Role of RNA surveillance proteins Upf1/CpaR, Upf2 and Upf3 in the translational regulation of yeast CPA1 gene.

Gene CPA1, encoding one of the subunits of carbamoylphosphate synthetase (CPSase A) is subject to a translational control by arginine of which the essential element is a 25 amino acid peptide encoded by the CPA1 messenger. The peptide is the product of an open reading frame located upstream (uORF) of the coding phase of the gene, within a 250 nucleotide leader. In the past, a series of mutations impairing the repression of gene CPA1 by arginine had been selected in vivo. Most of the mutations were located in the CPA1 uORF, but mutations unlinked to the CPA1 gene were also isolated and mapped in a gene called CPAR. In this work, we show that the CPAR gene is identical to the UPF1 gene, encoding a protein responsible for the premature termination step of RNA surveillance by nonsense-mediated mRNA decay (NMD). Deletion of UPF1, or deletion of UPF2 and UPF3, the other genes involved in the NMD pathway, enhances the synthesis of CPSase A, whether arginine is present or not in the growth medium. The regulatory effect of the NMD protein complex is only observed when the uORF is present in the CPA1 messenger, indicating that the arginine-peptide repression mechanism and the RNA surveillance pathway are complementary mechanisms. Our results indicate that the NMD destabilizes the 5' end of the CPA1 message and this decay is strongly enhanced when arginine is present in the growth medium.

Inositol polyphosphate kinase activity of Arg82/ArgRIII is not required for the regulation of the arginine metabolism in yeast.

Arg82, a nuclear regulator of diverse cellular processes in yeast, is an inositol polyphosphate kinase. Some defects such as the regulation of arginine metabolism observed in an arg82Delta, result from a lack of Mcm1 and Arg80 stability. We show here that neither the kinase activity of Arg82 nor inositol phosphates are required for the control of arginine metabolism. Arg82 mutations keeping kinase active affect the expression of arginine genes, whereas mutations in the kinase domain do not impair this metabolic control.

Functional analysis of the leader peptide of the yeast gene CPA1 and heterologous regulation by other fungal peptides.

The 25-amino-acid leader peptide present at the 5' end of yeast CPA1 mRNA is responsible for the translational repression of that gene by arginine. We show here that the active domain of the yeast peptide is highly specific and extends over amino acids 6-23. The region between amino acids 6-21 is well conserved between similar peptides present upstream of CPA1-homologous genes in other fungi. The Neurospora crassa arg-2 peptide represses the expression of CPA1, whereas the peptide from Aspergillus nidulans has only a weak regulatory effect. Such results suggest that the N- and C-terminal amino acids of the peptide may influence its regulatory activity. We also show that the transcription start sites of CPA1 are not modified when the cells are grown in the presence of arginine, nor in a strain carrying an inactive peptide.

In Saccharomyces cerevisiae, expression of arginine catabolic genes CAR1 and CAR2 in response to exogenous nitrogen availability is mediated by the Ume6 (CargRI)-Sin3 (CargRII)-Rpd3 (CargRIII) complex.

The products of three genes named CARGRI, CARGRII, and CARGRIII were shown to repress the expression of CAR1 and CAR2 genes, involved in arginine catabolism. CARGRI is identical to UME6 and encodes a regulator of early meiotic genes. In this work we identify CARGRII as SIN3 and CARGRIII as RPD3. The associated gene products are components of a high-molecular-weight complex with histone deacetylase activity and are recruited by Ume6 to promoters containing a URS1 sequence. Sap30, another component of this complex, is also required to repress CAR1 expression. This histone deacetylase complex prevents the synthesis of the two arginine catabolic enzymes, arginase (CAR1) and ornithine transaminase (CAR2), as long as exogenous nitrogen is available. Upon nitrogen depletion, repression at URS1 is released and Ume6 interacts with ArgRI and ArgRII, two proteins involved in arginine-dependent activation of CAR1 and CAR2, leading to high levels of the two catabolic enzymes despite a low cytosolic arginine pool. Our data also show that the deletion of the UME6 gene impairs cell growth more strongly than the deletion of the SIN3 or RPD3 gene, especially in the Sigma1278b background.

ArgRII, a component of the ArgR-Mcm1 complex involved in the control of arginine metabolism in Saccharomyces cerevisiae, is the sensor of arginine.

