Latifa Al-Haj

Alternative polyadenylation variants of the RNA binding protein, HuR: abundance, role of AU-rich elements and auto-Regulation

The RNA-binding protein, HuR, is involved in the stabilization of AU-rich element-containing mRNAs with products that are involved in cell-cycle progression, cell differentiation and inflammation. We show that there are multiple polyadenylation variants of HuR mRNA that differ in their abundance, using both bioinformatics and experimental approaches. A polyadenylation variant with distal poly(A) signal is a rare transcript that harbors functional AU-rich elements (ARE) in the 3′UTR. A minimal 60-nt region, but not a mutant form, fused to reporter-3′UTR constructs was able to downregulate the reporter activity. The most predominant and alternatively polyadenylated mature transcript does not contain the ARE. HuR itself binds HuR mRNA, and upregulated the activity of reporter from constructs fused with ARE-isoform and the HuR ARE. Wild-type tristetraprolin (TTP), but not the zinc finger mutant TTP, competes for HuR binding and upregulation of HuR mRNA. The study shows that the HuR gene codes for several polyadenylation variants differentially regulated by AU-rich elements, and demonstrates an auto-regulatory role of HuR.

Identification of a set of KSRP target transcripts upregulated by PI3K-AKT signaling

BackgroundKSRP is a AU-rich element (ARE) binding protein that causes decay of select sets of transcripts in different cell types. We have recently described that phosphatidylinositol 3-kinase/AKT (PI3K-AKT) activation induces stabilization and accumulation of the labile β-catenin mRNA through an impairment of KSRP function.ResultsAim of this study was to identify additional KSRP targets whose stability and steady-state levels are enhanced by PI3K-AKT activation. First, through microarray analyses of the AU-rich transcriptome in pituitary αT3-1 cells, we identified 34 ARE-containing transcripts upregulated in cells expressing a constitutively active form of AKT1. In parallel, by an affinity chromatography-based technique followed by microarray analyses, 12 mRNAs target of KSRP, additional to β-catenin, were identified. Among them, seven mRNAs were upregulated in cells expressing activated AKT1. Both steady-state levels and stability of these new KSRP targets were consistently increased by either KSRP knock-down or PI3K-AKT activation.ConclusionOur study identified a set of transcripts that are targets of KSRP and whose expression is increased by PI3K-AKT activation. These mRNAs encode RNA binding proteins, signaling molecules and a replication-independent histone. The increased expression of these gene products upon PI3K-AKT activation could play a role in the cellular events initiated by this signaling pathway.

Regulation of p21/CIP1/WAF-1 mediated cell-cycle arrest by RNase L and tristetraprolin, and involvement of AU-rich elements

The p21Cip1/WAF1 plays an important role in cell-cycle arrest. Here, we find that RNase L regulates p21-mediated G1 growth arrest in AU-rich elements-dependent manner. We found a significant loss of p21 mRNA expression in RNASEL−/− MEFs and that the overexpression of RNase L in HeLa cells induces p21 mRNA expression. The p21 mRNA half-life significantly changes as a result of RNase L modulation, indicating a post-transcriptional effect. Indeed, we found that RNase L promotes tristetraprolin (TTP/ZFP36) mRNA decay. This activity was not seen with dimerization- and nuclease-deficient RNase L mutants. Deficiency in TTP led to increases in p21 mRNA and protein. With induced ablation of RNase L, TTP mRNA and protein expressions were higher, while p21 expression became reduced. We further establish that TTP, but not C124R TTP mutant, binds to, and accelerates the decay of p21 mRNA. The p21 mRNA half-life was prolonged in TTP−/− MEFs. The TTP regulation of p21 mRNA decay required functional AU-rich elements. Thus, we demonstrate a novel mechanism of regulating G1 growth arrest by an RNase L-TTP-p21 axis.

RNase L downmodulation of the RNA-binding protein, HuR, and cellular growth

Ribonuclease L (RNase L) is an intracellular enzyme that is vital in innate immunity, but also is a tumor suppressor candidate. Here, we show that overexpression of RNase L decreases cellular growth and downmodulates the RNA-binding protein, HuR, a regulator of cell-cycle progression and tumorigenesis. The effect is temporal, occurring in specific cell-cycle phases and correlated with the cytoplasmic localization of RNase L. Both cellular growth and HuR were increased in RNASEL-null mouse fibroblast lines when compared to wild-type cells. Moreover, the stability of HuR mRNA was enhanced in RNASEL-null cells. The HuR 3′ untranslated region (UTR), which harbors U-rich and adenylate-uridylate-rich elements, was potently responsive to RNase L when compared to control 3′ UTR. Our results may offer a new explanation to the tumor suppressor function of RNase L.

