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Differential regulation of transcript stability is an effective means by which an organism can modulate gene expression. A well-characterized example is glutamine signalled degradation of specific transcripts in Aspergillus nidulans. In the case of areA, which encodes a wide-domain transcription factor mediating nitrogen metabolite repression, the signal is mediated through a highly conserved region of the 3′ UTR. Utilizing this RNA sequence we isolated RrmA, an RNA recognition motif protein. Disruption of the respective gene led to loss of both glutamine signalled transcript degradation as well as nitrate signalled stabilization of niaD mRNA. However, nitrogen starvation was shown to act independently of RrmA in stabilizing certain transcripts. RrmA was also implicated in the regulation of arginine catabolism gene expression and the oxidative stress responses at the level of mRNA stability. ΔrrmA mutants are hypersensitive to oxidative stress. This phenotype correlates with destabilization of eifE and dhsA mRNA. eifE encodes eIF5A, a translation factor within which a conserved lysine is post-translationally modified to hypusine, a process requiring DhsA. Intriguingly, for specific transcripts RrmA mediates both stabilization and destabilization and the specificity of the signals transduced is transcript dependent, suggesting it acts in consort with other factors which differ between transcripts.

The arginine catabolism gene otaA encoding ornithine transaminase (OTAse) is specifically induced by arginine and is under the control of the broad-domain carbon and nitrogen repression systems. Arginine induction is mediated by a product of arcA gene coding for Zn(2)C(6) activator. We have identified a region responsible for arginine induction in the otaA promoter (AnUAS(arg)). Deletions within this region result in non-inducibility of OTAse by arginine, whether in an arcA(+) strain or in the presence of the arcA(d)47 gain of function allele. AnUAS(arg) is very similar to the Saccharomyces cerevisiae UAS(arg), a sequence bound by the Zn(2)C(6) activator (ArgRIIp), acting in a complex with two MADS-box proteins (McmIp and ArgRIp). We demonstrate here that two CREA in vitro binding sites in the otaA promoter are functional in vivo. CREA is directly involved in carbon repression of the otaA gene and it also reduces its basal level of expression. Although AREA binds to the otaA promoter in vitro, it probably does not participate in nitrogen metabolite repression of the gene in vivo. We show here that another putative negatively acting GATA factor AREB participates directly or indirectly in otaA nitrogen repression. We also demonstrate that the high levels of OTAse activity are an important factor in the suppression of proline auxotrophic mutations. This suppression can be achieved neither by growing of the proline auxotroph under carbon/nitrogen derepressing conditions nor by introducing of a creA(d) mutation.

International audience; The filamentous fungus Aspergillus nidulans can utilize arginine both as a nitrogen and carbon source. Analysis of areA and areB single and double mutants has shown that the two GATA transcription factors AREA and AREB negatively regulate the expression of arginine catabolism genes agaA and otaA under nitrogen repressing conditions. AREA is necessary for the ammonium repression of agaA and otaA under carbon repressing conditions, while AREB is involved under carbon-limiting conditions. The ability of both AREA and AREB to sense the status of carbon metabolism is most probably dependent on NMRA, and not on the transcription factor CREA, which mediates general carbon catabolite repression in A. nidulans. NMRA is a co-repressor which has previously been shown to bind the C-terminus of AREA and inhibits its activity under conditions of nitrogen sufficiency, in response to high intracellular glutamine levels. We therefore propose a novel function for NMRA, the modulation of AREA and AREB activity in response to the carbon status of the cell.

Expression of the arginase structural gene (agaA) in Aspergillus nidulans is subject to complex transcriptional and post-transcriptional regulation. Arginase mRNA has a long 5'-UTR sequence. Analysis of this sequence in silico revealed its putative complex secondary structure, the presence of arginine-binding motifs (arginine aptamers) and a short intron with two potential 3' splicing sites. In this report we present evidence that L-arginine (i) binds directly to the arginase 5'-UTR; (ii) invokes drastic changes in the secondary structure of the 5'-UTR, unlike several other L-amino acids and D-arginine; and (iii) forces the selection of one of two 3' splice sites of an intron present in the 5'-UTR. We postulate that expression of the eukaryotic structural gene coding for arginase in A. nidulans is regulated at the level of mRNA stability, depending on riboswitch-mediated alternative splicing of the 5'-UTR intron.

We have cloned and analysed the arcA gene which encodes a transcriptional activator necessary for the high-level expression of two genes for enzymes of the arginine catabolic pathway in Aspergillus nidulans: agaA (for arginase) and otaA (for ornithine transaminase, OTAse). Here we present complete genomic and cDNA sequences for, and describe the pattern of expression of, the arcA gene. This gene contains one intron and encodes a polypeptide of 600 amino acids. The deduced protein belongs to the family of Zn(2)Cys(6) fungal regulatory proteins. ARCA is the first known protein of this family that has glycine instead of the conserved proline at the fifth position in the second, six-residue, loop of the Zn cluster domain. We have established that transcription of the arcA gene is not self-regulated and does not depend on arginine. Two mutations in arcA, one gain-of-function and one loss-of-function, have been sequenced and the effects of these mutations on the expression of the agaA gene at the transcriptional level are reported.

The arginase structural gene (agaA) from Aspergillus nidulans has been cloned and characterised. Depending on the growth conditions of the mycelium, transcripts of this gene have different 5'ends. These differences could result either from the presence of multiple transcription initiation sites or from differential processing of mRNA. The agaA mRNA has a long 5'UTR with a potentially complex secondary structure. Putative arginine binding aptamers were found in this UTR suggesting interesting possibilities for regulation of the agaA expression.

