Global analysis of the abundance of AU-rich mRNAs in response to glucocorticoid treatment.

Glucocorticoids (GC) like dexamethasone (Dex) are potent anti-inflammatory agents with diverse cellular functions including the potentiation of the activity of AU-rich elements (AREs). AREs are cis-acting instability sequence elements located in the 3'UTRs of many inflammatory mediator mRNAs. Here, available RNA-seq data were used to investigate the effect of GCs on the ARE-mRNA-transcriptome. At a global scale, ARE-mRNAs had a tendency to be downregulated after GC-treatment of the A549 lung cancer cell-line, but with notable cases of upregulation. mRNA stability experiments indicated that not only the downregulated, but also the upregulated ARE-mRNAs are destabilized by Dex-treatment. Several of the most upregulated ARE-mRNAs code for anti-inflammatory mediators including the established GC targets DUSP1 and ZFP36; both code for proteins that target ARE-containing mRNAs for destruction. GCs are widely used in the treatment of COVID-19 patients; we show that ARE-mRNAs are more likely to regulate in opposite directions between Dex-treatment and SARS-CoV-2 infections compared to non-ARE mRNAs. The effect of GC treatment on ARE-mRNA abundance was also investigated in blood monocytes of COVID-19 patients. The results were heterogeneous; however, in agreement with in vitro observations, ZFP36 and DUSP1 were often amongst the most differentially expressed mRNAs. The results of this study propose a universal destabilization of ARE-mRNAs by GCs, but a diverse overall outcome in vitro likely due to induced transcription or due to the heterogeneity of COVID-19 patient's responses in vivo.

Post-transcriptional regulation of BIRC5/survivin expression and induction of apoptosis in breast cancer cells by tristetraprolin.

Inhibition of apoptosis is one of the hallmarks of cancer and is a target of various therapeutic interventions. BIRC5 is an inhibitor of apoptosis that is aberrantly expressed in cancer leading to sustained growth of tumours. Post-transcriptional control mechanisms involving RNA-binding proteins and AU-rich elements (AREs) are fundamental to many cellular processes and changes in the expression or function of these proteins can promote an aberrant and pathological phenotype. BIRC5 mRNA has an ARE in its 3' UTR making it a candidate for regulation by the RNA binding proteins tristetraprolin (TTP) and HuR (ELAVL1). In this study, we investigated the binding of TTP and HuR by RNA-immunoprecipitation assays and found that these proteins were associated with BIRC5 mRNA to varying extents. Consequently, BIRC5 expression decreased when TTP was overexpressed and apoptosis was induced. In the absence of TTP, BIRC5 mRNA was stabilized, protein expression increased and the number of apoptotic cells declined. As an ARE-mRNA stabilizing protein, recombinant HuR led to upregulation of BIRC5 expression, whereas HuR silencing was concomitant with downregulation of BIRC5 mRNA and protein and increased cell death. Survival analyses demonstrated that increased TTP and low BIRC5 expression predicted an overall better prognosis compared to dysregulated TTP and high BIRC5. Thus, the results present a novel target of ARE-mediated post-transcriptional regulation.

Beyond the exome: utility of long-read whole genome sequencing in exome-negative autosomal recessive diseases.

BACKGROUND: Long-read whole genome sequencing (lrWGS) has the potential to address the technical limitations of exome sequencing in ways not possible by short-read WGS. However, its utility in autosomal recessive Mendelian diseases is largely unknown. METHODS: In a cohort of 34 families in which the suspected autosomal recessive diseases remained undiagnosed by exome sequencing, lrWGS was performed on the Pacific Bioscience Sequel IIe platform. RESULTS: Likely causal variants were identified in 13 (38%) of the cohort. These include (1) a homozygous splicing SV in TYMS as a novel candidate gene for lethal neonatal lactic acidosis, (2) a homozygous non-coding SV that we propose impacts STK25 expression and causes a novel neurodevelopmental disorder, (3) a compound heterozygous SV in RP1L1 with complex inheritance pattern in a family with inherited retinal disease, (4) homozygous deep intronic variants in LEMD2 and SNAP91 as novel candidate genes for neurodevelopmental disorders in two families, and (5) a promoter SNV in SLC4A4 causing non-syndromic band keratopathy. Surprisingly, we also encountered causal variants that could have been identified by short-read exome sequencing in 7 families. The latter highlight scenarios that are especially challenging at the interpretation level. CONCLUSIONS: Our data highlight the continued need to address the interpretation challenges in parallel with efforts to improve the sequencing technology itself. We propose a path forward for the implementation of lrWGS sequencing in the setting of autosomal recessive diseases in a way that maximizes its utility.