Cloning-free regulated monitoring of reporter and gene expression

BackgroundThe majority of the promoters, their regulatory elements, and their variations in the human genome remain unknown. Reporter gene technology for transcriptional activity is a widely used tool for the study of promoter structure, gene regulation, and signaling pathways. Construction of transcriptional reporter vectors, including use of cis-acting sequences, requires cloning and time-demanding manipulations, particularly with introduced mutations.ResultsIn this report, we describe a cloning-free strategy to generate transcriptionally-controllable linear reporter constructs. This approach was applied in common transcriptional models of inflammatory response and the interferon system. In addition, it was used to delineate minimal transcriptional activity of selected ribosomal protein promoters. The approach was tested for conversion of genes into TetO-inducible/repressible expression cassettes.ConclusionThe simple introduction and tuning of any transcriptional control in the linear DNA product renders promoter activation and regulated gene studies simple and versatile.

Cloning of cytokine 3' untranslated regions and posttranscriptional assessment using cell-based GFP assay.

Cytokine biosynthesis is tightly regulated by a number of processes, including gene expression control. Posttranscriptional control of cytokine gene expression offers a fine-tuning mechanism that contributes not only to transient biosynthesis of cytokines, but also helps in rapid and early initiation of the cytokine response. Deregulation of cytokine biosynthesis has been associated with a number of disease conditions, including autoimmune diseases, cancer, and others. Thus, there is a need for accurate measurement of posttranscriptional gene expression events in cytokine research. The method described here is a cell-based GFP assay that quantitatively measures posttranscriptional effects. This method is used for assessing the effects of modulators and conditions that lead to changes in posttranscriptional gene expression during cytokine production or for assessment of cytokine action on posttranscriptional events of gene expression.

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

An enzyme involved in nucleotide catabolism reduces inflammation through chromatin-mediated suppression of the expression of NF-κB target genes. Suppressing inflammation Inflammatory cytokines and chemokines, such as tumor necrosis factor (TNF) and interleukin-8 (IL-8), are important for effective immune responses; however, feedback mechanisms that inhibit the production of such factors are critical to prevent tissue damage. Al-Haj and Khabar found that the gene encoding the nucleotidase NT5C3A was induced in a type I interferon (IFN)–dependent manner in many cell types. Whereas knockdown of NT5C3A enhanced inflammatory cytokine production, overexpression of the enzyme enhanced the activity of the histone deacetylases SIRT1 and SIRT6, which in turn acted on histone proteins to reduce the expression of Il8, an NF-κB target gene. Together, these data suggest that NT5C3A mediates feedback inhibition of proinflammatory cytokine production by acting epigenetically to block NF-κB signaling output. 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.

145: The erthyrocyte cytosolic 5′-nucleotidase III is a broad tissue IFN-stimulated gene that limits NF-κB-mediated inflammatory response

Type-I interferon (IFN) has been shown to inhibit inflammatory processes such as NF- κ B activation and cytokine production through largely unknown mechanisms. We have found that pyrimidine 5′-nucleotidase (PN-I or NT5C3), previously designated as erythrocyte-specific gene product, is stimulated by IFN in a number of cell types, including normal, transformed, epithelial and fibroblast cells. Type-I IFN but not IFN- γ exerts dose-dependent stimulation of NT5C3 mRNA and protein. This activation requires Stat1/tyk2 factors and ISRE binding. NT5C3 was also induced by a number of viruses and pro-inflammatory cytokines. Ectopic over-expression of NT5C3 in HEK293 cells significantly reduced TNF- α -mediated NF- κ B reporter activity. IFN- α -mediated suppression of the chemokine IL-8 mRNA and protein expression in the IFN-sensitive HeLa cells is more pronounced with NT5C3 overexpression. Down-modulation of NT5C3 in HEK293 cells significantly increased both basal and TNF- α -induced NF- κ B reporter activation. In order to explore further mechanisms, we investigated upstream signaling components, and showed that NT5C3 reduced IKK β -mediated activation of NF- κ B reporter.- IKK β is a kinase required to phosphorylate and degrade NF-kB inhibitor (IK β ). Results were further confirmed with NT5C3 silencing showing enhancement of NF- κ B reporter activity in cells overexpressing wild-type IKK β . These activities were not seen in the case of IKK β (S177E) mutant experiments suggesting that NT5C3 may interfere with IKK β related activity such as its phosphorylation. Indeed, we found that NT5C3 could reduce phosphorylated levels of IKK β during TNF- α activation. Global gene expression profiling.-and further QPCR/WB confirmatory tests.- indicated that serine/threonine protein phosphatase (PPP2R1A), which dephosphorylates IKK β expression, was enhanced in NT5C3-expressing cells. IFN-mediated suppression of IL-8 expression is diminished in both NT5C3- or PPP2R1A-silenced cells. Overall, these results suggest that NT5C3 is broad ISG involved in dampening inflammation through changes in PPP2R1A/IKK β /NF- κ B network with reduction in pro-inflammatory cytokines highlighting a novel mode for the anti-inflammatory action of Type-I IFN.