HuR binds a cyclic nucleotide-dependent, stabilizing domain in the 3' untranslated region of Na(+)/glucose cotransporter (SGLT1) mRNA.

Differentiation-dependent expression of the Na(+)/glucose cotransporter (SGLT1) is accompanied by a large, cAMP-dependent increase in stability of its mRNA. Stabilization is mediated by protein binding to a critical uridine-rich element (URE) in its 3' untranslated region. In the present study, we demonstrate that HuR, an RNA binding protein of the embryonic lethal abnormal vision family, binds the SGLT1 URE. HuR binding was increased after elevation of intracellular cAMP levels and was dependent on protein phosphorylation. This interaction was prevented by a substitution mutation previously shown to block cAMP-dependent reporter message stabilization. These results implicate HuR as a key mediator of cAMP-dependent SGLT1 mRNA stabilization.     

Cloning and functional expression of a mammalian Na+/nucleoside cotransporter. A member of the SGLT family.

Complementary DNAs encoding seven different proteins related to the rabbit intestinal Na+/glucose cotransporter, SGLT1, were isolated from a rabbit renal cDNA library at relatively high stringency. The messages for RK-B to RK-F were single mRNA species at 2.3 kilobases (kb) in heart and kidney. The message for RK-A was 4 kb and was found in brain, lung, intestine, liver, and kidney. RK-I mRNA was approximately 3 kb and was found in all tissues tested. The most abundant clone, RK-C, constituted nucleotides 66-2150 of the sodium-nucleoside cotransporter, SNST1. The 672-amino acid protein encoded by SNST1 is 61% identical and 80% similar in sequence to SGLT1. Expression of SNST1c in Xenopus oocytes resulted in nucleoside-stimulated 22Na uptake and sodium-dependent [3H]uridine uptake. The uptake of [3H]uridine was inhibited by a range of nucleosides, including the anti-human immunodeficiency virus drug, dideoxycytidine. The results of this study show that there is a family of SGLT1-related proteins found in a wide variety of tissues and that one of these is a Na+/nucleoside cotransporter.     

Calcium modulates promoter occupancy by the Entamoeba histolytica Ca2+-binding transcription factor URE3-BP

The Entamoeba histolytica upstream regulatory element 3-binding protein (URE3-BP) binds to the URE3 sequence of the Gal/GalNAc-inhibitable lectin hgl5 and ferredoxin 1 (fdx) gene promoters. This binding can be inhibited in vitro by addition of calcium. Two EF-hand motifs, which are associated with the ability to bind calcium, are present in the amino acid sequence of URE3-BP. Mutation of the second EF-hand motif in URE3-BP resulted in the loss of calcium inhibition of DNA binding as monitored by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays revealed that URE3-BP was physically bound to the hgl5 and fdx promoters in vivo. Parasite intracellular calcium concentrations were altered by changes in extracellular calcium. Promoter occupancy was lost when intracellular calcium levels were increased by coordinate increases in extracellular calcium. Increased intracellular calcium also resulted in decreased levels of URE3-BP mRNA. Together these results demonstrate that changes in extracellular calcium result in changes in URE3-BP mRNA and in the ability of URE3-BP to bind to URE3-containing promoters. Modulation of URE3-BP by calcium may represent an important mechanism of control of gene expression in E. histolytica.     

A novel transforming growth factor-beta 1 responsive cytoplasmic trans-acting factor binds selectively to the 3'-untranslated region of mammalian ribonucleotide reductase R2 mRNA: role in message stability.

Ribonucleotide reductase is a highly regulated enzyme that provides the four deoxyribonucleotides required for DNA synthesis. Our studies showed that TGF-beta 1 treatment of BALB/c 3T3 mouse fibroblasts markedly elevated ribonucleotide reductase R2 mRNA levels, and also increased the half-life of R2 message by 4-fold from 1.5 h in untreated cells to 6 h in treated cells. We describe a novel 75 Kd sequence-specific cytoplasmic factor (p75) that binds selectively to a 83-nucleotide 3'-untranslated region of R2 mRNA and did not bind to the 5'UTR, the coding region of the R2 message or to the 3'UTRs of other mRNAs (from c-myc, GM-CSF and the iron responsive element from the transferrin receptor mRNA), or to the homopolymer poly(A) sequence. p75-RNA binding activity, which requires new protein synthesis, is not present in untreated cells, but is induced following TGF-beta 1 stimulation. The in vivo kinetics of appearance of p75 binding activity paralleled the accumulation of R2 mRNA. Insertion of the 3'-untranslated region into the chloramphenicol acetyltransferase (CAT) message confers TGF-beta 1 induced stability of RNA in stably transfected cells, while the same insert carrying a deletion of the 83-nucleotide fragment had little affect on RNA levels. Furthermore, in vitro decay reactions that contained the 83-nucleotide RNA or deletion of this fragment caused a significant decrease in TGF-beta 1 stabilization of R2 message. A model is presented of R2 message regulation in which TGF-beta 1 mediated stabilization of R2 message involves a specific interaction of a p75-trans-acting factor with a cis-element(s) stability determinant within the 83-nucleotide sequence which is linked to a reduction in the rate of R2 mRNA degradation.     

Functional mapping of destabilizing elements in the protein-coding region of the Drosophila fushi tarazu mRNA

The instability of the fushi tarazu (ftz) mRNA is essential for the proper development of the Drosophila embryo. Previously, we identified a 201-nucleotide instability element (FIE3) in the 3' untranslated region (UTR) of the ftz mRNA. Here we report on the identification of two additional elements in the protein-coding region of the message: the 63-nucleotide-long FIE5-1 and the 69-nucleotide-long FIE5-2. The function of both elements was position-dependent; the same elements destabilized RNAs when present within the coding region but did not when embedded in the 3' UTR of the hybrid mRNAs. We conclude that ftz mRNA has three redundant instability elements, two in the protein-coding region and one in the 3' UTR. Although each instability element is sufficient to destabilize a heterologous mRNA, the destabilizing activity of the two 5'-elements depended on their position within the message.     

