technique, and site-directed mutagenesis, we examined the domains involved in HuR multimerization. Results obtained with the combination of the split HuR luciferase assay with the bioluminescence resonance energy transfer technique suggested that multiple (at least three) HuR molecules come together during HuR multimerization in glioma cells. Using these data, we developed a model of HuR multimerization in glioma cells. We also demonstrate that exposing glioma cells to the HuR inhibitor tanshinone group compound 15,16-dihydrotanshinone-I or to the newly identified compound 5 disrupts HuR multimerization modules and reduces tumor cell survival and proliferation. In summary, our findings provide new insights into HuR multimerization in glioma and highlight possible pharmacological approaches for targeting HuR domains involved in cancer cell-specific multimerization.

Cytoplasmic overexpression of RNA-binding protein HuR is a marker of poor prognosis in meningioma, and HuR knockdown decreases meningioma cell growth and resistance to hypoxia.

HuR regulates cytoplasmic mRNA stability and translatability, and the HuR expression level has been shown to correlate with poor disease outcome in several cancer types; however, the prognostic value and potential pro-oncogenic properties of HuR in meningioma remain unclear. Thus, in the present study, we analysed 85 meningioma tissue samples to establish the relationship between HuR expression, tumour cell proliferation, and/or patient survival. In addition, we examined the anti-proliferative effects of HuR knockdown in two meningioma cell lines (IOMM-Lee and Ben-Men-1) and conducted transcriptome-wide analyses (IOMM-Lee cells) to elucidate the molecular consequences of HuR knockdown. The results of the present study showed HuR cytoplasmic expression to correlate positively with tumour grade (p = 1.2 × 10-8 ) and negatively with progression-free and overall survival (p = 0.01) time in human meningioma tissues. In vitro, siHuR-induced HuR knockdown was shown to reduce the growth of both Ben-Men-1 (p = 2 × 10-8 ) and IOMM-Lee (p = 4 × 10-9 ) cells. Transcriptome analyses revealed HuR knockdown in IOMM-Lee cells to deregulate the HIF1A signalling pathway (p = 1.5 × 10-6 ) and to up-regulate the expression of genes essential for the assembly of the cytoplasmic mRNA processing body, global genome nucleotide-excision repair, poly(A)-specific ribonuclease activity, the positive regulation of apoptosis and of cell cycle arrest, and the negative regulation of RNA splicing [p(FDR) < 0.001]. Interestingly, HuR knockdown under hypoxic culture conditions further potentiated the effects of HuR knockdown on cell growth, apoptosis, and HIF1A expression. We thus conclude that cytoplasmic HuR expression is a marker of poor prognosis in meningioma and that HuR is a promising potential therapeutic target for use in tumours refractory to standard therapies. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.