Developmental expression of AUF1 and HuR, two c-myc mRNA binding proteins.

Several proteins that may regulate c-myc mRNA post-transcriptionally were previously isolated and characterized. Two of them, HuR and AUF1, bind specifically to the 3' untranslated region (UTR) of c-myc mRNA. Because c-myc is regulated post-transcriptionally in various mouse tissues, including quiescent tissues, fetal liver and regenerating liver, we investigated whether HuR and AUF1 expression was also regulated in these tissues. Concerning AUF1, we analysed the expression of various mRNA and protein isoforms. We discovered a new AUF1 mRNA variant with a long AU-rich 3' UTR. We show that AUF1 expression, regardless of the RNA isoform considered, and HuR mRNA expression parallel c-myc expression in quiescent tissues and during liver development; their expression is high in lymphoid tissues and fetal liver and low in adult liver. However, no upregulation of HuR or AUF1 accompanies the upregulation of c-myc mRNA following partial hepatectomy. We discuss our results in relation to the current hypothesis that HuR and AUF1 act as mRNA destabilizing factors.

enod40, a gene expressed during nodule organogenesis, codes for a non-translatable RNA involved in plant growth.

Rhizobium meliloti can interact symbiotically with Medicago plants, thereby inducing root nodules. However, certain Medicago plants can form nodules spontaneously, in the absence of rhizobia. A differential screening was performed using spontaneous nodule versus root cDNAs from Medicago sativa ssp. varia. Transcripts of a differentially expressed clone, Msenod40, were detected in all differentiating cells of nodule primordia and spontaneous nodules, but were absent in fully differentiated cells. Msenod40 showed homology to a soybean early nodulin gene, Gmenod40, although no significant open reading frame (ORF) or coding capacity was found in the Medicago sequence. Furthermore, in the sequences of cDNAs and a genomic clone (Mtenod40) isolated from Medicago truncatula, a species containing a unique copy of this gene, no ORFs were found either. In vitro translation of purified Mtenod40 transcripts did not reveal any protein product. Evaluation of the RNA secondary structure indicated that both msenod40 and Gmenod40 transcripts showed a high degree of stability, a property shared with known non-coding RNAs. The Mtenod40 RNA was localized in the cytoplasm of cells in the nodule primordium. Infection with Agrobacterium tumefaciens strains bearing antisense constructs of Mtenod40 arrested callus growth of Medicago explants, while overexpressing Mtenod40 embryos developed into teratomas. These data suggest that the enod40 genes might have a role in plant development, acting as 'riboregulators', a novel class of untranslated RNAs associated with growth control and differentiation.

Regulation of the rat alpha-fetoprotein gene expression in liver. Both the promoter region and an enhancer element are liver-specific and negatively modulated by dexamethasone.

Cis-acting elements involved in the control of rat alpha-fetoprotein gene expression in the liver and its modulation by glucocorticoid hormones were detected after transfection of chloramphenicol acetyltransferase constructs and their transient expression into two hepatoma cell lines. The proximal promoter region (-324 to -15) was found to contain all the information necessary for tissue-specific expression. It is also involved in the negative gene modulation by glucocorticoids and includes an activating regulatory domain allowing efficient expression in the HepG2 cells. Three regions within 7 kilobase pairs of the 5' extragenic sequences are capable of stimulating the chloramphenicol acetyltransferase activity driven by the alpha-fetoprotein promoter sequence. One of these regions, at about -2.5 kilobase pairs, contains a short indivisible 170-base pair DNA element that fulfills all the criteria of a tissue-specific enhancer, i.e. orientation and position independence, as well as cell-specific stimulation of gene expression driven by a homologous or heterologous promoter. The enhancing properties of this element are totally abolished by glucocorticoids. DNase I footprinting experiments indicate that several rat liver nuclear proteins interact with this enhancer element.

Albumin and alpha-fetoprotein gene expression in various nonhepatic rat tissues.

Albumin and alpha-fetoprotein (AFP), two major serum proteins, are synthesized predominantly in the liver and yolk sac of mammals. In the present paper we report on the developmental expression of the corresponding genes in nonhepatic rat tissues. Significant quantities of mature albumin and AFP mRNAs were revealed in kidney, pancreas, heart, and lung of fetal and/or newborn rats using dot blot and Northern blot assays. Very low levels of these mRNA sequences were also detected in adult kidney and pancreas using sensitive RNA-cDNA solution hybridization assays. In situ hybridization analysis revealed that the albumin and AFP gene transcripts are present in the tubular cells of the 20-day-old fetal kidney. In order to elucidate further the mechanisms governing this expression, we studied the chromatin structure and methylation pattern in the 5'-end of these two genes. A faint band, corresponding to a specific DNase I-hypersensitive site upstream from the albumin gene, was detected in the fetal and neonatal kidney nuclei but not in adult kidney. For both genes, a site CG, demethylation of which is correlated with expression in liver and hepatoma cell lines, is highly methylated in fetal kidney even though AFP and albumin genes are expressed. Taken together, these results show the presence of a cell population in the rat kidney that actively transcribes both the albumin and AFP genes. The expression of these genes may be mediated by mechanisms differing in at least some steps from those exerted in the liver.

Activation of alpha-fetoprotein synthesis in rat hepatoma cells with reduced sensitivity to dexamethasone.

The Faza 967 'differentiated', dexamethasone (DEX)-sensitive cell line of Reuber rat hepatoma cells does not synthesize detectable amounts of alpha-fetoprotein (AFP), whereas it does produce albumin. AFP production was activated in 'differentiated' variants of Faza 967 cells with reduced glucocorticoid sensitivity upon culture for several months in the presence of high concentrations of dexamethasone. The stability of AFP production differed among the variants, while albumin synthesis did not change, thus indicating that the regulation of these two genes is not co-ordinated. Using molecular hybridization techniques, we found that the AFP message could not be detected in the non-AFP-producing cells, suggesting that the lack of AFP synthesis most probably originates from a transcriptional block of the AFP gene. AFP-producing and non-AFP-producing variants of Faza 967 cells constitute a valuable model system for studying the regulatory mechanisms involved in the activation and inactivation of the gene coding for the oncodevelopmental protein, AFP.

The mechanism of action of methionyl-tRNA synthetase from Escherichia coli. Inhibition by adenosine and 8-aminoadenosine of the amino-acid activation reaction.

Adenosine and 8-aminoadenosine, both competitive inhibitors of ATP-Mg2+ in the ATP-PPi exchange reaction catalyzed by methionyl-tRNA synthetase, are used to investigate the active center for methionyl-adenylate formation. Resolution of the kinetics parameters of the reaction indicates that methionine markedly enhances the affinity of the nucleosides for the enzyme, providing evidence for coupling between the sites for amino acid and the nucleoside moiety of ATP. Furthermore, occupation of both of these sites is a prerequisite for binding of pyrophosphate. Introduction of an amino group in position 8 of the adenine ring strongly increases the affinity constants for the nucleoside and for pyrophosphate in the coupled reactions described above.