ß-catenin attenuation leads to up-regulation of activating NKG2D ligands and tumor regression in BrafV600E-driven thyroid cancer cells.

Introduction: BRAFV600E mutations frequently occur in papillary thyroid cancer (PTC). ß-catenin, encoded by CTNNB1, is a key downstream component of the canonical Wnt signaling pathway and is often overexpressed in PTC. BRAFV600E-driven PTC tumors rely on Wnt/ß-catenin signaling to sustain growth and progression. Methods: In the present study, we investigated the tumorigenicity of thyroid cancer cells derived from BRAFV600E PTC mice following Ctnnb1 ablation (BVE-Ctnnb1null). Results: Remarkably, the tumorigenic potential of BVE-Ctnnb1null tumor cells was lost in nude mice. Global gene expression analysis of BVE-Ctnnb1null tumor cells showed up-regulation of NKG2D receptor activating ligands (H60a, H60b, H60c, Raet1a, Raet1b, Raet1c, Raet1d, Raet1e, and Ulbp1) and down-regulation of inhibitory MHC class I molecules H-2L and H-2K2 in BVE-Ctnnb1null tumor cells. In vitro cytotoxicity assay demonstrated that BVE-Ctnnb1wt tumor cells were resistant to NK cell-mediated cytotoxicity, whereas BVE-Ctnnb1null tumor cells were sensitive to NK cell-mediated killing. Furthermore, the overexpression of any one of these NKG2D ligands in the BVE-Ctnnb1wt cell line resulted in a significant reduction of tumor growth in nude mice. Conclusions: Our results indicate that active ß-catenin signaling inhibits NK cell-mediated immune responses against thyroid cancer cells. Targeting the ß-catenin signaling pathway may have significant therapeutic benefits for BRAF-mutant thyroid cancer by not only inhibiting tumor growth but also enhancing host immune surveillance.

Differential upregulation of AU-rich element-containing mRNAs in COVID-19.

BACKGROUND: AU-rich elements (AREs) are located in the 3'UTRs of 22% of human mRNAs, including most transiently expressed inflammatory mediators. By default, AREs mark mRNAs for decay and translational inhibition, but this activity can be temporarily inhibited in case of infection to allow the onset of inflammation. Morbidity and mortality in COVID-19 patients have been associated with dysregulated inflammation, a process that may include aberrant ARE activity. RESULTS: RNA-seq data from available transcriptomic studies were analyzed to investigate a possible differential expression of mRNAs that contain AREs in the context of SARS-CoV-2 infections. ARE-mRNAs turned out to be significantly overrepresented among the upregulated mRNAs after SARS-CoV-2 infection (up to 42%). In contrast, ARE-mRNAs were underrepresented (16%) in the downregulated group. Consequently, at a global scale, ARE-mRNAs are significantly more upregulated after SARS-CoV-2 infection compared to non-ARE mRNAs. This observation was apparent in lung cell line models such as A549 and Calu-3 and with infections with other respiratory viruses and cell lines. Most importantly, at the clinical level, the elevated ARE-mRNA response appeared strongest in blood cells of COVID-19 patients with mild disease. It diminished with disease severity and was least apparent in patients in need of intubation and respiratory-related death. Gene function and clustering analysis suggest that the ARE-response is rather global and the upregulated ARE-mRNAs in patients with mild disease do not particularly cluster in specific functional groups. CONCLUSIONS: Compared to the rest of the transcriptome, ARE-containing mRNAs are preferentially upregulated in response to viral infections at a global level. In the context of COVID-19, they are most upregulated in mild disease. Due to their large number, their levels measured by RNA-seq may provide a reliable indication of COVID-19 severity.

