Meriel G. Jones

The 2008 update of the Aspergillus nidulans genome annotation: A community effort

The identification and annotation of protein-coding genes is one of the primary goals of whole-genome sequencing projects, and the accuracy of predicting the primary protein products of gene expression is vital to the interpretation of the available data and the design of downstream functional applications. Nevertheless, the comprehensive annotation of eukaryotic genomes remains a considerable challenge. Many genomes submitted to public databases, including those of major model organisms, contain significant numbers of wrong and incomplete gene predictions. We present a community-based reannotation of the Aspergillus nidulans genome with the primary goal of increasing the number and quality of protein functional assignments through the careful review of experts in the field of fungal biology.

Characterization of nitrogen metabolite signalling in Aspergillus via the regulated degradation of areA mRNA

AreA is the principal transcription factor involved in determining nitrogen utilization in Aspergillus nidulans. NH4+ and Gln are utilized preferentially but in their absence, AreA acts to facilitate the expression of genes involved in metabolizing alternative nitrogen sources. It is crucial to the function of AreA that its expression is tightly modulated by the quality and availability of nitrogen sources. One signalling mechanism involves regulated degradation of the areA transcript in response to NH4+ and Gln, which provides the first direct means of monitoring nitrogen signalling in this fungus. Here we assess the specificity of the transcript degradation response, determining that it responds qualitatively to a variety of additional nitrogen sources including Asn. Furthermore, the response to Gln and NH4+ requires the same discrete region of the areA 3′‐UTR but both NH4+ and Asn need to be metabolized to Gln before they are effective as a signal. However, NH4+ signalling is independent of AreA activity, unlike Gln and Asn signalling. A mutation in the structural gene for NADP‐linked glutamate dehydrogenase, gdhA, which disrupts metabolism of NH4+ to Glu, is additive with mutations in two distinct regions of areA that disrupt the previously identified signalling mechanisms. The triple mutant is both strongly derepressed and expresses very high levels of nitrate reductase activity. These data suggest nitrogen metabolism in A. nidulans is in part regulated in response to the intracellular levels of Gln via the regulated degradation of areA mRNA, but the intracellular Gln level is not the sole determinant of nitrogen metabolite repression.

A defined sequence within the 3′ UTR of the areA transcript is sufficient to mediate nitrogen metabolite signalling via accelerated deadenylation

Nitrogen metabolism in Aspergillus nidulans is regulated by AREA, a member of the GATA family of transcription factors. One mechanism that modulates AREA activity involves the rapid degradation of the areA transcript when sufficient NH4+ or Gln are available. This signalling mechanism has been shown to require a region of 218 nucleotides within the 3′ untranslated region of areA mRNA. We demonstrate that this region functions independently in a heterologous transcript and acts to accelerate degradation of the poly(A) tail, which in turn leads to rapid transcript degradation in response to the addition of NH4+ or Gln to the growth medium. areA transcript degradation is inhibited by cycloheximide, but this is not a general consequence of translational inhibition. We believe that this is the first reported example in which specific physiological signals, acting through a defined sequence within a transcript, have been shown to promote accelerated poly(A) degradation, which in turn triggers transcript degradation.

Genetic analysis of the TOR pathway in Aspergillus nidulans.

ABSTRACT We identified five genes encoding components of the TOR signaling pathway within Aspergillus nidulans. Unlike the situation in Saccharomyces cerevisiae, there is only a single Tor kinase, as in plant and animal systems, and mutant phenotypes suggest that the TOR pathway plays only a minor role in regulating nitrogen metabolism.

The Aspergillus nidulans GATA transcription factor gene areB encodes at least three proteins and features three classes of mutation

In Aspergillus nidulans, the principal transcription factor regulating nitrogen metabolism, AREA, belongs to the GATA family of DNA‐binding proteins. In seeking additional GATA factors, we have cloned areB, which was originally identified via a genetic screen for suppressors of areA loss‐of‐function mutations. Based on our analysis, areB is predicted to encode at least three distinct protein products. These arise from the use of two promoters, differential splicing and translation initiating at AUG and non‐AUG start codons. All the putative products include a GATA domain and a putative Leu zipper. These regions show strong sequence similarity to regulatory proteins from Saccharomyces cerevisiae (Dal80p and Gzf3p), Penicillium chrysogenum (NREB) and Neurospora crassa (ASD4). We have characterized three classes of mutation in areB; the first are loss‐of‐function mutations that terminate the polypeptides within or before the GATA domain. The second class truncates the GATA factor either within or upstream of the putative Leu zipper but retains the GATA domain. The third class fuses novel gene sequences to areB with the potential to produce putative chimeric polypeptides. These novel gene fusions transform the putative negative‐acting transcription factor into an activator that can partially replace areA.

