Control of RNA Stability in Immunity.

Posttranscriptional control of mRNA regulates various biological processes, including inflammatory and immune responses. RNA-binding proteins (RBPs) bind cis-regulatory elements in the 3' untranslated regions (UTRs) of mRNA and regulate mRNA turnover and translation. In particular, eight RBPs (TTP, AUF1, KSRP, TIA-1/TIAR, Roquin, Regnase, HuR, and Arid5a) have been extensively studied and are key posttranscriptional regulators of inflammation and immune responses. These RBPs sometimes collaboratively or competitively bind the same target mRNA to enhance or dampen regulatory activities. These RBPs can also bind their own 3' UTRs to negatively or positively regulate their expression. Both upstream signaling pathways and microRNA regulation shape the interactions between RBPs and target RNA. Dysregulation of RBPs results in chronic inflammation and autoimmunity. Here, we summarize the functional roles of these eight RBPs in immunity and their associated diseases.

HuR facilitates miR-93-5p-induced activation of MAP3K2 translation via MAP3K2 3'UTR ARE2 in hepatocellular carcinoma.

MicroRNAs (miRNAs) can positively regulate gene expression through an unconventional RNA activation mechanism involving direct targeting 3' untranslated regions (UTRs). Our prior study found miR-93-5p activates mitogen-activated protein kinase kinase kinase 2 (MAP3K2) in hepatocellular carcinoma (HCC) via its 3'UTR. However, the underlying mechanism remains elusive. Here, we identified two candidate AU-rich element (ARE) motifs (ARE1 and ARE2) adjacent to the miR-93-5p binding site located within the MAP3K2 3'UTR using AREsite2. Luciferase reporter and translation assays validated that only ARE2 participated in MAP3K2 activation. Integrative analysis revealed that human antigen R (HuR), an ARE2-associated RNA-binding protein (RBP), physically and functionally interacted with the MAP3K2 3'UTR. Consequently, an HuR-ARE2 complex was shown to facilitate miR-93-5p-mediated upregulation of MAP3K2 expression. Furthermore, bioinformatics analysis and studies of HCC cells and specimens highlighted an oncogenic role for HuR and positive HuR-MAP3K2 expression correlation. HuR is also an enhancing factor in the positive feedback circuit comprising miR-93-5p, MAP3K2, and c-Jun demonstrated in our prior study. The newly identified HuR-ARE2 involvement enriches the mechanism of miR-93-5p-driven MAP3K2 activation and suggests new therapeutic strategies warranted for exploration in HCC.

Necessity of HuR/ELAVL1 for the activation-induced cytidine deaminase-dependent decrease in topoisomerase 1 in antibody diversification.

Activation-induced cytidine deaminase (AID)-dependent DNA cleavage is the initial event of antibody gene-diversification processes such as class switch recombination (CSR) and somatic hypermutation (SHM). We previously reported the requirement of an AID-dependent decrease of topoisomerase 1 (Top1) for efficient DNA cleavage, but the underlying molecular mechanism has remained elusive. This study focuses on HuR/ELAVL1, a protein that binds to AU-rich elements in RNA. HuR-knockout (KO) CH12 cells derived from murine B lymphoma cells were found to have lower CSR and hypermutation efficiencies due to decreased AID-dependent DNA cleavage levels. The HuR-KO CH12 cells do not show impairment in cell cycles and Myc expression, which have been reported in HuR-reduced spleen B cells. Furthermore, drugs that scavenge reactive oxygen species (ROS) do not rescue the lower CSR in HuR-KO CH12 cells, meaning that ROS or decreased c-Myc protein amount is not the reason for the deficiencies of CSR and hypermutation in HuR-KO CH12 cells. We show that HuR binds to Top1 mRNA and that complete deletion of HuR abolishes AID-dependent repression of Top1 protein synthesis in CH12 cells. Additionally, reduction of CSR to IgG3 in HuR-KO cells is rescued by knockdown of Top1, indicating that elimination of the AID-dependent Top1 decrease is the cause of the inefficiency of DNA cleavage, CSR and hypermutation in HuR-KO cells. These results show that HuR is required for initiation of antibody diversification and acquired immunity through the regulation of AID-dependent DNA cleavage by repressing Top1 protein synthesis.