Repression of arginine anabolic genes and induction of arginine catabolic genes are mediated by a three-component protein complex, interacting with specific DNA sequences in the presence of arginine. Although ArgRI and Mcm1, two MADS-box proteins, and ArgRII, a zinc cluster protein, contain putative DNA binding domains, alone they are unable to bind the arginine boxes in vitro. Using purified glutathione S-transferase fusion proteins, we demonstrate that ArgRI and ArgRII1-180 or Mcm1 and ArgRII1-180 are able to reconstitute an arginine-dependent binding activity in mobility shift analysis. Binding efficiency is enhanced when the three recombinant proteins are present simultaneously. At physiological concentration, the full-length ArgRII is required to fulfill its functions; however, when ArgRII is overexpressed, the first 180 amino acids are sufficient to interact with ArgRI, Mcm1, and arginine, leading to the formation of an ArgR-Mcm1-DNA complex. Several lines of evidence indicate that ArgRII is the sensor of the effector arginine and that the binding site of arginine would be the region downstream from the zinc cluster, sharing some identity with the arginine binding domain of bacterial arginine repressors.

Recruitment of the yeast MADS-box proteins, ArgRI and Mcm1 by the pleiotropic factor ArgRIII is required for their stability.

Regulation of arginine metabolism requires the integrity of four regulatory proteins, ArgRI, ArgRII, ArgRIII and Mcm1. To characterize further the interactions between the different proteins, we used the two-hybrid system, which showed that ArgRI and Mcm1 interact together, and with ArgRII and ArgRIII, without an arginine requirement. To define the interacting domains of ArgRI and Mcm1 with ArgRIII, we fused portions of ArgRI and Mcm1 to the DNA-binding domain of Gal4 (GBD) and created mutations in GBD-ArgRI and GBD-Mcm1. The putative alpha helix present in the MADS-box domain of ArgRI and Mcm1 is their major region of interaction with ArgRIII. Interactions between the two MADS-box proteins and ArgRIII were confirmed using affinity chromatography. The requirement for ArgRIII in the control of arginine metabolism can be bypassed in vitro as well as in vivo by overproducing ArgRI or Mcm1, which indicates that ArgRIII is not present in the protein complex formed with the 'arginine boxes'. We show that the impairment of arginine regulation in an argRIII deletant strain is a result of a lack of stability of ArgRI and Mcm1. A mutation in ArgRI, impairing its interaction with ArgRIII, leads to an unstable ArgRI protein in a wild-type strain. ArgRIII integrity is crucial for Mcm1 function, as shown by the marked decreased expression of five genes controlled by Mcm1. However, ArgRIII is likely to recruit other proteins in the yeast cell, as overexpression of Mcm1 does not compensate some physiological defects observed in an argRIII deletant strain.

The nucleotide sequence of Saccharomyces cerevisiae chromosome XII.

The yeast Saccharomyces cerevisiae is the pre-eminent organism for the study of basic functions of eukaryotic cells. All of the genes of this simple eukaryotic cell have recently been revealed by an international collaborative effort to determine the complete DNA sequence of its nuclear genome. Here we describe some of the features of chromosome XII.

Integration of the multiple controls regulating the expression of the arginase gene CAR1 of Saccharomyces cerevisiae in response to differentnitrogen signals: role of Gln3p, ArgRp-Mcm1p, and Ume6p.

Expression of the catabolic gene encoding arginase in Saccharomyces cerevisiae, CAR1, is controlled by multiple nitrogen signals, such as the presence of the inducer, arginine, and the nature and amount of the nitrogen source. The present study has determined or confirmed the identity of the proteins involved in these different controls, as well as their targets in the CAR1 promoter. We show that Gln3p activates CAR1 expression through the GATAA sequences in the absence of an optimal nitrogen source, such as ammonia, glutamine or asparagine. Ume6p, which also controls the expression of early meiotic genes, represses CAR1 expression through a sequence called URS, as a function of nitrogen availability. Thus, the responses to the quality of the nitrogen source and to nitrogen starvation are achieved through different cis- and trans-regulatory elements. At least one of the multiple Rap1p and Abf1p binding sites is required for the basal transcription of the gene. The UAS(arg), containing the previously defined "arginine boxes" is the region that responds to the inducer through the action of the ArgRp-Mcm1p proteins, and its deletion alone significantly affects growth on arginine as sole nitrogen source. The functional UAS(arg) is about 60 nucleotides long, and contains two sequences homologous to the binding site for MADS-box proteins, to which ArgRIp and Mcm1p belong. No obvious palindromic sequence similar to the binding site of Gal4p, Ppr1p or Put3p is present in the UAS(arg), although ArgRIIp contains a Zn(II)2Cys6 motif. Interestingly, we have found that induction of CAR1 expression by arginine in the presence of an optimal nitrogen source is counteracted by Gln3p, independently of its action at the GATAA sequences.

