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.