Lethal eosinophilic crystalline pneumonia in mice expressing a stabilized Csf2 mRNA.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that stimulates the proliferation and differentiation of granulocyte and macrophage precursors. The mouse gene-encoding GM-CSF, Csf2, is regulated at both transcriptional and post-transcriptional levels. An adenine-uridine-rich element (ARE) within the 3'-untranslated region of Csf2 mRNA was shown in cell transfection studies to confer instability on this transcript. To explore the physiological importance of this element in an intact animal, we generated mice with a knock-in deletion of the 75-nucleotide ARE. Mice heterozygous for this ARE deletion developed severe respiratory distress and death within about 12 weeks of age. There was dense infiltration of lung alveolar spaces by crystal-containing macrophages. Increased stability of Csf2 mRNA was confirmed in bone marrow-derived macrophages, and elevated GM-CSF levels were observed in serum and lung. These mice did not exhibit notable abnormalities in blood or bone marrow, and transplantation of bone marrow from mutant mice into lethally irradiated WT mice did not confer the pulmonary phenotype. Mice with a conditional deletion of the ARE restricted to lung type II alveolar cells exhibited an essentially identical lethal lung phenotype at the same ages as the mice with the whole-body deletion. In contrast, mice with the same conditional ARE deletion in myeloid cells, including macrophages, exhibited lesser degrees of macrophage infiltration into alveolar spaces much later in life, at approximately 9 months of age. Post-transcriptional Csf2 mRNA stability regulation in pulmonary alveolar epithelial cells appears to be essential for normal physiological GM-CSF secretion and pulmonary macrophage homeostasis.

Multiple roles for AU-rich RNA binding proteins in the development of haematologic malignancies and their resistance to chemotherapy.

Post-transcriptional regulation by RNA binding proteins can determine gene expression levels and drive changes in cancer cell proteomes. Identifying mechanisms of protein-RNA binding, including preferred sequence motifs bound in vivo, provides insights into protein-RNA networks and how they impact mRNA structure, function, and stability. In this review, we will focus on proteins that bind to AU-rich elements (AREs) in nascent or mature mRNA where they play roles in response to stresses encountered by cancer cells. ARE-binding proteins (ARE-BPs) specifically impact alternative splicing, stability, decay and translation, and formation of RNA-rich biomolecular condensates like cytoplasmic stress granules (SGs). For example, recent findings highlight the role of ARE-BPs - like TIAR and HUR - in chemotherapy resistance and in translational regulation of mRNAs encoding pro-inflammatory cytokines. We will discuss emerging evidence that different modes of ARE-BP activity impact leukaemia and lymphoma development, progression, adaptation to microenvironment and chemotherapy resistance.

RNA regulation of inflammatory responses in glia and its potential as a therapeutic target in central nervous system disorders.

A major hallmark of neuroinflammation is the activation of microglia and astrocytes with the induction of inflammatory mediators such as IL-1ß, TNF-α, iNOS, and IL-6. Neuroinflammation contributes to disease progression in a plethora of neurological disorders ranging from acute CNS trauma to chronic neurodegenerative disease. Posttranscriptional pathways of mRNA stability and translational efficiency are major drivers for the expression of these inflammatory mediators. A common element in this level of regulation centers around the adenine- and uridine-rich element (ARE) which is present in the 3' untranslated region (UTR) of the mRNAs encoding these inflammatory mediators. (ARE)-binding proteins (AUBPs) such as Human antigen R (HuR), Tristetraprolin (TTP) and KH- type splicing regulatory protein (KSRP) are key nodes for directing these posttranscriptional pathways and either promote (HuR) or suppress (TTP and KSRP) glial production of inflammatory mediators. This review will discuss basic concepts of ARE-mediated RNA regulation and its impact on glial-driven neuroinflammatory diseases. We will discuss strategies to target this novel level of gene regulation for therapeutic effect and review exciting preliminary studies that underscore its potential for treating neurological disorders.

