The proximal region of the 3'-untranslated region of cyclooxygenase-2 is recognized by a multimeric protein complex containing HuR,TIA-1, TIAR,and the heterogeneous nuclear ribonucleoprotein U

Cyclooxygenase-2 (COX-2) is an early response gene induced in renal mesangial cells by interleukin-1beta (IL-1beta). The 3'-untranslated region (3'-UTR) of COX-2 mRNA plays an important role in IL-1beta induction by regulating message stability and translational efficiency. The first 60 nucleotides of the 3'-UTR of COX-2 are highly conserved and contain multiple copies of the regulatory sequence AUUUA. Introduction of the 60-nucleotide sequence into the 3'-UTR of a heterologous reporter gene resulted in a 70% decrease in reporter gene expression. Electrophoretic mobility shift assays (EMSAs) demonstrated that mesangial cell nuclear fractions contain a multimeric protein complex that bound this region of COX-2 mRNA in a sequence-specific manner. We identified four members of the protein-RNA complex as HuR, TIA-1, TIAR, and the heterogeneous nuclear ribonucleoprotein U (hnRNP U). Treatment of mesangial cells with IL-1beta caused an increase in cytosolic HuR, which was accompanied by an increase in COX-2 mRNA that co-immunoprecipitated with cytosolic HuR. Therefore, we propose that HuR binds to the proximal region of the 3'-UTR of COX-2 following stimulation by IL-1beta and increases the expression of COX-2 mRNA by facilitating its transport out of the nucleus.     

用于鉴定microRNA靶基因的新型双萤光素酶报告基因系统的构建及应用

目的:构建用于鉴定microRNA靶基因的报告基因系统。方法:在pGL3-Basic载体的luc基因上游插入CMV启动子,下游插入用于克隆靶基因3’UTR的多克隆位点,构建报告基因载体pMIR-luciferase;将pMIR-lu-ciferase载体的luc基因替换成Rluc基因,构建内参载体pMIR-control;将补体因子H(CFH)的3’UTR插入pMIR-luciferase载体的多克隆位点处,构建含有CFH 3’UTR的报告基因载体;用pIRES2-EGFP载体构建microRNA146a真核表达载体;将含有CFH 3’UTR的报告基因载体、microRNA146a真核表达载体及内参载体共转染HepG2细胞,进行报告基因的活性检测。结果:构建了报告基因载体、内参载体和microRNA146a真核表达载体,经酶切和测序鉴定正确;microRNA146a真核表达载体转染细胞72 h后,经荧光显微镜观察确认载体转染及表达;用实时定量PCR检测,microRNA146a的表达水平显著上调(P<0.01);用构建的报告基因系统检测,结果表明microRNA146a显著地抑制了含CFH 3’UTR的报告基因的活性(P<0.05)。结论:构建了一种新型的报告基因载体系统,该系统可用于miRNA靶基因的鉴定。     

Transforming growth factor-beta1 enhances Ha-ras-induced expression of cyclooxygenase-2 in intestinal epithelial cells via stabilization of mRNA

Oncogenic ras induces the expression of cyclooxygenase-2 (COX-2) in a variety of cells. Here we investigated the role of transforming growth factor-beta (TGF-beta) in the Ras-mediated induction of COX-2 in intestinal epithelial cells (RIE-1). RIE-1 cells were transfected with an inducible Ha-Ras(Val12) cDNA and are referred as RIE-iRas cells. the addition of 5 mM isopropyl-1-thio-beta-D-galactopyranoside (IPTG) induced the expression of Ha-Ras(Val12), closely followed by an increase in the expression of COX-2. Neutralizing anti-TGF-beta antibody partially blocked the Ras-induced increase in COX-2. Combined treatment with IPTG and TGF-beta1 resulted in a 20-50-fold increase in the levels of COX-2 mRNA. The t1/2 of COX-2 mRNA was increased from 13 to 24 min by Ha-Ras induction alone. The addition of TGF-beta1 further stabilized the COX-2 mRNA (t1/2 > 50 min). Stable transfection of a luciferase reporter construct containing the COX-2 3'-untranslated region (3'-UTR) revealed that TGF-beta1 treatment and Ras induction each stabilized the COX-2 3'-UTR. Combined treatment with IPTG and TGF-beta1 synergistically increased the luciferase activity. Furthermore, a conserved AU-rich region located in the proximal COX-2 3'-UTR is required for maximal stabilization of COX-2 3'-UTR by Ras or TGF-beta1 and is necessary for the synergistic stabilization of COX-2 3'-UTR by oncogenic Ras and TGF-beta1.     

Post-transcriptional regulation in higher eukaryotes: the role of the reporter gene in controlling expression

We have investigated whether reporter genes influence cytoplasmic regulation of gene expression in tobacco and Chinese hamster ovary (CHO) cells. Two genes, uidA encoding beta-glucuronidase (GUS) from Escherichia coli and Luc, encoding firefly luciferase (LUC), were used to analyze the ability of a cap, polyadenylated tail, and the 5'- and 3'-untranslated regions (UTR) from tobacco mosaic virus (TMV) to regulate expression. The regulation associated with the 5' cap structure and the TMV 5'-UTR, both of which enhance translational efficiency, was reporter gene-independent. The poly(A) tail and the TMV 3'-UTR, which is functionally equivalent to a poly(A) tail, increase translational efficiency as well as mRNA stability. The regulation associated with these 3' ends was highly reporter gene-dependent; their effect on GUS expression was almost an order of magnitude greater than that on LUC expression. In tobacco, the tenfold reporter gene effect on poly(A) tail or TMV 3'-UTR function could not be explained by a differential impact on mRNA stability; GUS and LUC mRNA half-life increased only twofold when either the poly(A) tail or TMV 3'-UTR was present. In CHO cells, however, GUS mRNA was stabilized to a greater extent by a poly(A) tail or the TMV 3'-UTR than was LUC mRNA.     

