Expression analysis of RNA14, a gene involved in mRNA 3' end maturation in yeast: characterization of the rna14-5 mutant strain.

In Saccharomyces cerevisiae, Rna14 protein is involved in both cleavage and polyadenylation of mRNA in the nucleus. Previous work has demonstrated that this protein is also localized in mitochondria. Moreover, all known rna14 mutants can be separated into two distinct classes: the poly(A)-negative class, which contains mutants that are deficient in mRNA 3'-end processing, and the poly(A)-positive class, which includes those mutants that are not impaired in any of the steps in mRNA metabolism investigated. This suggests that in addition to its involvement in mRNA polyadenylation, Rna14p could have a second function related to mitochondrial metabolism. Here we investigated the regulation of RNA14 by characterizing the rna14-5 mutant, which is the only poly(A)-positive allele that also overproduces the RNA14 mRNA. We showed that both deregulation of RNA14 transcription and modification of RNA14 mRNA stability contribute to the strong accumulation of the transcripts in this mutant. Surprisingly, the RNA14 promoter itself is not essential for this phenotype of the rna14-5 mutant. However, the 3' UTR of the mRNA is necessary for overproduction of the transcripts, although it is not sufficient to deregulate a reporter gene by itself. Site-directed mutagenesis experiments provided additional data suggesting that the rna14-5 mutation acts at the protein level rather than modifying the properties of the RNA14 transcripts themselves. A tentative model accounting for the data is discussed, in light of the proposed extranuclear function of the Rna14p and its mitochondrial localization.

Deletion of the PAT1 gene affects translation initiation and suppresses a PAB1 gene deletion in yeast.

The yeast poly(A) binding protein Pab1p mediates the interactions between the 5' cap structure and the 3' poly(A) tail of mRNA, whose structures synergistically activate translation in vivo and in vitro. We found that deletion of the PAT1 (YCR077c) gene suppresses a PAB1 gene deletion and that Pat1p is required for the normal initiation of translation. A fraction of Pat1p cosediments with free 40S ribosomal subunits on sucrose gradients. The PAT1 gene is not essential for viability, although disruption of the gene severely impairs translation initiation in vivo, resulting in the accumulation of 80S ribosomes and in a large decrease in the amounts of heavier polysomes. Pat1p contributes to the efficiency of translation in a yeast cell-free system. However, the synergy between the cap structure and the poly(A) tail is maintained in vitro in the absence of Pat1p. Analysis of translation initiation intermediates on gradients indicates that Pat1p acts at a step before or during the recruitment of the 40S ribosomal subunit by the mRNA, a step which may be independent of that involving Pab1p. We conclude that Pat1p is a new factor involved in protein synthesis and that Pat1p might be required for promoting the formation or the stabilization of the preinitiation translation complexes.

PCF11 encodes a third protein component of yeast cleavage and polyadenylation factor I.

Cleavage and polyadenylation factor I (CF I) is one of four factors required in vitro for yeast pre-mRNA 3'-end processing. Two protein components of this factor, encoded by genes RNA14 and RNA15, have already been identified. We describe here another gene, PCF11 (for protein 1 of CF I), that genetically interacts with RNA14 and RNA15 and which presumably codes for a third protein component of CF I. This gene was isolated in a two-hybrid screening designed to identify proteins interacting with Rna14 and Rna15. PCF11 is an essential gene encoding for a protein of 626 amino acids having an apparent molecular mass of 70 kDa. Thermosensitive mutations in PCF11 are synergistically lethal with thermosensitive alleles of RNA14 and RNA15. The Pcf11-2 thermosensitive strain shows a shortening of the poly(A) tails and a strong decrease in the steady-state level of actin transcripts after a shift to the nonpermissive temperature as do the thermosensitive alleles of RNA14 and RNA15. Extracts from the pcf11-1 and pcf11-2 thermosensitive strains and the wild-type strain, when Pcf11 is neutralized by specific antibodies, are deficient in cleavage and polyadenylation. Moreover, fractions obtained by anion-exchange chromatography of extracts from the wild-type strain contain both Pcf11 and Rna15 in the same fractions, as shown by immunoblotting with a Pcf11-specific antibody.

Mutations in STS1 suppress the defect in 3' mRNA processing caused by the rna15-2 mutation in Saccharomyces cerevisiae.