Post-transcriptional screen of cancer amplified genes identifies ERBB2/Her2 signaling as AU-rich mRNA stability-promoting pathway.

Amplification of specific cancer genes leads to their over-expression contributing to tumor growth, spread, and drug resistance. Little is known about the ability of these amplified oncogenes to augment the expression of cancer genes through post-transcriptional control. The AU-rich elements (ARE)-mediated mRNA decay is compromised for many key cancer genes leading to their increased abundance and effects. Here, we performed a post-transcriptional screen for frequently amplified cancer genes demonstrating that ERBB2/Her2 overexpression was able to augment the post-transcriptional effects. The ERBB1/2 inhibitor, lapatinib, led to the reversal of the aberrant ARE-mediated process in ERBB2-amplified breast cancer cells. The intersection of overexpressed genes associated with ERBB2 amplification in TCGA datasets with ARE database (ARED) identified ERBB2-associated gene cluster. Many of these genes were over-expressed in the ERBB2-positive SKBR3 cells compared to MCF10A normal-like cells, and were under-expressed due to ERBB2 siRNA treatment. Lapatinib accelerated the ARE-mRNA decay for several ERBB2-regulated genes. The ERBB2 inhibitor decreased both the abundance and stability of the phosphorylated inactive form of the mRNA decay-promoting protein, tristetraprolin (ZFP36/TTP). The ERBB2 siRNA was also able to reduce the phosphorylated ZFP36/TTP form. In contrast, ectopic expression of ERBB2 in MCF10A or HEK293 cells led to increased abundance of the phosphorylated ZFP36/TTP. The effect of ERBB2 on TTP phosphorylation appeared to be mediated via the MAPK-MK2 pathway. Screening for the impact of other amplified cancer genes in HEK293 cells also demonstrated that EGFR, AKT2, CCND1, CCNE1, SKP2, and FGFR3 caused both increased abundance of phosphorylated ZFP36/TTP and ARE-post-transcriptional reporter activity. Thus, specific amplified oncogenes dysregulate post-transcriptional ARE-mediated effects, and targeting the ARE-mediated pathway itself may provide alternative therapeutic approaches.

ß-Catenin Attenuation Inhibits Tumor Growth and Promotes Differentiation in a BRAFV600E-Driven Thyroid Cancer Animal Model.

BRAFV600E mutation is the most frequent genetic alteration in papillary thyroid cancer (PTC). ß-Catenin (Ctnnb1) is a key downstream component of canonical Wnt signaling pathway and is frequently overexpressed in PTC. BRAF V600E-driven tumors have been speculated to rely on Wnt/ß-catenin signaling to sustain its growth, although many details remain to be elucidated. In this study, we investigated the role of ß-catenin in BrafV600E -driven thyroid cancer in a transgenic mouse model. In Braf V600E mice with wild-type (WT) Ctnnb1 (BVE-Ctnnb1WT or BVE), overexpression of ß-catenin was observed in thyroid tumors. In Braf V600E mice with Ctnnb1 knockout (BVE-Ctnnb1null), thyroid tumor growth was slowed with significant reduction in papillary architecture. This was associated with increased expression of genes involved in thyroid hormone synthesis, elevated 124iodine uptake, and serum T4. The survival of BVE-Ctnnb1null mice was increased by more than 50% during 14-month observation. Mechanistically, downregulation of MAPK, PI3K/Akt, and TGFß pathways and loss of epithelial-mesenchymal transition (EMT) were demonstrated in the BVE-Ctnnb1null tumors. Treatment with dual ß-catenin/KDM4A inhibitor PKF118-310 dramatically improved the sensitivity of BVE-Ctnnb1WT tumor cells to BRAFV600E inhibitor PLX4720, resulting in significant growth arrest and apoptosis in vitro, and tumor regression and differentiation in vivo These findings indicate that ß-catenin signaling plays an important role in thyroid cancer growth and resistance to BRAFV600E inhibitors. Simultaneously targeting both Wnt/ß-catenin and MAPK signaling pathways may achieve better therapeutic outcome in BRAFV600E inhibitor-resistant and/or radioiodine-refractory thyroid cancer.