Lengthening of 3' Untranslated Regions of mRNAs by Alternative Polyadenylation Is Associated With Tumor Progression and Poor Prognosis of Clear Cell Renal Cell Carcinoma.

Alternative polyadenylation (APA) is emerging as a major posttranscriptional mechanism for gene regulation in cancer. A prevailing hypothesis is that shortening of the 3' untranslated region (3'UTR) increases oncoprotein expression because of the loss of miRNA-binding sites (MBSs). We showed that the longer 3'UTR is associated with a more advanced tumor stage in patients with clear cell renal cell carcinoma (ccRCC). More surprisingly, 3'UTR shortening is correlated with better overall survival in patients with ccRCC. Furthermore, we identified a mechanism by which longer transcripts lead to increased oncogenic protein and decreased tumor-suppressive protein expression compared to the shorter transcripts. In our model, shortening of 3'UTRs by APA may increase the mRNA stability of the majority of the potential tumor-suppressor genes due to the loss of MBSs and AU-rich elements (AREs). Unlike potential tumor-suppressor genes, the potential oncogenes display much lower MBS and ARE density and globally much higher m6A density in distal 3'UTRs. As a result, 3'UTRs shortening decreases the mRNA stability of potential oncogenes and enhances the mRNA stability of potential tumor-suppressor genes. Our findings highlight the cancer-specific pattern of APA regulation and extend our understanding of the mechanism of APA-mediated 3'UTR length changes in cancer biology.

Tristetraprolin posttranscriptionally downregulates PFKFB3 in cancer cells.

The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases 3 (PFKFB3) catalyzes the first committed rate-limiting step of glycolysis and is upregulated in cancer cells. The mechanism of PFKFB3 expression upregulation in cancer cells has not been fully elucidated. The PFKFB3 3'-UTR is reported to contain AU-rich elements (AREs) that are important for regulating PFKFB3 mRNA stability. However, the mechanisms by which PFKFB3 mRNA stability is determined by its 3'-UTR are not well known. We demonstrated that tristetraprolin (TTP), an ARE-binding protein, has a critical function regulating PFKFB3 mRNA stability. Our results showed that PFKFB3 mRNA contains three AREs in the 3'-UTR. TTP bound to the 3rd ARE and enhanced the decay of PFKFB3 mRNA. Overexpression of TTP decreased PFKFB3 expression and ATP levels but increased GSH level in cancer cells. Overexpression of PFKFB3 cDNA without the 3'-UTR rescued ATP level and GSH level in TTP-overexpressing cells. Our results suggested that TTP post-transcriptionally downregulated PFKFB3 expression and that overexpression of TTP may contribute to suppression of glycolysis and energy production of cancer cells in part by downregulating PFKFB3 expression.

Enhanced oncolytic activity of E4orf6-deficient adenovirus by facilitating nuclear export of HuR.

An AU-rich element (ARE) is RNA element that enhances the rapid decay of mRNA. The RNA binding protein HuR stabilizes ARE-mRNA by exporting it to the cytoplasm. In most of cancer cells, HuR is exported to the cytoplasm and ARE-mRNA is stabilized. In addition, the viral gene product E4orf6 exports HuR to stabilize ARE-mRNA in adenovirus-infected cells and the stabilization is required for full virus replication. Previously we showed the oncolytic activity of E4orf6-deleted adenovirus dl355, which can replicate in cancer cells where ARE-mRNA is stabilized. In this study, we examined whether the further enhancement of HuR export can stimulate the replication and the oncolytic activity of dl355. We found that ethanol treatment promoted the cytoplasmic relocalization of HuR in cancer cells. In addition, the replication efficiency of dl355 increased in ethanol-treated cells, and in response, the cytolytic activity of the virus also increased in vitro and in vivo. Upregulation of a cleaved-PARP level in infected cells mediated by ethanol is suggesting that ethanol activated the apoptosis induced by dl355. IVa2 mRNA, the only ARE-mRNA among transcripts of adenovirus was augmented by ethanol treatment. These data indicate that the enhancement of ARE-mRNA stabilization as a result of ethanol treatment upregulates the oncolytic activity of dl355 and suggests that the combined use of an oncolytic adenovirus and ethanol treatment may be a good strategy for cancer therapy.