In a search for proteins associated with Rna15p in processing the 3' ends of messenger RNAs, we have looked for suppressors that correct, even partially, the thermosensitive growth defect of the rna15-2 mutant. Mutations in a single locus that we named SSM5, were able to suppress both the thermosensitivity of cell growth and the mRNA 3' processing defect associated with the rna15-2 mutation, but only slightly alleviated the thermosensitive growth defect of an rna14-1 mutant. The ssm5-1 mutant is sensitive to hydroxyurea at 37 degrees C, a drug that inhibits DNA synthesis. By screening for complementation of the hydroxyurea-sensitive phenotype we cloned the corresponding wild-type gene and found that it corresponds to the essential gene STS1 (also named DBF8). Sts1p has an apparent molecular weight of 30 kDa and was confirmed to be a cytosolic protein by immunofluorescence analysis. Western blot analysis indicates that the thermosensitive mutant strains rna15-2, rna14-1 and pap1-1 present a very low level of the Rna15p at 37 degrees C. The ssm5-1 mutation restores the level of Rna15p in the rna15-2 ssm5-1 double mutant. Use of the two-hybrid system suggests that Sts1p does not interact directly with Rna15p, but may be active as a homodimer. The present data suggest that Sts1p may play a role in the transport of Rna15p from the cytoplasm to the nucleus.

SLS1, a new Saccharomyces cerevisiae gene involved in mitochondrial metabolism, isolated as a syntheticlethal in association with an SSM4 deletion.

SSM4 was isolated as a suppressor of rna14-1, a mutant involved in nuclear mRNA maturation. In order to isolate genes interacting with SSM4, we have searched for mutants that are syntheticlethal in association with an SSM4 deletion. Among the mutants obtained, one, named sls1-1, shows a pet- phenotype. We have cloned and sequenced this gene. It encodes a protein with a calculated molecular mass of 73 kDa. This protein contains a mitochondrial targeting presequence but does not show homology with other known proteins. Deletion of SLS1 does not affect cell viability on glucose but is lethal on a non-fermentable medium. The Sls1p protein does not appear to be involved in mitochondrial DNA replication, transcription, or in RNA splicing maturation or stability. We have also tagged this protein and localized it in mitochondria. Treatment with alkaline carbonate does not extract this protein from mitochondria, suggesting strongly that it is a mitochondrial integral membrane protein. Thus, the SLS1 gene, encodes a mitochondrial integral membrane protein and is paradoxically synlethal in association with a deletion of the SSM4 gene, which encodes an integral nuclear membrane protein.

Inactivation of SSM4, a new Saccharomyces cerevisiae gene, suppresses mRNA instability due to rna14 mutations.

Decay rates of mRNAs depend on many elements and among these, the role of the poly(A) tail is now well established. In the yeast Saccharomyces cerevisiae, thermosensitive mutations in two genes, RNA14 and RNA15, result in mRNAs having shorter poly(A) tails and reduced half-life. To identify other components interacting in the same process, we have used a genetic approach to isolate mutations that suppress the thermosensitivity of an rna14 mutant strain. Mutations in a single locus, named SSM4, not only suppress the cell growth phenotype but also the mRNA instability and extend the short mRNA poly(A) tails. The frequency of appearance and the recessive nature of these mutations suggested that the suppressor effect was probably due to a loss of function. We failed to clone the SSM4 gene directly by complementation, owing to its absence from gene banks; it later emerged that the gene is toxic to Escherichia coli, but we have nevertheless been able to clone the SSM4 sequence by Ty element transposition tagging. Disruption of the SSM4 gene does not affect cell viability and suppresses the rna14 mutant phenotypes. The protein encoded by the SSM4 gene has a calculated molecular mass of 151 kDa and does not contain any known motif or show homology with known proteins. The toxicity of the SSM4 gene in E. coli suggests that a direct biochemical activity is associated with the corresponding protein.

Heterospecific cloning of Arabidopsis thaliana cDNAs by direct complementation of pyrimidine auxotrophic mutants of Saccharomyces cerevisiae. I. Cloning and sequence analysis of two cDNAs catalysing the second, fifth and sixth steps of the de novo pyrimidine biosynthesis pathway.