The ornithine transaminase (otaA) gene of Aspergillus nidulans has been cloned by transformation of the A. nidulans pro – ota – mutant strain with a cosmid gene library. The otaA gene contains two introns and potentially codes for a 453-aa-long protein. The deduced amino-acid sequence is homologous to known ornithine transaminases from Saccharomyces cerevisiae, Plasmodium falciparum, Vigna aconitifolia, rat, mouse and man, particularly in the pyridoxal phosphate-binding domain. The expression of the otaA gene is specifically induced by arginine, and is also under the control of nitrogen-metabolite and carbon-catabolite repression. Regulation of the gene occurs at both transcriptional and post-transcriptional levels. The promoter region of otaA contains putative AREA and CREA binding-sites. Fusion proteins containing AREA or CREA DNA-binding domains bind some of these sites. CREA binding-sites correspond very well to the CREA-binding consensus sequence which is SYGGRG. AREA binding-sites are composed of GATT sequences which are not typical binding sites for the GATA - binding family of transcription factors.

An Aspergillus nidulans DNA fragment composed of two adjacent Sal I subfragments (1.8 and 0.85 kb) that carries an argB complementing the yeast arg3 mutation has been isolated from two different gene libraries. Hybridization results and immunological tests indicate that the cloned fragment contains the A. nidulans structural gene coding for ornithine carbamoyltransferase (OTCase). Using the cloned gene as a probe, the specific mRNA was identified. The level of this RNA observed in A. nidulans strains grown under various conditions correlated with the level of the OTCase activity, suggesting transcriptional control of OTCase synthesis. Expression of the cloned gene in Saccharomyces cerevisiae does not depend on its orientation in the vector. In Escherichia coli , the cloned gene does not function; however arg - transformants revert to phototrophy with high frequency possibly due to DNA rearrangements within the recombinant plasmid.

The synthesis of two enzymes of the arginine catabolic pathway, arginase and ornithine δ-transaminase (OTAse), in Aspergillus nidulans was found to be sensitive to both glucose and ammonium repression. The glucose and nitrogen starvation result in the identical derepression of OTAse synthesis and have no effects on arginase synthesis. Glucose and ammonium affect the kinetics of induction of both enzymes, however, the effect of ammonium is much stronger. Evidence was obtained for the direct involvement of ammonium in the repression phenomenon. The relations between glucose and ammonium repression are discussed.

Two kinds of mutants of Aspergillus nidulans with altered response of arginine catabolic enzymes to glucose and ammonium repression were obtained. Mutations in the suF locus result in the insensitivity of these enzymes to glucose and to one type of ammonium repression. Mutations in the AniA locus result in hypersensitivity to both types of repression. The enzymes studied can be induced by arginine in AniA mutants only when glucose or the nitrogen source is removed from the medium. The suF mutations are recessive while AniA are dominant. Double suF AniA mutants retain only the suF properties. The functions of both genes and their interrelations are discussed.

The kaeAKAE1 (suDpro) gene, which was identified in Aspergillus nidulans as a suppressor of proline auxotrophic mutations, encodes the orthologue of Saccharomyces cerevisiae Kae1p, a member of the evolutionarily conserved KEOPS/EKC (Kinase, Endopeptidase and Other Proteins of Small size/Endopeptidase-like and Kinase associated to transcribed Chromatin) complex. In yeast, this complex has been shown to be involved in tRNA modification, transcription, and genome maintenance. In A. nidulans, mutations in kaeA result in several phenotypic effects, the derepression of arginine catabolism genes, and changes in the expression levels of several others, including genes involved in amino acid and siderophore metabolism, sulfate transport, carbon/energy metabolism, translation, and transcription regulation, such as rcoATUP1, which encodes the global transcriptional corepressor.

The transcriptional organization and sequence of the Aspergillus nidulans argB gene, encoding ornithine carbamoyl transferase (OCTase; E.C. 2.1.3.3.), was determined. Transcription of the gene begins within a methionine-initiated open translation reading frame, indicating that a second methionine codon of the open reading frame is used for translation initiation. The predicted length of the OCTase precursor peptide is 359 amino acids, and it contains a highly basic amino terminus that is probably involved in mitochondrial targeting. There is extensive homology between Aspergillus OCTase and mammalian and bacterial OCTases and weaker homology between the Aspergillus polypeptide and bacterial arginine carbamoyl transferase.

Two regulatory mutants for arginine catabolism isolated as proline suppressors were tested for the synthesis of ornithine transcarbamylase (OTC), the arginine anabolic enzyme. Mutations at one locus, suD, result in the insensitivity of OTC synthesis to effectors responsible for the enzyme level in the wild strain. The common genetic regulation of both catabolic and anabolic pathways of arginine is postulated.

The induction of arginase in the fungus Aspergillus nidulans depends on the synthesis of messenger and its subsequent translation, as is shown by selective inhibition of the two processes by proflavine and actidione. In mycelium grown at 30°C messenger synthesis starts with no measurable delay but the enzyme appears 6 min after arginine addition. The stability of the enzyme is accompanied by the lability of its messenger, the coding capacity of which decays with the half-life of 2.7 min. The synthesis of messenger can be separated from its translation by including actidione (5 μg/ml) in an arginine-supplemented culture. After the withdrawal of inducer and inhibitor the expression of arginase messenger takes place. The 4.5-fold increased stability of messenger observed under conditions of blocked translation may be responsible for its accumulation in presence of actidione.

SUMMARY: Proline suppressor mutant su-19 was analysed for arginase and ornithine δ-transaminase (OTA) activity. Both enzymes are constitutively produced by this mutant. Five other suppressor loci not linked with su-19 or to each other are involved in regulation of the two enzymes. A mutant showing no OTA activity was isolated. Properties of this mutant and its effect in various strains of the pro su type were studied.