Helicobacter pylori inhibits GKN1 expression via the CagA/p-ERK/AUF1 pathway.

BACKGROUND: Recent studies have shown that gastrokine 1 (GKN1), an important tumor suppressor gene, is downregulated in Helicobacter pylori (H. pylori) infected gastric mucosa and gastric cancer. However, the underlying mechanism is poorly understood. Herein, we investigated the potential mechanism of H. pylori-induced GKN1 downregulation. MATERIALS AND METHODS: GKN1 and AU-rich element RNA-binding factor 1 (AUF1) expressions were assessed by quantitative real-time PCR, Western blot, or immunohistochemistry in H. pylori-infected tissues and H. pylori co-cultured cell lines. The regulation of AUF1 on GKN1 was determined by RNA pulldown assay, RNA immunoprecipitation, mRNA turnover, and luciferase activity assays. The involvement of phosphorylated extra-cellular signal-regulated kinase (p-ERK) or CagA in H. pylori-induced AUF1 expression was verified using p-ERK inhibitor or CagA knockout H. pylori. In addition, the cell proliferation and migration capacities of AUF1-knockdown cells were investigated. RESULTS: GKN1 expression progressively decreased from H. pylori-infected gastritis to gastric cancer tissues. H. pylori co-culture also induced significant GKN1 reduction in GES-1 and BGC-823 cells. Besides, the mRNA level of GKN1 and AUF1 in human gastric mucosa showed negative correlation significantly. AUF1 knockdown resulted in upregulation of GKN1 expression and promoted GKN1 mRNA decay by binding the 3' untranslated region of GKN1 mRNA H. pylori-induced AUF1 expression was associated with p-ERK activation and CagA. Furthermore, knockdown of AUF1 significantly inhibited cell viability, migration ability, and arrested fewer cells in S-phase. CONCLUSION: Our data demonstrated that H. pylori infection downregulated GKN1 expression via the CagA/p-ERK/AUF1 pathway. AUF1 promoted gastric cancer at least partly through downregulating GKN1, which presented a novel potential target for the treatment of gastric cancer.

Rotavirus Induces Formation of Remodeled Stress Granules and P Bodies and Their Sequestration in Viroplasms To Promote Progeny Virus Production.

Rotavirus replicates in unique virus-induced cytoplasmic inclusion bodies called viroplasms (VMs), the composition and structure of which have yet to be understood. Based on the analysis of a few proteins, earlier studies reported that rotavirus infection inhibits stress granule (SG) formation and disrupts P bodies (PBs). However, the recent demonstration that rotavirus infection induces cytoplasmic relocalization and colocalization with VMs of several nuclear hnRNPs and AU-rich element-binding proteins (ARE-BPs), which are known components of SGs and PBs, suggested the possibility of rotavirus-induced remodeling of SGs and PBs, prompting us to analyze a large number of the SG and PB components to understand the status of SGs and PBs in rotavirus-infected cells. Here we demonstrate that rotavirus infection induces molecular triage by selective exclusion of a few proteins of SGs (G3BP1 and ZBP1) and PBs (DDX6, EDC4, and Pan3) and sequestration of the remodeled/atypical cellular organelles, containing the majority of their components, in the VM. The punctate SG and PB structures are seen at about 4 h postinfection (hpi), coinciding with the appearance of small VMs, many of which fuse to form mature large VMs with progression of infection. By use of small interfering RNA (siRNA)-mediated knockdown and/or ectopic overexpression, the majority of the SG and PB components, except for ADAR1, were observed to inhibit viral protein expression and virus growth. In conclusion, this study demonstrates that VMs are highly complex supramolecular structures and that rotavirus employs a novel strategy of sequestration in the VM and harnessing of the remodeled cellular RNA recycling bins to promote its growth.IMPORTANCE Rotavirus is known to replicate in specialized virus-induced cytoplasmic inclusion bodies called viroplasms (VMs), but the composition and structure of VMs are not yet understood. Here we demonstrate that rotavirus interferes with normal SG and PB assembly but promotes formation of atypical SG-PB structures by selective exclusion of a few components and employs a novel strategy of sequestration of the remodeled SG-PB granules in the VMs to promote virus growth by modulating their negative influence on virus infection. Rotavirus VMs appear to be complex supramolecular structures formed by the union of the triad of viral replication complexes and remodeled SGs and PBs, as well as other host factors, and designed to promote productive virus infection. These observations have implications for the planning of future research with the aim of understanding the structure of the VM, the mechanism of morphogenesis of the virus, and the detailed roles of host proteins in rotavirus biology.