Cloning and sequencing of Schizosaccharomyces pombe car1 gene encoding arginase. Expression of the arginine anabolic and catabolic genes in response to arginine and related metabolites.

We report here the cloning and sequencing of the gene encoding arginase (car1) from Schizosaccharomyces pombe. Since no arginase-less strain exists in this organism, we cloned the gene by functional complementation of a car1 mutant strain from Saccharomyces cerevisiae. The S. pombe car1 gene encodes a 323 amino acids polypeptide sharing identity with arginases from different organisms. Measurements of arg3, arg11 and car1 mRNA under different growth conditions confirm the very weak repression by arginine of the two anabolic genes and show that the induction of arginase synthesis operates at a transcriptional level. The promoter of S. pombe car1 gene does not contain the 'arginine boxes' defined as the target of the ARGR-MCM1 proteins in the promoters of the arginine co-regulated genes in S. cerevisiae. The heterologous expression of S. pombe car1 gene in S. cerevisiae is independent of the ARGRII gene product (ArgRIIp/Arg81p). Determination of arginine, ornithine and citrulline intracellular concentrations shows the efficiency of the different controls operating in S. cerevisiae, and also indicates that in S. pombe enzyme compartmentation is not always sufficient to control the arginine metabolic flux.

Pleiotropic function of ArgRIIIp (Arg82p), one of the regulators of arginine metabolism in Saccharomyces cerevisiae. Role in expression of cell-type-specific genes.

ArgRIIIp (Arg82p), together with ArgRIp (Arg80p), ArgRIIp (Arg81p) and Mcm1p, regulates the expression of arginine anabolic and catabolic genes. An argRIII mutant constitutively expresses five anabolic enzymes and is impaired in the induction of the synthesis of two catabolic enzymes. A genomic disruption of the ARGRIII gene not only leads to an argR phenotype, but also prevents cell growth at 37 degrees C. The disrupted strain is sterile especially in an alpha background and transcription of alpha- and a-specific genes (MF alpha 1 and STE2) is strongly reduced. By gel retardation assays we show that the binding of the Mcm1p present in a crude protein extract from an argRIII mutant strain to the P(PAL) sequence is impaired. Sporulation of alpha/a argRIII::URA3 homozygous diploids is also affected. Overexpression of Mcm1p in an argRIII-disrupted strain restores the mating competence of the strain, the ability to form a protein complex with P(PAL) DNA in vitro, and the regulation of arginine metabolism. However, overexpression of Mcm1p does not complement the sporulation deficiency of the argRIII-disrupted strain, nor does it complement its growth defect at 37 degrees C. Western blot analysis indicates that Mcm1p is less abundant in a strain devoid of ArgRIIIp than in wild type.

UME6 is a key regulator of nitrogen repression and meiotic development.

This report describes the identification, cloning, and molecular analysis of UME6 (CAR80/CARGRI), a key transcriptional regulator of early meiotic gene expression. Loss of UME6 function results in the accumulation of fully derepressed levels (70- to 100-fold increase above basal level) of early meiotic transcripts during vegetative growth. In contrast, mutations in five previously identified UME loci (UME1 to UME5), result in low to moderate derepression (2- to 10-fold increase) of early meiotic genes. The behavior of insertion and deletion alleles indicates that UME6 is dispensable for mitotic division but is required for meiosis and spore germination. Despite the high level of meiotic gene expression during vegetative growth, the generation times of ume6 mutant haploid and diploid cells are only slightly reduced. However, both ascus formation and spore viability are affected more severely. The UME6 gene encodes a 91-kD protein that contains a C6 zinc cluster motif similar to the DNA-binding domain of GAL4. The integrity of this domain is required for UME6 function. It has been reported recently that a mutation in CAR80 fails to complement an insertion allele of UME6. CAR80 is a gene required for nitrogen repression of the arginine catabolic enzymes. Here, through sequence analysis, we demonstrate that UME6 and CAR80 are identical. Analyses of UME6 mRNA during both nitrogen starvation and meiotic development indicate that its transcription is constitutive, suggesting that regulation of UME6 activity occurs at a post-transcriptional level.

A segment of mRNA encoding the leader peptide of the CPA1 gene confers repression by arginine on a heterologous yeast gene transcript.