Kinome inhibition reveals a role for polo-like kinase 1 in targeting post-transcriptional control in cancer.

Dysfunctions in post-transcriptional control are observed in cancer and chronic inflammatory diseases. Here, we employed a kinome inhibitor library (n = 378) in a reporter system selective for 3'-untranslated region-AU-rich elements (ARE). Fifteen inhibitors reduced the ARE-reporter activity; among the targets is the polo-like kinase 1 (PLK1). RNA-seq experiments demonstrated that the PLK1 inhibitor, volasertib, reduces the expression of cytokine and cell growth ARE mRNAs. PLK1 inhibition caused accelerated mRNA decay in cancer cells and was associated with reduced phosphorylation and stability of the mRNA decay-promoting protein, tristetraprolin (ZFP36/TTP). Ectopic expression of PLK1 increased abundance and stability of high molecular weight of ZFP36/TTP likely of the phosphorylated form. PLK1 effect was associated with the MAPK-MK2 pathway, a major regulator of ARE-mRNA stability, as evident from MK2 inhibition, in vitro phosphorylation, and knockout experiments. Mutational analysis demonstrates that TTP serine 186 is a target for PLK1 effect. Treatment of mice with the PLK1 inhibitor reduced both ZFP36/TTP phosphorylation in xenograft tumor tissues, and the tumor size. In cancer patients' tissues, PLK1/ARE-regulated gene cluster was overexpressed in solid tumors and associated with poor survival. The data showed that PLK1-mediated post-transcriptional aberration could be a therapeutic target.

Bi-phased regulation of the post-transcriptional inflammatory response by Tristetraprolin levels.

AU-rich elements (AREs) are cis-acting instability and translation inhibition elements that are present in the 3'UTR of most inducible inflammatory mRNAs such as TNF and Cxcl2. mRNAs that contain AREs are, by default, repressed and only transiently expressed in response to stimuli. They are targeted by the inducible RNA-binding protein Tristetraprolin (TTP) which blocks their translation and facilitates their decay, thereby contributing to the quick termination of their expression. The exogenous over-expression of TTP in HEK293 cells can unexpectedly lead to the upregulation and extended expression of a nanoLuciferase reporter that contains the ARE of TNF. Here we show that, a moderate downregulation of the highly expressed endogenous TTP after LPS induction by siRNA in macrophages can lead to a reduction in the release of TNF and Cxcl2. We propose that, in contrast to their canonical function, very high levels of induced TTP at the onset of the inflammatory response can enhance the expression of ARE-mRNAs at the post-transcriptional level, independently of phosphorylation status. As the inflammatory response progresses, TTP levels diminish but they continuously regain their ability to reduce the expression of ARE-mRNAs to reach a turning point of 'optimal TTP level' with a maximum ability to repress ARE-mRNA expression. Below this level, a further reduction in TTP levels now leads to the loss of canonical-TTP function resulting in increased ARE-mRNA expression. These novel findings should contribute to the understanding of feedback loops that control the kinetics of the inflammatory response.

Post-Transcriptional Inflammatory Response to Intracellular Bacterial c-di-AMP.