Cytoplasmic Relocalization and Colocalization with Viroplasms of Host Cell Proteins, and Their Role in Rotavirus Infection.

Rotavirus replicates in the cytoplasm of infected cells in unique virus-induced cytoplasmic inclusion bodies called viroplasms (VMs), which are nucleated by two essential viral nonstructural proteins, NSP2 and NSP5. However, the precise composition of the VM, the intracellular localization of host proteins during virus infection, and their association with VMs or role in rotavirus growth remained largely unexplored. Mass spectrometry analyses revealed the presence of several host heterogeneous nuclear ribonucleoproteins (hnRNPs), AU-rich element-binding proteins (ARE-BPs), and cytoplasmic proteins from uninfected MA104 cell extracts in the pulldown (PD) complexes of the purified viroplasmic proteins NSP2 and NSP5. Immunoblot analyses of PD complexes from RNase-treated and untreated cell extracts, analyses of coimmunoprecipitation complexes using RNase-treated infected cell lysates, and direct binding assays using purified recombinant proteins further demonstrated that the interactions of the majority of the hnRNPs and ARE-BPs with viroplasmic proteins are RNA independent. Time course immunoblot analysis of the nuclear and cytoplasmic fractions from rotavirus-infected and mock-infected cells and immunofluorescence confocal microscopy analyses of virus-infected cells revealed a surprising sequestration of the majority of the relocalized host proteins in viroplasms. Analyses of ectopic overexpression and small interfering RNA (siRNA)-mediated downregulation of expression revealed that host proteins either promote or inhibit viral protein expression and progeny virus production in virus-infected cells. This study demonstrates that rotavirus induces the cytoplasmic relocalization and sequestration of a large number of nuclear and cytoplasmic proteins in viroplasms, subverting essential cellular processes in both compartments to promote rapid virus growth, and reveals that the composition of rotavirus viroplasms is much more complex than is currently understood.IMPORTANCE Rotavirus replicates exclusively in the cytoplasm. Knowledge on the relocalization of nuclear proteins to the cytoplasm or the role(s) of host proteins in rotavirus infection is very limited. In this study, it is demonstrated that rotavirus infection induces the cytoplasmic relocalization of a large number of nuclear RNA-binding proteins (hnRNPs and AU-rich element-binding proteins). Except for a few, most nuclear hnRNPs and ARE-BPs, nuclear transport proteins, and some cytoplasmic proteins directly interact with the viroplasmic proteins NSP2 and NSP5 in an RNA-independent manner and become sequestered in the viroplasms of infected cells. The host proteins differentially affected viral gene expression and virus growth. This study demonstrates that rotavirus induces the relocalization and sequestration of a large number of host proteins in viroplasms, affecting host processes in both compartments and generating conditions conducive for virus growth in the cytoplasm of infected cells.