An Arabidopsis thaliana cDNA library was used to complement Saccharomyces cerevisiae pyrimidine auxotrophic mutants. Mutants in all but one (carbamylphosphate synthetase) of the six steps in the de novo pyrimidine biosynthetic pathway could be complemented. We report here the cloning, sequencing and computer analysis of two cDNAs encoding the aspartate transcarbamylase (ATCase; EC 2.1.3.2) and orotate phosphoribosyltransferase-orotidine-5'-phosphate decarboxylase (OPRTase-OMPdecase; EC 2.4.2.10, EC 4.1.1.23) enzymes. These results confirm the presence in A. thaliana of a bifunctional gene whose product catalyses the last two steps of the pyrimidine biosynthetic pathway, as previously suggested by biochemical studies. The ATCase encoding cDNA sequence (PYRB gene) shows an open reading frame (ORF) of 1173 bp coding for 390 amino acids. The cDNA encoding OPRTase-OMPdecase (PYRE-F gene) shows an ORF of 1431 bp coding for 476 amino acids. Computer analysis of the deduced amino acid sequences of both cDNAs shows the expected high similarity with the ATCase, ornithine transcarbamylase (OTCase; EC 2.1.3.3), OPRTase and OMPdecase families. This heterospecific cloning approach increases our understanding of the genetic organization and interspecific functional conservation of the pyrimidine biosynthetic pathway and underlines its usefulness as a model for evolutionary studies.

Maturational changes induced by 1 alpha,25-dihydroxyvitamin D3 in type II cells from fetal rat lung explants.

Specific binding sites for 1 alpha,25 dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] localized to type II pneumocytes have been evidenced in fetal rat lung at the end of gestation, suggesting a role for vitamin D3 in the control of lung maturation. In this study, we describe the morphological changes that occur in lung explants from 18-day-old rat fetuses grown for 1 and 2 days in control conditions and in the presence of 1 alpha,25(OH)2D3 (10(-9) M) or dexamethasone (10(-7) M). Point counting and planimetric measurements on light and electron micrographs show that 1 alpha,25-(OH)2D3 1) dramatically decreases the mean glycogen content of type II cell profiles between days 1 and 2 of the culture, suggesting an acceleration of the glycogenolytic processes normally occurring at that stage and 2) does not change the intracellular osmiophilic lamellar body (OLB) content of cell profiles, but increases the amount of intraluminal surfactant by 126% when expressed as surfactant clusters surface area/section surface area and by 129% when expressed on a per cell basis, suggesting a stimulation of surfactant synthesis and secretion. By contrast, dexamethasone increases the mean intracellular OLB content of type II cell profiles by 306% and decreases the relative surface area of secreted material by 53 and 73%. In conclusion, 1 alpha,25(OH)2D3 accelerates the physiological maturation of fetal rat type II pneumocytes and could represent a key factor for the onset of normal lung function at birth.

Cloning and sequencing of a human cDNA coding for dihydroorotate dehydrogenase by complementation of the corresponding yeast mutant.

Dihydroorotate dehydrogenase (DHOdehase, EC 1.3.3.1) catalyses the fourth enzymatic step in de novo pyrimidine biosynthesis. A truncated human cDNA encoding this enzyme was isolated from a HeLa cell cDNA library by functional complementation of a corresponding deletion mutant from the yeast, Saccharomyces cerevisiae. The complementing clone contained a 1.5-kb poly(A)(+)-tailed insert with a 1191-bp open reading frame, hybridising with a unique human mRNA of 1.6 kb. The deduced amino acid sequence has 54%, 46% and 42% identity with Arabidopsis thaliana, Schizosaccharomyces pombe and Escherichia coli DHOdehases, respectively. In contrast, it has only 21% identity with the S. cerevisiae enzyme, which probably reflects the cytosolic location of the enzyme in the latter organism.

Complementation of Saccharomyces cerevisiae auxotrophic mutants by Arabidopsis thaliana cDNAs.

An Arabidopsis thaliana cDNA bank has been constituted in a Saccharomyces cerevisiae expression vector based on the phosphoglycerate kinase (PGK) promoter and terminator. This bank was used to complement eight S. cerevisiae auxotrophic markers. All of them were corrected. These results confirm the quality of the bank and the feasibility of cloning plant genes by yeast mutant complementation. The cDNA complementing the ura1 yeast mutant was sequenced, analysed and shown to encode a dihydroorotic (DHO) dehydrogenase sequence.

1,25-dihydroxyvitamin D3 receptors in rat lung during the perinatal period: regulation and immunohistochemical localization.