Cyclic-di-AMP (c-di-AMP) is a bacterial second messenger that is produced by intracellular bacterial pathogens in mammalian host macrophages. Previous reports have shown that c-di-AMP is recognized by intracellular pattern recognition receptors of the innate immune system and stimulate type I interferon response. Here we report that the response to c-di-AMP includes a post-transcriptional component that is involved in the induction of additional inflammatory cytokines including IL-6, CXCL2, CCL3, and CCL4. Their mRNAs contain AU-rich elements (AREs) in their 3' UTR that promote decay and repress translation. We show that c-di-AMP leads to the phosphorylation of p38 MAPK as well as the induction of the ARE-binding protein TTP, both of which are components of a signaling pathway that modulate the expression of ARE-containing mRNAs at the post-transcriptional level. Pharmacological inhibition of p38 reduces the c-di-AMP-dependent release of induced cytokines, while TTP knockdown increases their release and mRNA stability. C-di-AMP can specifically increase the expression of a nano-Luciferase reporter that contains AREs. We propose a non-canonical intracellular mode of activation of the p38 MAPK pathway with the subsequent enhancement in the expression of inflammatory cytokines. C-di-AMP is widely distributed in bacteria, including infectious intracellular pathogens; hence, understanding of its post-transcriptional gene regulatory effect on the host response may provide novel approaches for therapy.

The AU-rich element landscape across human transcriptome reveals a large proportion in introns and regulation by ELAVL1/HuR.

Adenylate-uridylate (AU)-rich elements (AREs) are sequence instability elements that are known to be located in the 3' untranslated regions (UTR) in thousands of human transcripts. AREs regulate the expression of many genes at the post-transcriptional level, and they are essential for many normal cellular functions. We conducted a transcriptome-wide screen for AREs and found that they are most abundant in introns, with up to 25% of introns containing AREs corresponding to 58% of human genes. Clustering studies of ARE size, complexity, and distribution revealed that, in introns, longer AREs with two or more overlapping repeats are more abundant than in the 3'UTR, and only introns can contain very long AREs with 6-14 overlapping AUUUA pentamers. We found that intronic sites of the ARE binding proteins HuR/ELAVL1, ZFP36/TTP, AUF1, and BRF1/ZFP36L1 overlap with the intronic AREs with HuR being most abundant. Accordingly, RNA-IP experiments demonstrated a specific association of HuR with reporter and endogenous pre-mRNAs that contain intronic AREs. Moreover, HuR knockdown led to a significant general reduction in the mRNA levels of genes that contain intronic AREs and to a specific reduction in the expression of ARE-intronic reporters. The data represent bioinformatics analysis for key RNA-binding proteins interactions with intronic AREs and provide experimental evidence for HuR binding to AREs. The widespread distribution of intronic AREs and their particular association with HuR and HuR binding sites indicates that more than half of human genes can be regulated post-transcriptionally by AREs.

The intracellular pyrimidine 5'-nucleotidase NT5C3A is a negative epigenetic factor in interferon and cytokine signaling.

The enzyme pyrimidine 5'-nucleotidase (NT5C3A), which mediates nucleotide catabolism, was previously thought to be restricted to blood cells. We showed that expression of the gene encoding NT5C3A was induced by type I interferons (IFNs) in multiple cell types and that NT5C3A suppressed cytokine production through inhibition of the nuclear factor κB (NF-κB) pathway. NT5C3A expression required both an intronic IFN-stimulated response element and the IFN-stimulated transcription factor IRF1. Overexpression of NT5C3A, but not of its catalytic mutants, suppressed IL-8 production by HEK293 cells. Whereas knockdown of NT5C3A enhanced tumor necrosis factor (TNF)-stimulated IL-8 production, it reduced the IFN-mediated suppression of Il8 expression. Overexpression of NT5C3A increased the abundance of NAD+ and the activation of the sirtuins SIRT1 and SIRT6, which are NAD+-dependent deacetylases. NT5C3A-stimulated sirtuin activity resulted in deacetylation of histone H3 and the NF-κB subunit RelA (also known as p65), both of which were associated with the proximal region of the Il8 promoter, thus repressing the transcription of Il8 Together, these data identify an anti-inflammatory pathway that depends on the catalytic activity of NT5C3A and functions as a negative feedback regulator of inflammatory cytokine signaling.

