RESUMO
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.
Assuntos
Regulação Fúngica da Expressão Gênica , Genes Fúngicos , RNA Helicases/genética , RNA Fúngico/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transativadores/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Proteínas Adaptadoras de Transdução de Sinal , Arginina/fisiologia , Sequência de Bases , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/biossíntese , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Deleção de Genes , Regulação Enzimológica da Expressão Gênica , Dados de Sequência Molecular , Fases de Leitura Aberta , Iniciação Traducional da Cadeia Peptídica , Mutação Puntual , RNA Fúngico/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
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.
Assuntos
Aspergillus nidulans/genética , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante) , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/fisiologia , Genes Fúngicos/fisiologia , Neurospora crassa/genética , Sinais Direcionadores de Proteínas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Proteínas Fúngicas/biossíntese , Dados de Sequência Molecular , Biossíntese de Proteínas , RNA Fúngico/biossíntese , RNA Fúngico/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
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.