The expression of the yeast gene CPA1, which encodes the small subunit of the arginine pathway carbamoylphosphate synthetase, is repressed by arginine at a translational level. CPA1 mRNA contains a 250-nucleotide-long leader which includes a 25-codon upstream open reading frame (uORF). Oligonucleotide site-directed mutagenesis of this uORF as well as sequencing of constitutive cis-dominant mutations has suggested that the leader peptide product of the CPA1 uORF is an essential negative element for repression of the CPA1 gene by arginine. In this work, a series of deletions affecting the regions 5' and 3' to the uORF in the leader sequence was constructed. The arginine-dependent repression of CPA1 was little affected in these constructions, indicating that these regions are not essential for the regulatory response. This conclusion was further supported by the finding that inserting the mRNA segment encoding the leader peptide sequence of CPA1 in the leader sequence of another gene, namely, GCN4, places this gene under arginine repression. Similarly, the behavior of fusions of the leader sequence of CPA1 with those of ARG4 or GAL10 confirmed that the regions of this leader located upstream and downstream from the uORF are dispensable for the regulation by arginine. Finally, a set of substitution mutations which modify the uORF nucleotide sequence while leaving unchanged the corresponding amino acid sequence was constructed. The mutations did not affect the repression of CPA1 by arginine. The data presented in this paper consequently agree with the conclusion that the leader peptide itself is the main element required for the translational repression of CPA1.

Sequencing and functional analysis of a 32,560 bp segment on the left arm of yeast chromosome II. Identification of 26 open reading frames, including the KIP1 and SEC17 genes.

We report here the DNA sequence of a segment (alpha 1006.13: YBLO5) of chromosome II of Saccharomyces cerevisiae, extending over 32.5 kb. The segment contains 26 open reading frames (ORFs) from YBLO501 to YBLO526. YBL0505 corresponds to the SEC17 gene and YBL0521 to the KIP1 gene. YBL0516 contains an intron, YBL0513 shows homology with the RAT protein phosphatase and YBL0526 contains a zinc-finger motif. Disruption of 14 genes by insertion of a URA3 cassette has been performed and these mutants were analysed for their mating and sporulation ability, and for their growth on different carbon sources. YBL0515 and YBL0526 ORFs seem to be involved in the sporulation process.

Genetic evidence for a role for MCM1 in the regulation of arginine metabolism in Saccharomyces cerevisiae.

ARGRI, ARGRII, and ARGRIII regulatory proteins control the expression of arginine anabolic and catabolic genes in Saccharomyces cerevisiae. We have shown that MCM1 is part of the ARGR regulatory complex, by in vitro binding experiments, at the ARGR5,6 promoter. The participation of MCM1 in the regulation of arginine metabolism is confirmed by the behavior of an mcm1-gcn4 mutant, which is affected in the repression of arginine anabolic genes. In this mcm1 mutant, synthesis of the catabolic enzymes is rather constitutive, but this derepression requires the integrity of the ARGR system and of the target sequences of these proteins in the CAR1 promoter. Our in vitro binding experiments confirm the presence of MCM1 in the protein complex interacting with the promoters of the catabolic CAR1 and CAR2 genes. This is the first in vivo transcription role ascribed to MCM1 other than its role in the transcription of cell-type-specific genes.

The complete sequence of a 9,543 bp segment on the left arm of chromosome III reveals five open reading frames including glucokinase and the protein disulfide isomerase.

We report here the DNA sequence of a 9.5 kb segment of chromosome III. The sequence was determined by subcloning the segment into subfragments generated by appropriate restriction enzymes followed by oligonucleotide-directed sequencing. The segment contains at least five open reading frames, YCL311, YCL312, YCL313, YCL314, YCL315. YCL311 and YCL315 extend in the adjacent fragments, A4H and A6C respectively. YCL312 encodes glucokinase, and YCL313 the protein disulfide isomerase. Disruption of YCL311, 314 and 315 by insertion of a URA3 cassette does not lead to a detectable phenotype, whereas disruption of YCL313 provokes cell lethality.

Cloning and sequencing of arg3 and arg11 genes of Schizosaccharomyces pombe on a 10-kb DNA fragment. Heterologous expression and mitochondrial targeting of their translation products.