ARED-Plus: an updated and expanded database of AU-rich element-containing mRNAs and pre-mRNAs.

Here we present an updated version of the AU-Rich Element Database (ARED-Plus) that is freely available at http://brp.kfshrc.edu.sa/ared. AREs are conserved sequence elements that were first discovered in the 3'UTR of mammalian transcripts. Over the past years, we compiled a series of ARE databases that revealed the extent and wide distribution of ARE-containing genes. For this update, we adopted an optimized search algorithm with improved specificity and sensitivity in ARE selection. The designation of the different ARE clusters was simplified by directly correlating the number of the ARE cluster to the number of overlapping AUUUA pentamers. Additionally, the new database was expanded to include genes with intronic AREs (pre-mRNAs) and their characteristics since recent observations reported their abundance and biological significance. Several enhancements were incorporated such as customized column view, additional search options and live search functionalities. The new version includes links to AREsite and AREScore, two related ARE assessment algorithms for further evaluation of the ARE characteristics. ARED-Plus now contains an updated repertoire of AREs in the human transcriptome that may be useful in several research fields.

Identification of a novel genetic locus underlying tremor and dystonia.

BACKGROUND: Five affected individuals with syndromic tremulous dystonia, spasticity, and white matter disease from a consanguineous extended family covering a period of over 24 years are presented. A positional cloning approach utilizing genome-wide linkage, homozygozity mapping and whole exome sequencing was used for genetic characterization. The impact of a calmodulin-binding transcription activator 2, (CAMTA2) isoform 2, hypomorphic mutation on mRNA and protein abundance was studied using fluorescent reporter expression cassettes. Human brain sub-region cDNA libraries were used to study the expression pattern of CAMTA2 transcript variants. RESULTS: Linkage analysis and homozygozity mapping localized the disease allele to a 2.1 Mb interval on chromosome 17 with a LOD score of 4.58. Whole exome sequencing identified a G>A change in the transcript variant 2 5'UTR of CAMTA2 that was only 6 bases upstream of the translation start site (c.-6G > A) (NM_001171166.1) and segregated with disease in an autosomal recessive manner. Transfection of wild type and mutant 5'UTR-linked fluorescent reporters showed no impact upon mRNA levels but a significant reduction in the protein fluorescent activity implying translation inhibition. CONCLUSIONS: Mutation of CAMTA2 resulting in post-transcriptional inhibition of its own gene activity likely underlies a novel syndromic tremulous dystonia.

A novel mechanism for variable phenotypic expressivity in Mendelian diseases uncovered by an AU-rich element (ARE)-creating mutation.

BACKGROUND: Variable expressivity is a well-known phenomenon in which patients with mutations in one gene display varying degrees of clinical severity, potentially displaying only subsets of the clinical manifestations associated with the multisystem disorder linked to the gene. This remains an incompletely understood phenomenon with proposed mechanisms ranging from allele-specific to stochastic. RESULTS: We report three consanguineous families in which an isolated ocular phenotype is linked to a novel 3' UTR mutation in SLC4A4, a gene known to be mutated in a syndromic form of intellectual disability with renal and ocular involvement. Although SLC4A4 is normally devoid of AU-rich elements (AREs), a 3' UTR motif that mediates post-transcriptional control of a subset of genes, the mutation we describe creates a functional ARE. We observe a marked reduction in the transcript level of SLC4A4 in patient cells. Experimental confirmation of the ARE-creating mutation is shown using a post-transcriptional reporter system that reveals consistent reduction in the mRNA-half life and reporter activity. Moreover, the neo-ARE binds and responds to the zinc finger protein ZFP36/TTP, an ARE-mRNA decay-promoting protein. CONCLUSIONS: This novel mutational mechanism for a Mendelian disease expands the potential mechanisms that underlie variable phenotypic expressivity in humans to also include 3' UTR mutations with tissue-specific pathology.

Hallmarks of cancer and AU-rich elements.