Evolutionary conservation of an atypical glucocorticoid‐responsive element in the human tyrosine hydroxylase gene

The human tyrosine hydroxylase (hTH) gene has a 42 bp evolutionarily conserved region designated (CR) II at ?7.24 kb, which bears 93% homology to the region we earlier identified as containing the glucocorticoid response element, a 7 bp activator protein‐1 (AP‐1)‐like motif in the rat TH gene. We cloned this hTH‐CRII region upstream of minimal basal hTH promoter in luciferase (Luc) reporter vector, and tested glucocorticoid responsiveness in human cell lines. Dexamethasone (Dex) stimulated Luc activity of hTH‐CRII in HeLa cells, while mifepristone, a glucocorticoid receptor (GR) antagonist, prevented Dex stimulation. Deletion of the 7 bp 5′‐TGACTAA at ?7243 bp completely abolished the Dex‐stimulated Luc activity of hTH‐CRII construct. The AP‐1 agonist, tetradeconoyl‐12,13‐phorbol acetate (TPA), also stimulated hTH promoter activity, and Dex and TPA together further accentuated this response. Chromatin immunoprecipitation assays revealed the presence of both GR and AP‐1 proteins, especially Jun family members, at this hTH promoter site. Dex did not stimulate hTH promoter activity in a catecholaminergic cell line, which had low endogenous GR levels, but did activate the response when GR was expressed exogenously. Thus, our studies have clearly identified a glucocorticoid‐responsive element in a 7 bp AP‐1‐like motif in the promoter region at ?7.24 kb of the human TH gene.     

豌豆根瘤菌结瘤基因启动子内二级结构区与转录活性有关

用羟胺随机改变豌豆根瘤菌(Rhizobium leguminosarum)结瘤基因 nodD上游的调控区间的核苷酸顺序,将它们插入广谱转录质粒pMP220的LacZ基因前,启动LacZ基因的表达、突变表明与结瘤基因的转录活力有关.DNA序列分析发现,这些突变都在距nodD基因起始密码子约80bp的二级结构区内.     

3'-UTR polymorphism in the human CYP2A6 gene affects mRNA stability and enzyme expression

Cytochrome P450 2A6 (CYP2A6) is the major nicotine C-oxidase in human and participates in the metabolism of drugs and precarcinogens. The CYP2A6 gene is highly polymorphic and more than 22 different alleles have been described. We here focused on the polymorphism in the 3'-UTR region, in particular the common CYP2A6*1B allele, carrying an unequal crossover element from the pseudogene CYP2A7. Analysis of CYP2A6 expression in a human liver bank (n=46) revealed that the protein level and catalytic activity using coumarin as a substrate were all higher, following a linear gene-dose relationship, in livers carrying one or two copies of CYP2A6*1B, as compared to other CYP2A6 allelic variants. Different variants of the CYP2A6 3'-UTR were cloned into a modified pGL3 plasmid downstream of the luciferase reporter gene. The plasmids, having the proximal promoter of CYP2A6 gene, were transfected into HeLa cells or injected into the tail veins of male CD1 mice. In both systems, the 3'-UTR CYP2A6*1B constructs caused higher reporter gene activity and the CYP2A7 3'-UTR construct lower activity, compared to the CYP2A6*1 3'-UTR constructs. Two SNPs differentiating the 3'-UTR between CYP2A7 and CYP2A6*1B were found to be of importance for the expression in both systems. Analysis of reporter enzyme degradation in HeLa cells showed that luciferase-3'-UTR-CYP2A6*1A had a half-life of approximately 4.9h as compared to 6.3h for luciferase-3'-UTR-CYP2A6*1B. In conclusion, we identified polymorphic motifs in the CYP2A6 3'-UTR of importance for CYP2A6 mRNA stabilization and enzyme expression. Such polymorphism has been described to influence the in vivo rate of nicotine elimination and possibly the cigarette consumption and risk of smoking induced lung cancer.     

Differences in the translation efficiency and mRNA stability mediated by 5'-UTR splice variants of human SP-A1 and SP-A2 genes

Surfactant protein A (SP-A) plays an important role in host defense, modulation of inflammatory processes, and surfactant-related functions of the lung. The human SP-A (hSP-A) locus consists of two functional genes, SP-A1 and SP-A2. Several hSP-A 5'-untranslated region (UTR) splice variants for each gene have been characterized and shown to be translated in vitro and in vivo. In this report, we investigated the role of hSP-A 5'-UTR splice variants on SP-A production and molecular mechanisms involved. We used in vitro transient expression of hSP-A 5'-UTR constructs containing luciferase as the reporter gene and quantitative real-time PCR to study hSP-A 5'-UTR-mediated gene expression. We found that 1) the four (A'D', ABD, AB'D', and A'CD') 5'-UTR splice variants under study enhanced gene expression, by increasing luciferase activity from 2.5- to 19.5-fold and luciferase mRNA from 4.3- to 8.8-fold compared with the control vector that lacked hSP-A 5'-UTR; 2) all four 5'-UTR splice variants studied regulated mRNA stability. The ABD variant exhibited the lowest rate of mRNA decay compared with the other three constructs (A'D', AB'D', and A'CD'). These three constructs also exhibited significantly lower rate of mRNA decay compared with the control vector; 3) based on the indexes of translational efficiency (luciferase activity/mRNA), ABD and AB'D' exhibited higher translational efficiency compared with the control vector, whereas the translational efficiency of each A'D' and A'CD' was lower than that of the control vector. These findings indicate that the hSP-A 5'-UTR splice variants play an important role in both SP-A translation and mRNA stability.