HuR translocation to the cytoplasm of cancer cells in actin-independent manner.

Human antigen R (HuR) is a RNA-binding protein, which binds to the AU-rich element (ARE) in the 3'-untranslated region (3'-UTR) of certain mRNA and is involved in the export and stabilization of ARE-mRNA. HuR constitutively relocates to the cytoplasm in many cancer cells, however the mechanism of intracellular HuR trafficking is poorly understood. To address this question, we examined the functional role of the cytoskeleton in HuR relocalization. We tested the effect of actin depolymerizing macrolide latrunculin A or myosin II ATPase activity inhibitor blebbistatin for HuR relocalization induced by the vasoactive hormone Angiotensin II in cancer and control normal cells. Western blot and confocal imaging data revealed that both inhibitors attenuated the cytoplasmic HuR in normal cells but no such alteration was observed in cancer cells. Concomitant with changes in intracellular HuR localization, both inhibitors markedly decreased the accumulation and half-lives of HuR target ARE-mRNAs in normal cells, whereas no change was observed in cancer cells. Furthermore, co-immunoprecipitation experiments with HuR proteins revealed clear physical interaction with ß-actin only in normal cells. The current study is the first to verify that cancer cells can implicate a microfilament independent HuR transport. We hypothesized that when cytoskeleton structure is impaired, cancer cells can acquire an alternative HuR trafficking strategy.

Autotaxin Expression Is Regulated at the Post-transcriptional Level by the RNA-binding Proteins HuR and AUF1.

Autotaxin (ATX) is a key enzyme that converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), a lysophospholipid mediator that regulates cellular activities through its specific G protein-coupled receptors. The ATX-LPA axis plays an important role in various physiological and pathological processes, especially in inflammation and cancer development. Although the transcriptional regulation of ATX has been widely studied, the post-transcriptional regulation of ATX is largely unknown. In this study, we identified conserved adenylate-uridylate (AU)-rich elements in the ATX mRNA 3'-untranslated region (3'UTR). The RNA-binding proteins HuR and AUF1 directly bound to the ATX mRNA 3'UTR and had antagonistic functions in ATX expression. HuR enhanced ATX expression by increasing ATX mRNA stability, whereas AUF1 suppressed ATX expression by promoting ATX mRNA decay. HuR and AUF1 were involved in ATX regulation in Colo320 human colon cancer cells and the LPS-stimulated human monocytic THP-1 cells. HuR knockdown suppressed ATX expression in B16 mouse melanoma cells, leading to inhibition of cell migration. This effect was reversed by AUF1 knockdown to recover ATX expression or by the addition of LPA. These results suggest that the post-transcriptional regulation of ATX expression by HuR and AUF1 modulates cancer cell migration. In summary, we identified HuR and AUF1 as novel post-transcriptional regulators of ATX expression, thereby elucidating a novel mechanism regulating the ATX-LPA axis.

Gastrin induces parathyroid hormone-like hormone expression in gastric parietal cells.

Parietal cells play a fundamental role in stomach maintenance, not only by creating a pathogen-free environment through the production of gastric acid, but also by secreting growth factors important for homeostasis of the gastric epithelium. The gastrointestinal hormone gastrin is known to be a central regulator of both parietal cell function and gastric epithelial cell proliferation and differentiation. Our previous gene expression profiling studies of mouse stomach identified parathyroid hormone-like hormone (PTHLH) as a potential gastrin-regulated gastric growth factor. Although PTHLH is commonly overexpressed in gastric tumors, its normal expression, function, and regulation in the stomach are poorly understood. In this study we used pharmacologic and genetic mouse models as well as human gastric cancer cell lines to determine the cellular localization and regulation of this growth factor by the hormone gastrin. Analysis of PthlhLacZ/+ knock-in reporter mice localized Pthlh expression to parietal cells in the gastric corpus. Regulation by gastrin was demonstrated by increased Pthlh mRNA abundance after acute gastrin treatment in wild-type mice and reduced expression in gastrin-deficient mice. PTHLH transcripts were also observed in normal human stomach as well as in human gastric cancer cell lines. Gastrin treatment of AGS-E gastric cancer cells induced a rapid and robust increase in numerous PTHLH mRNA isoforms. This induction was largely due to increased transcriptional initiation, although analysis of mRNA half-life showed that gastrin treatment also extended the half-life of PTHLH mRNA, suggesting that gastrin regulates expression by both transcriptional and posttranscriptional mechanisms.NEW & NOTEWORTHY We show that the growth factor parathyroid hormone-like hormone (PTHLH) is expressed in acid-secreting parietal cells of the mouse stomach. We define the specific PTHLH mRNA isoforms expressed in human stomach and in human gastric cancer cell lines and show that gastrin induces PTHLH expression via transcription activation and mRNA stabilization. Our findings suggest that PTHLH is a gastrin-regulated growth factor that might contribute to gastric epithelial cell homeostasis.

