Defining a novel ribonucleotide reductase r1 mRNA cis element that binds to an unique cytoplasmic trans-acting protein.

Ribonucleotide reductase is a highly regulated rate-limiting enzyme activity in DNA synthesis, responsible for reducing ribonucleotides to their deoxyribonucleotide forms. Using 3'-end labeled RNA and band-shift and UV cross-linking analyses, we have identified a cis-element, 5'-CAAACUUC-3', within the 3'-untranslated region of the mammalian ribonucleotide reductase R1 mRNA, which binds a cytoplasmic protein in BALB/c 3T3 mouse cells, to form a 57 kDa RNA-protein complex. Sequence-specific binding was observed, and binding was prevented by several different mutations within the cis-element. We suggest that this cis-trans interaction plays a role in R1 mRNA stability.     

A cis-acting element and a trans-acting factor involved in the wound-induced expression of a horseradish peroxidase gene

The mechanisms that control the wound-induced expression of the prxC2 gene for horseradish peroxidase (HRP) have been investigated. Analysis of the regulatory properties of 5′-deleted promoters showed that a positive element involved in the response to wounding was located between −307 and −99 bp from the site of initiation of translation. In in vitro binding assays of tobacco nuclear proteins and DNA fragments of prxC2 promoter, the binding site was the Box 1 from −296 to −283 containing the CACGTG motif. To identify the functional role of Box 1, the prxC2 promoter that has been digested from the 5′ end to −289 with a disrupted Box 1 was fused to a reporter gene for β-glucuronidase (GUS). No induction of GUS activity was observed in transgenic tobacco plants with the prxC2(−289)/GUS construct. These data indicated that the expression of prxC2 in response to wounding required the Box 1 sequence from −296 to −283. Furthermore, a tobacco cDNA expression library was screened and a cDNA clone for a protein, designated TFHP-1, that bound specifically to the Box 1 sequence was identified. The putative TFHP-1 protein contains a basic region and leucine zipper (bZip) motif and a helix—loop—helix (HLH) motif. The mRNA for TFHP-1 was abundant in roots and stems, and it was not induced by wounding in leaves. In tobacco protoplasts, antisense TFHP-1 suppressed the expression of prxC2 (−529)/GUS.     

Different mechanisms are used by insulin to repress three genes that contain a homologous thymine-rich insulin response element

Insulin rapidly and completely inhibits expression of the hepatic insulin-like growth factor binding protein-1 (IGFBP-1), phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) genes. This inhibition is mediated through a phosphatidyl inositol 3-kinase-dependent regulation of a DNA element, termed the thymine-rich insulin response element, found within the promoters of each of these genes. This has led to the conclusion that these three promoters are regulated by insulin using the same molecular mechanism. However, we recently found that the regulation of the IGFBP1 but not the PEPCK or G6Pase genes by insulin was sensitive to rapamycin, an inhibitor of mTOR. Here, we present further evidence that different regulatory pathways mediate the insulin regulation of these promoters. Importantly, we identify a protein phosphatase activity in the pathway connecting mTOR to the IGFBP-1 promoter. These data have major implications for the development of molecular therapeutics for the treatment of insulin-resistant states such as diabetes and hypertension.     

Identification of a cis-acting element and a novel trans-acting factor of the glutamine synthetase gene in liver cells

In the mammalian liver the expression of the enzyme glutamine synthetase (GS) is restricted to a small population of hepatocytes. In cells expressing the enzyme up to 3.5% of total cellular protein is GS. In order to identify enhancer elements contributing to this extraordinarily high level of expression we focused on a region roughly 2.5 kbp upstream of the GS promoter. Gel mobility shift assays revealed binding of an unknown protein within the most distal part of this region and reportergene assays demonstrated that roughly 60 bp downstream from position -2503 are indispensable for protein binding and the full effect of the enhancer. In UV cross-link analysis a 38 kDa nuclear protein that binds to the sequence was identified in rat hepatocytes. This nuclear protein, designated as upstream binding factor of the GS gene (UFGS) seems to play an important role in high-level expression of GS in liver.     

Brain and muscle creatine kinase genes contain common TA-rich recognition protein-binding regulatory elements.

We have previously reported that the rat brain creatine kinase (ckb) gene promoter contains an AT-rich sequence that is a binding site for a protein called TARP (TA-rich recognition protein). This AT-rich segment is a positively acting regulatory element for the ckb promoter. A similar AT-rich DNA segment is found at the 3' end of the 5' muscle-specific enhancer of the rat muscle creatine kinase (ckm) gene and has been shown to be necessary for full muscle-specific enhancer activity. In this report, we show that TARP binds not only to the ckb promoter but also to the AT-rich segment at the 3' end of the muscle-specific ckm enhancer. A second, weaker TARP-binding site was identified in the ckm enhancer and lies at the 5' end of the minimal enhancer segment. TARP was found in both muscle cells (C2 and L6 myotubes) and nonmuscle (HeLa) cells and appeared to be indistinguishable from both sources, as judged by gel retardation and footprinting assays. The TARP-binding sites in the ckm enhancer and the ckb promoter were found to be functionally interchangeable. We propose that TARP is active in both muscle and nonmuscle cells and that it is one of many potential activators that may interact with muscle-specific regulators to determine the myogenic phenotype.     

Repression of bovine papilloma virus replication is mediated by a virally encoded trans-acting factor

Cells transformed with bovine papilloma virus type 1 mutants in the E6 or E6/7 genes are resistant to high-copy-number amplification of wild-type DNA after supertransfection. Transient and stable replication assays demonstrate this effect. If the supertransfected DNA has a mutation in a newly defined gene (M), this cellular immunity to high-copy-number replication is overcome, resulting in transient replication of the input DNA. In contrast, the resident plasmid does not participate in amplification and is maintained at a constant low copy number. Cotransformation of M- mutants and wild-type DNA into these cells leads to shutoff of replication of both genomes. Thus, M- mutants define a trans-acting negative modulator that regulates viral replication. This function is distinct from the positive factors required for replication. We propose a model that explains why the loss of E6 and E6/7 function leads to immunity of the infected cell.     

Subcellular relocalization of a trans-acting factor regulates XIAP IRES-dependent translation

Translation of the X-linked inhibitor of apoptosis (XIAP) proceeds by internal ribosome entry site (IRES)-mediated initiation, a process that is physiologically important because XIAP expression is essential for cell survival under conditions of compromised cap-dependent translation, such as cellular stress. The regulation of internal initiation requires the interaction of IRES trans-acting factors (ITAFs) with the IRES element. We used RNA-affinity chromatography to identify XIAP ITAFs and isolated the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). We find that hnRNP A1 interacts with XIAP IRES RNA both in vitro and in vivo and that hnRNP A1 negatively regulates XIAP IRES activity. Moreover, XIAP IRES-dependent translation is significantly reduced when hnRNP A1 accumulates in the cytoplasm. Osmotic shock, a cellular stress that causes cytoplasmic accumulation of hnRNP A1, also leads to a decrease in XIAP levels that is abrogated by knockdown of hnRNP A1 expression. These results suggest that the subcellular localization of hnRNP A1 is an important determinant of its ability to negatively regulate XIAP IRES activity, suggesting that the subcellular distribution of ITAFs plays a critical role in regulating IRES-dependent translation. Our findings demonstrate that cytoplasmic hnRNP A1 is a negative regulator of XIAP IRES-dependent translation, indicating a novel function for the cytoplasmic form of this protein.