We have previously reported that in contrast to what has been described in adult lung, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] has specific binding sites in rat fetal lung at the end of gestation, and it stimulates in vitro the phospholipid biosynthesis and surfactant release from fetal rat type II pneumocytes. In the present study an immunohistochemical technique using a rat monoclonal antibody (9A7 gamma) and binding studies were carried out on fresh lung tissues from fetal and newborn rats during the perinatal period to identify the cell(s) directly responsive to 1,25-(OH)2D3 in fetal lung and to look for a down-regulation of the 1,25-(OH)2D3 receptors in the perinatal period. We also searched for a regulation of 1,25-(OH)2D3 binding to fetal lung by 1,25-(OH)2D3 itself and by factors known to affect lung maturation or be involved in parturition. Our results suggest that 1) fetal type II pneumocytes are target cells for 1,25-(OH)2D3; 2) a physiological down-regulation of the 1,25-(OH)2D3 receptors in rat lung occurs in the perinatal period, starting a few hours before birth and lasting at least up to the fifth day of life; and 3) the capacity of rat fetal lung to bind 1,25-(OH)2D3 can be modulated in vitro by different hormones; a small inhibitory effect is observed with oxytocin (100 microU/ml), while PRL (10(-8) M), T4 (10(-6)-10(-10) M), 1,25-(OH)2D3 (10(-9)-10(-10) M), and, to a lesser extent, dexamethasone (10(-7) M) induce a 2- to 4-fold increase in the number of 1,25-(OH)2D3 receptors without altering the binding affinity of receptor for 1,25-(OH)2D3.

Effects of the antiglucocorticoid RU 486 on the initiation of ultrastructural type-II cell differentiation in fetal rat lung.

The possible early role of endogenous glucocorticosteroids on the further ultra-structural development of the fetal rat lung epithelium was investigated in vitro using a potent antiglucocorticoid drug, RU 486. Lung primordia were explanted on day 13 of gestation and cultured for 4-6 days in the presence of fetal calf serum, with or without RU 486 in excess. The results obtained show that osmiophilic lamellar bodies specific for morphologically differentiated type-II cells did appear in a number of epithelial cells, even though the glucocorticosteroids possibly present in the culture medium, or transferred at explantation were antagonized by RU 486. The number of lamellar bodies stored in these pneumocytes was not different from that in controls. In contrast, their total surface area per cell profile was transiently decreased. Taken together the reported data strongly suggest that endogenous glucocorticosteroids are not necessary for the initiation of type-II cell differentiation.

1,25(OH)2D3 stimulates phospholipid biosynthesis and surfactant release in fetal rat lung explants.

Lung tissue from 18-day-old rat fetuses was cultured in the presence of 1,25-dihydroxyvitamin D3 - 1,25(OH)2D3 - (10(-9) M) and dexamethasone (10(-7) M) for 48 h. 1,25(OH)2D3 increased the lung content in phospholipids more specifically related to lung surfactant, phosphatidylcholine and phosphatidylglycerol. This increase was similar to that observed with dexamethasone. In addition, unlike dexamethasone, 1,25(OH)2D3 stimulated the surfactant release into luminal spaces, as evidenced by light and electron microscopy. Thus, vitamin D3 might represent an additional factor controlling fetal lung maturation by stimulating phospholipid synthesis and surfactant release from type II cells.

Effects of the antiglucocorticoid RU 486 on the maturation of fetal rat lung surfactant.

The role of endogenous glucocorticoids in the control of surfactant system maturation was investigated in the fetal rat using an antiglucocorticoid molecule synthesized by Roussel-UCLAF, RU 486. The drug was administered to the mother from day 16 of gestation on. In a preliminary step, the transplacental transfer of RU 486 and its antiglucocorticoid effects on fetal target tissues were verified by evidencing RU 486-receptor complexes in fetal liver and lung, by measuring liver glycogen content, and by evaluating fetal blood corticosterone. The maturational state of fetal lungs was assessed biochemically on days 19, 20, and 21 of gestation by measuring their glycogen content, the phospholipid content of whole lung tissue and isolated surfactant fraction, and the incorporation of [methyl-3H]choline into DSPC. Morphological development was studied by analyzing electron micrographs of type II cells. The measured parameters clearly indicated a slowing of maturational processes in lungs of fetuses from RU 486-treated mothers, thereby demonstrating that endogenous glucocorticoids are actually involved in the control of lung maturation. In addition, the obtained results showed that endogenous corticosteroids specifically acted on the surfactant system of the fetal lung.