Pou1f1, the key transcription factor related to somatic growth in tilapia (Orechromis niloticus), is regulated by two independent post-transcriptional regulation mechanisms.

This study aims to determine the post-transcriptional regulation mechanism of the transcription factor pou1f1 (pou class 1 homeobox 1), which is the key gene for pituitary development, somatic growth in vertebrates, and transcription of several hormone genes in teleost fish. MicroRNA miR-223-3p was identified as a bona fide target of pou1f; overexpression of miR-223-3p in primary pituitary cells led to the down-regulation of pou1f1 and downstream genes, and inhibition of miR-223-3p led to the up-regulation of pou1f1 in Nile tilapia dispersed primary pituitary cells. An adenylate-uridylate-rich element (AU-Rich element) was found in the 3'UTR of pou1f1 mRNA, and deletion of the AU-Rich element led to slower mRNA decay and therefore more protein output. A potential mutual relationship between miR-223-3p and the AU-rich element was also investigated, and the results demonstrated that with or without the AU-Rich element, miR-223-3p induced the up-regulation of a reporter system under serum starvation conditions, indicating that miR-223-3p and the AU-Rich element function independent of each other. This study is the first to investigate the post-transcriptional mechanism of pou1f1, which revealed that miR-223-3p down-regulated pou1f1 and downstream gene expressions, and the AU-Rich element led to rapid decay of pou1f1 mRNA. MicroRNA miR-223-3p and the AU-Rich element co-regulated the post-transcriptional expression of pou1f1 independently in Nile tilapia, demonstrating that pou1f1 is under the control of a dual post-transcription regulation mechanism.

Brf1 posttranscriptionally regulates pluripotency and differentiation responses downstream of Erk MAP kinase.

AU-rich element mRNA-binding proteins (AUBPs) are key regulators of development, but how they are controlled and what functional roles they play depends on cellular context. Here, we show that Brf1 (zfp36l1), an AUBP from the Zfp36 protein family, operates downstream of FGF/Erk MAP kinase signaling to regulate pluripotency and cell fate decision making in mouse embryonic stem cells (mESCs). FGF/Erk MAP kinase signaling up-regulates Brf1, which disrupts the expression of core pluripotency-associated genes and attenuates mESC self-renewal without inducing differentiation. These regulatory effects are mediated by rapid and direct destabilization of Brf1 targets, such as Nanog mRNA. Enhancing Brf1 expression does not compromise mESC pluripotency but does preferentially regulate mesendoderm commitment during differentiation, accelerating the expression of primitive streak markers. Together, these studies demonstrate that FGF signals use targeted mRNA degradation by Brf1 to enable rapid posttranscriptional control of gene expression in mESCs.

Saturated fatty acids induce post-transcriptional regulation of HAMP mRNA via AU-rich element-binding protein, human antigen R (HuR).