Post-transcriptional control of gene expression is aberrant in cancer cells. Sustained stabilization and enhanced translation of specific mRNAs are features of tumor cells. AU-rich elements (AREs), cis-acting mRNA decay determinants, play a major role in the posttranscriptional regulation of many genes involved in cancer processes. This review discusses the role of aberrant ARE-mediated posttranscriptional processes in each of the hallmarks of cancer, including sustained cellular growth, resistance to apoptosis, angiogenesis, invasion, and metastasis. WIREs RNA 2017, 8:e1368. doi: 10.1002/wrna.1368 For further resources related to this article, please visit the WIREs website.

Systematic Analysis of AU-Rich Element Expression in Cancer Reveals Common Functional Clusters Regulated by Key RNA-Binding Proteins.

Defects in AU-rich elements (ARE)-mediated posttranscriptional control can lead to several abnormal processes that underlie carcinogenesis. Here, we performed a systematic analysis of ARE-mRNA expression across multiple cancer types. First, the ARE database (ARED) was intersected with The Cancer Genome Atlas databases and others. A large set of ARE-mRNAs was over-represented in cancer and, unlike non-ARE-mRNAs, correlated with the reversed balance in the expression of the RNA-binding proteins tristetraprolin (TTP, ZFP36) and HuR (ELAVL1). Serial statistical and functional enrichment clustering identified a cluster of 11 overexpressed ARE-mRNAs (CDC6, KIF11, PRC1, NEK2, NCAPG, CENPA, NUF2, KIF18A, CENPE, PBK, TOP2A) that negatively correlated with TTP/HuR mRNA ratios and was involved in the mitotic cell cycle. This cluster was upregulated in a number of solid cancers. Experimentally, we demonstrated that the ARE-mRNA cluster is upregulated in a number of tumor breast cell lines when compared with noninvasive and normal-like breast cancer cells. RNA-IP demonstrated the association of the ARE-mRNAs with TTP and HuR. Experimental modulation of TTP or HuR expression led to changes in the mitosis ARE-mRNAs. Posttranscriptional reporter assays confirmed the functionality of AREs. Moreover, TTP augmented mitotic cell-cycle arrest as demonstrated by flow cytometry and histone H3 phosphorylation. We found that poor breast cancer patient survival was significantly associated with low TTP/HuR mRNA ratios and correlated with high levels of the mitotic ARE-mRNA signature. These results significantly broaden the role of AREs and their binding proteins in cancer, and demonstrate that TTP induces an antimitotic pathway that is diminished in cancer. Cancer Res; 76(14); 4068-80. ©2016 AACR.

Human Cytokinome Analysis for Interferon Response.

UNLABELLED: Cytokines are a group of small secreted proteins that mediate a diverse range of immune and nonimmune responses to inflammatory and microbial stimuli. Only a few of these cytokines mount an antiviral response, including type I, II, and III interferons (IFNs). During viral infections and under inflammatory conditions, a number of cytokines and chemokines are coproduced with IFN; however, no systematic study exists on the interactions of the cytokine repertoire with the IFN response. Here, we performed the largest cytokine and chemokine screen (the human cytokinome, with >240 members) to investigate their modulation of type I and type II IFN responses in a cell line model. We evaluated the cytokine activities in both IFN-stimulated response element (ISRE) and IFN-γ activation sequence (GAS) reporter systems. Several cytokine clusters that augment either or both ISRE- and GAS-mediated responses to IFNs were derived from the screen. We identified novel modulators of IFN response-betacellulin (BTC), interleukin 11 (IL-11), and IL-17F-that caused time-dependent induction of the IFN response. The ability to induce endogenous IFN-ß and IFN-stimulated genes varies among these cytokines and was largely dependent on Stat1, as assessed by Stat1 mutant fibroblasts. Certain cytokines appear to augment the IFN-ß response through the NF-κB pathway. The novel IFN-like cytokines augmented the antiviral activity of IFN-α against several RNA viruses, including encephalomyocarditis virus, vesicular stomatitis virus, and influenza virus, in susceptible cell lines. Overall, the study represents a large-scale analysis of cytokines for enhancing the IFN response and identified cytokines capable of enhancing Stat1, IFN-induced gene expression, and antiviral activities. IMPORTANCE: Innate immunity to viruses is an early defense system to ward off viruses. One mediator is interferon (IFN), which activates a cascade of biochemical events that aim to control the virus life cycle. In our work, we examined more than 200 cytokines, soluble mediators produced within the body as a result of infection, for the ability to enhance IFN action. We identified enhanced interactions with specific IFNs and cytokines. We also revealed that betacellulin, IL-17, and IL-11 cytokines have the novel property of enhancing the antiviral action of IFN against several viruses. These results demonstrate that the human genome codes for previously unknown proteins with unrelated functions that can augment the innate immunity to viruses. Knowing these interactions not only helps our understanding of immunity to viruses and emerging diseases, but can also lead to devising possible new therapeutics by enhancing the mediator of antiviral action itself, IFN.