Distinct domains of AU-rich elements exert different functions in mRNA destabilization and stabilization by p38 mitogen-activated protein kinase or HuR

AU-rich elements (AREs) control the expression of numerous genes by accelerating the decay of their mRNAs. Rapid decay and deadenylation of beta-globin mRNA containing AU-rich 3' untranslated regions of the chemoattractant cytokine interleukin-8 (IL-8) are strongly attenuated by activating the p38 mitogen-activated protein (MAP) kinase/MAP kinase-activated protein kinase 2 (MK2) pathway. Further evidence for a crucial role of the poly(A) tail is provided by the loss of destabilization and kinase-induced stabilization in ARE RNAs expressed as nonadenylated forms by introducing a histone stem-loop sequence. The minimal regulatory element in the IL-8 mRNA is located in a 60-nucleotide evolutionarily conserved sequence with a structurally and functionally bipartite character: a core domain with four AUUUA motifs and limited destabilizing function on its own and an auxiliary domain that markedly enhances destabilization exerted by the core domain and thus is essential for the rapid removal of RNA targets. A similar bipartite structure and function are observed for the granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE. Stabilization in response to p38/MK2 activation is seen with the core domain alone and also after mutation of the AUUUA motifs in the complete IL-8 ARE. Stabilization by ARE binding protein HuR requires different sequence elements. Binding but no stabilization is observed with the IL-8 ARE. Responsiveness to HuR is gained by exchanging the auxiliary domain of the IL-8 ARE with that of GM-CSF or with a domain of the c-fos ARE, which results in even stronger responsiveness. These results show that distinct ARE domains differ in function with regard to destabilization, stabilization by p38/MK2 activation, and stabilization by HuR.     

A 40-bp RNA element that mediates stabilization of vascular endothelial growth factor mRNA by HuR

VEGF is a critical mediator of hypoxia-induced angiogenesis in numerous physiological and pathophysiological conditions. The hypoxic induction of VEGF is due in large part to an increase in the stability of its mRNA. We recently demonstrated that the stabilization of VEGF mRNA by hypoxia is dependent upon the RNA-binding protein HuR. This report describes the identification of a 40-bp functional HuR binding site in the VEGF mRNA 3'-untranslated region. This element can confer HuR-mediated stabilization of a heterologous gene in vitro and in vivo. Furthermore, the element is sufficient to confer an increase in the hypoxic induction of a heterologous gene. Deletion of the HuR binding site within this 40-bp element as mapped by RNase T1 and lead footprinting uncouples a stabilizing sequence from a destabilizing sequence, thus providing a novel RNA-protein regulatory model that might be exploited to manipulate VEGF expression and hypoxia-induced angiogenesis.     

Molecular evidence for two renal Na+/glucose cotransporters.

Previous studies have shown that two kinetically and genetically distinct Na+/glucose cotransporters exist in mammalian kidney. We have recently cloned and sequenced one of the rabbit renal Na+/glucose cotransporters (SGLT1) and have found that it is identical in sequence to the intestinal Na+/glucose cotransporter. Northern blots showed that SGLT1 mRNA was found predominantly in the outer medulla of rabbit kidney. Injection of mRNA from outer medulla and outer cortex into Xenopus oocytes resulted in equal expression of Na(+)-dependent sugar uptake, indicating that the outer cortex sample contained mRNA encoding both SGLT1 and a second Na+/glucose cotransporter. Western blots using antipeptide antibodies against SGLT1 showed that the SGLT1 protein is more abundant in outer medulla than outer cortex. However, brush border membrane vesicles prepared from outer cortex had a greater capacity for Na(+)-dependent glucose transport, indicating the presence of a second transporter in the vesicles from outer cortex. It appears that the cloned renal Na+/glucose cotransporter, SGLT1, is the 'high affinity, low capacity' transporter found predominantly in outer medulla. There is evidence that a second transporter, the 'low affinity, high capacity' transporter, is in outer cortex. Finally, the cDNA and protein sequences of the two renal Na+/glucose cotransporters are predicted to differ by more than 20%.     

Differential regulation of three glucose transporter genes in a renal epithelial cell line

Three hexose transporter genes, the Na(+)/glucose cotransporters SGLT1 and SGLT3 (formerly SAAT1/pSGLT2) and the facilitative transporter GLUT1, are expressed in a renal epithelial cell line with proximal tubule characteristics. A number of studies have demonstrated that SGLT1 expression is coupled to the cellular differentiation state and is also negatively regulated by its substrate glucose. In the present study, we demonstrate that SGLT3 mRNA expression is relatively unaffected by conditions promoting dedifferentiation (reseeding to a subconfluent density, activation of protein kinase C) or differentiation (confluent cell density, activation of protein kinase A) nor was expression sensitive to hyperglycemic glucose levels in the medium. We further demonstrate that protein kinase A and protein kinase C exert opposing effects on GLUT1 and SGLT1 mRNA levels in polarized cell monolayers, indicating that GLUT1 mRNA is also highly regulated in polarized epithelial cells by agents affecting cell differentiation. The relatively constitutive expression of SGLT3 mRNA suggests a novel role for this low-affinity Na(+)/glucose cotransporter, to provide concentrative glucose uptake under hyperglycemic conditions where expression of high-affinity glucose cotransporter SGLT1 mRNA is significantly downregulated.