The Schizosaccharomyces pombe arginine anabolic genes encoding ornithine carbamoyltransferase (arg3) and acetylglutamate kinase/acetylglutamyl-phosphate reductase (arg11) were cloned by functional complementation of S. pombe arg3 and arg11 mutant strains from S. pombe DNA genomic libraries. Restriction analysis and sequencing of the two clones showed that both genes are located on a common DNA fragment. The arg3 gene encodes a 327-amino-acid polypeptide presenting a strong identity to Saccharomyces cerevisiae and human ornithine carbamoyltransferases. The arg11 gene encodes a 884-amino-acid polypeptide. The acetylglutamate kinase and acetylglutamate-phosphate reductase domains have been defined by their identity with the S. cerevisiae ARG5,6 protein. The cloned arg11 gene from S. pombe does not complement an arg5,6 mutation in S. cerevisiae, nor does the ARG5,6 gene complement the S. pombe arg11- mutation. In contrast, both ornithine-carbamoyltransferase-encoding genes function in S. pombe. However, the S. pombe arg3 gene complements only weakly an arg3 S. cerevisiae strain, which is in agreement with the low level of expression of the S. pombe gene in S. cerevisiae. The subcellular localization of both ornithine carbamoyltransferases in the two yeasts indicates that, in contrast to the S. pombe enzyme, more than 95% of the S. cerevisiae enzyme remains in the S. pombe cytoplasm. The low expression of S. pombe ornithine carbamoyltransferases in S. cerevisiae did not allow its localization. The promoters of S. pombe arg3 and arg11 genes do not present striking similarities among themselves nor with the promoters of the equivalent genes of S. cerevisiae.

Determination of amino acid sequences involved in the processing of the ARG5/ARG6 precursor in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the ARG5/ARG6 locus encodes both acetylglutamate kinase and acetylglutamyl-phosphate reductase, localized in the mitochondria. Genetic analysis, determination of the nucleotide sequence of the ARG5/ARG6 gene and identification of the transcript indicate that it encodes a single translation product containing two enzyme activities. However, analysis of cellular extracts revealed that the activities are completely separable. In this work, we define different domains in the ARG5/ARG6 polypeptide; a mitochondrial target sequence and the acetylglutamate-kinase and acetylglutamyl-phosphate-reductase domains. We show that deletions in the N-terminal end of the protein and point mutations in the junction region between the acetylglutamate-kinase and acetylglutamyl-phosphate-reductase domains lead to the accumulation of large precursor. Our data support the idea that import of the ARG5/ARG6 precursor into the mitochondria is required for its processing into two mature enzymes.

Determination of the DNA-binding sequences of ARGR proteins to arginine anabolic and catabolic promoters.

ARGRI, ARGRII, and ARGRIII proteins regulate the expression of arginine anabolic and catabolic genes. The integrity of these three proteins is required to observe the formation of a DNA-protein complex with the different promoters of arginine coregulated genes. A study of deletions and point mutations created in the 5' noncoding region of ARG3, ARG5,6, CAR1, and CAR2 genes shows that at least two regions, called BoxA and BoxB, are required for proper regulation of these genes by arginine and ARGR proteins. By gel retardation assay and DNase I footprinting analysis, we have determined precisely the target of the ARGR proteins. Sequences in and around BoxA are necessary for ARGR binding to these four promoters in vitro, whereas sequences in and around BoxB are clearly protected against DNase I digestion only for CAR1. Sequences present at BoxA and BoxB are well conserved among the four promoters. Moreover, pairing can occur between sequences at BoxA and BoxB which could lead to the creation of secondary structures in ARG3, ARG5,6, CAR1, and CAR2 promoters, favoring the binding of ARGR proteins in vivo.

In vitro studies of the binding of the ARGR proteins to the ARG5,6 promoter.

ARGRI, ARGRII, and ARGRIII regulatory proteins control the expression of arginine anabolic and catabolic genes in Saccharomyces cerevisiae. We show here that they are also required in vitro to observe a protein-DNA complex with the promoter of the ARG5,6 gene. The specific binding of ARGR proteins in vitro is stimulated by arginine. Antibodies raised against a synthetic MCM1 polypeptide retard the migration of ARGR-DNA complex on gel mobility shift assays. This result suggests that MCM1 could be an additional regulatory element of arginine metabolism.

Dissection of the bifunctional ARGRII protein involved in the regulation of arginine anabolic and catabolic pathways.

ARGRII is a regulatory protein which regulates the arginine anabolic and catabolic pathways in combination with ARGRI and ARGRIII. We have investigated, by deletion analysis and fusion to LexA protein, the different domains of ARGRII protein. In contrast to other yeast regulatory proteins, 92% of ARGRII is necessary for its anabolic repression function and 80% is necessary for its catabolic activator function. We can define three domains in this protein: a putative DNA-binding domain containing a zinc finger motif, a region more involved in the repression activity located around the RNase-like sequence, and a large activation domain.