Iron is implicated in fatty liver disease pathogenesis. The human hepcidin gene, HAMP, is the master switch of iron metabolism. The aim of this study is to investigate the regulation of HAMP expression by fatty acids in HepG2 cells. For these studies, both saturated fatty acids (palmitic acid (PA) and stearic acid (SA)) and unsaturated fatty acid (oleic acid (OA)) were used. PA and, to a lesser extent, SA, but not OA, up-regulated HAMP mRNA levels, as determined by real-time PCR. To understand whether PA regulates HAMP mRNA at the transcriptional or post-transcriptional level, the transcription inhibitor actinomycin D was employed. PA-mediated induction of HAMP mRNA expression was not blocked by actinomycin D. Furthermore, PA activated HAMP 3'-UTR, but not promoter, activity, as shown by reporter assays. HAMP 3'-UTR harbors a single AU-rich element (ARE). Mutation of this ARE abolished the effect of PA, suggesting the involvement of ARE-binding proteins. The ARE-binding protein human antigen R (HuR) stabilizes mRNA through direct interaction with AREs on 3'-UTR. HuR is regulated by phosphorylation-mediated nucleo-cytoplasmic shuttling. PA activated this process. The binding of HuR to HAMP mRNA was also induced by PA in HepG2 cells. Silencing of HuR by siRNA abolished PA-mediated up-regulation of HAMP mRNA levels. PKC is known to phosphorylate HuR. Staurosporine, a broad-spectrum PKC inhibitor, inhibited both PA-mediated translocation of HuR and induction of HAMP expression. Similarly, rottlerin, a novel class PKC inhibitor, abrogated PA-mediated up-regulation of HAMP expression. In conclusion, lipids mediate post-transcriptional regulation of HAMP throughPKC- and HuR-dependent mechanisms.

Exercise, skeletal muscle and inflammation: ARE-binding proteins as key regulators in inflammatory and adaptive networks.

The role of inflammation in skeletal muscle adaptation to exercise is complex and has hardly been elucidated so far. While the acute inflammatory response to exercise seems to promote skeletal muscle training adaptation and regeneration, persistent, low-grade inflammation, as seen in a multitude of chronic diseases, is obviously detrimental. The regulation of cytokine production in skeletal muscle cells has been relatively well studied, yet little is known about the compensatory and anti-inflammatory mechanisms that resolve inflammation and restore tissue homeostasis. One important strategy to ensure sequential, timely and controlled resolution of inflammation relies on the regulated stability of mRNAs encoding pro-inflammatory mediators. Many key transcripts in early immune responses are characterized by the presence of AU-rich elements (AREs) in the 3'-untranslated regions of their mRNAs, allowing efficient fine-tuning of gene expression patterns at the post-transcriptional level. AREs exert their function by recruiting particular RNA-binding proteins, resulting, in most cases, in de-stabilization of the target transcripts. The best-characterized ARE-binding proteins are HuR, CUGBP1, KSRP, AUF1, and the three ZFP36 proteins, especially TTP/ZFP36. Here, we give a general introduction into the role of inflammation in the adaptation of skeletal muscle to exercise. Subsequently, we focus on potential roles of ARE-binding proteins in skeletal muscle tissue in general and specifically exercise-induced skeletal muscle remodeling. Finally, we present novel data suggesting a specific function of TTP/ZFP36 in exercise-induced skeletal muscle plasticity.

Identification of novel proteins binding the AU-rich element of α-prothymosin mRNA through the selection of open reading frames (RIDome).

We describe here a platform for high-throughput protein expression and interaction analysis aimed at identifying the RNA-interacting domainome. This approach combines the selection of a phage library displaying "filtered" open reading frames with next-generation DNA sequencing. The method was validated using an RNA bait corresponding to the AU-rich element of α-prothymosin, an RNA motif that promotes mRNA stability and translation through its interaction with the RNA-binding protein ELAVL1. With this strategy, we not only confirmed known RNA-binding proteins that specifically interact with the target RNA (such as ELAVL1/HuR and RBM38) but also identified proteins not previously known to be ARE-binding (R3HDM2 and RALY). We propose this technology as a novel approach for studying the RNA-binding proteome.