Posttranscriptional control of the chemokine receptor CXCR4 expression in cancer cells.

CXCR4 is a chemokine receptor that is overexpressed in certain cancer types and involved in migration toward distant organs. The molecular mechanisms underlying CXCR4 expression in invasive cancer, particularly posttranscriptional regulation, are poorly understood. Here, we find that CXCR4 harbors AU-rich elements (AREs) in the 3'-untranslated region (3'-UTR) that bind and respond to the RNA-binding proteins, tristetraprolin (TTP/ZFP36) and HuR (ELAVL1). Different experimental approaches, including RNA immunoprecipitation, 3'-UTR reporter, RNA shift and messenger RNA (mRNA) half-life studies confirmed functionality of the CXCR4 ARE. Wild-type TTP, but not the zinc finger mutant, C124R, was able to bind CXCR4 mRNA and ARE. In the invasive breast cancer phenotype, aberrant expression of CXCR4 is linked to both TTP deficiency and HuR overexpression. HuR silencing led to decreased CXCR4 mRNA stability and expression, and significant reduction in migration of the cells toward the CXCR4 ligand, CXCL12. Derepression of TTP using miR-29a inhibitor led to significant reduction in CXCR4 mRNA stability, expression and migration capability of the cells. The study shows that CXCR4 is regulated by ARE-dependent posttranscriptional mechanisms that involve TTP and HuR, and that aberration in this pathway helps cancer cells migrate toward the CXCR4 ligand. Targeting posttranscriptional control of CXCR4 expression may constitute an alternative approach in cancer therapy.

Sustained stabilization of Interleukin-8 mRNA in human macrophages.

The mRNAs of most inflammatory mediators are short-lived due to AU-rich elements (AREs) in their 3'-untranslated regions. AREs ensure a low basal level of expression during homeostasis and a transient nature of expression during the inflammatory response. Here, we report that the mRNA of the pro-inflammatory chemokine IL-8, which contains an archetypal ARE, is unexpectedly constitutively abundant and highly stable in primary human monocytes and macrophages. Using the pre-monocyte-like THP-1 cell line that can differentiate into macrophage-like cells, we show that a low level of unstable IL-8 mRNA in undifferentiated cells (half-life<30 min) becomes constitutively elevated and the mRNA is dramatically stabilized in differentiated THP-1 cells with a half-life of more than 15 h similar to primary monocytes and macrophages. In contrast, the level and stability of TNF-α mRNA also containing an ARE is only slightly affected by differentiation; it remains low and unstable in primary macrophages and differentiated THP-1 cells with an estimated half-life of less than 20 min. This differentiation-dependent stabilization of IL-8 mRNA is p38 MAPK-independent and is probably coupled with reduced protein translation. Reporter assays in THP-1 cells suggest that the ARE alone is not sufficient for the constitutive stabilization in macrophage-like cells and imply an effect of the natural biogenesis of the transcript on the stabilization of the mature form. We present a novel, cell type-dependent sustained stabilization of an ARE-containing mRNA with similarities to situations found in disease.