CUCU Modification of mRNA Promotes Decapping and Transcript Degradation in Aspergillus nidulans

ABSTRACT In eukaryotes, mRNA decay is generally initiated by the shortening of the poly(A) tail mediated by the major deadenylase complex Ccr4-Caf1-Not. The deadenylated transcript is then rapidly degraded, primarily via the decapping-dependent pathway. Here we report that in Aspergillus nidulans both the Caf1 and Ccr4 orthologues are functionally distinct deadenylases in vivo: Caf1 is required for the regulated degradation of specific transcripts, and Ccr4 is responsible for basal degradation. Intriguingly disruption of the Ccr4-Caf1-Not complex leads to deadenylation-independent decapping. Additionally, decapping is correlated with a novel transcript modification, addition of a CUCU sequence. A member of the nucleotidyltransferase superfamily, CutA, is required for this modification, and its disruption leads to a reduced rate of decapping and subsequent transcript degradation. We propose that 3′ modification of adenylated mRNA, which is likely to represent a common eukaryotic process, primes the transcript for decapping and efficient degradation.

Opposing signals differentially regulate transcript stability in Aspergillus nidulans

A good model for gene regulation, requiring the organism to monitor a complex and changing environment and respond in a precise and rapid way, is nitrogen metabolism in Aspergillus nidulans. This involves co‐ordinated expression of hundreds of genes, many dependent on the transcription factor AreA, which monitors the nitrogen state of the cell. AreA activity is in part modulated by differential degradation of its transcript in response to intracellular glutamine. Here we report that glutamine triggers synchronized degradation of a large subset of transcripts involved in nitrogen metabolism. Among these are all four genes involved in the assimilation of nitrate. Significantly, we show that two of these transcripts, niaD and niiA, are stabilized by intracellular nitrate, directly reinforcing transcriptional regulation. Glutamine‐signalled degradation and the nitrate‐dependent stabilization of the niaD transcript are effected at the level of deadenylation and are dependent on its 3′ UTR. When glutamine and nitrate are both present, nitrate stabilization is predominant, ensuring that nitrate and the toxic intermediate nitrite are removed from the cell. Regulated transcript stability is therefore an integral part of the adaptive response. This represents the first example of distinct physiological signals competing to differentially regulate transcripts at the level of deadenylation.

mRNA 3′ Tagging Is Induced by Nonsense-Mediated Decay and Promotes Ribosome Dissociation

ABSTRACT For a range of eukaryote transcripts, the initiation of degradation is coincident with the addition of a short pyrimidine tag at the 3′ end. Previously, cytoplasmic mRNA tagging has been observed for human and fungal transcripts. We now report that Arabidopsis thaliana mRNA is subject to 3′ tagging with U and C nucleotides, as in Aspergillus nidulans. Mutations that disrupt tagging, including A. nidulans cutA and a newly characterized gene, cutB, retard transcript degradation. Importantly, nonsense-mediated decay (NMD), a major checkpoint for transcript fidelity, elicits 3′ tagging of transcripts containing a premature termination codon (PTC). Although PTC-induced transcript degradation does not require 3′ tagging, subsequent dissociation of mRNA from ribosomes is retarded in tagging mutants. Additionally, tagging of wild-type and NMD-inducing transcripts is greatly reduced in strains lacking Upf1, a conserved NMD factor also required for human histone mRNA tagging. We argue that PTC-induced translational termination differs fundamentally from normal termination in polyadenylated transcripts, as it leads to transcript degradation and prevents rather than facilitates further translation. Furthermore, transcript deadenylation and the consequent dissociation of poly(A) binding protein will result in PTC-like termination events which recruit Upf1, resulting in mRNA 3′ tagging, ribosome clearance, and transcript degradation.

An Electrophoretic Study of Enzymes as a Tool in the Taxonomy of the Dermatophytes

Zymogram patterns from 84 strains of dermatophyte fungi were obtained using polyacrylamide gradient gel electrophoresis of total cell protein extracts. The enzymes investigated were alpha-naphthyl acetate esterase, acid phosphatase, lactate dehydrogenase, malate dehydrogenase, tetrazolium oxidase and catalase. These patterns were used to construct similarity matrices and dendrograms using computerized techniques. The results showed that zymograms allowed some species to be readily recognized despite morphological variation. This was also seen in the dendrograms where, in addition, groupings based on ecological or sexual criteria could be distinguished.

Distinct roles for Caf1, Ccr4, Edc3 and CutA in the co-ordination of transcript deadenylation, decapping and P-body formation in Aspergillus nidulans

Transcript degradation is a key step in gene regulation. In eukaryotes, mRNA decay is generally initiated by removal of the poly(A) tail mediated by the Ccr4–Caf1–Not complex. Deadenylated transcripts are then rapidly degraded, primarily via the decapping‐dependent pathway. Components of this pathway can be localized into highly dynamic cytoplasmic foci, the mRNA processing (P)‐bodies. We have undertaken confocal fluorescence microscopy to monitor P‐bodies in Aspergillus nidulans. As in other organisms a dynamic shift in P‐body formation occurs in response to diverse physiological signals. Significantly, both this cellular response and the signalled degradation of specific transcripts are dependent on the nuclease activity of Caf1 but not Ccr4. P‐body formation is disrupted in A. nidulans strains deleted for Edc3, an enhancer of decapping, or CutA, which encodes a nucleotidyltransferase that triggers mRNA decapping by the addition of a CUCU tag to the poly(A) tail. As with ΔcutA, Δedc3 led to reduced rates of transcript degradation. These data link P‐bodies to both the optimization and regulation of transcript degradation.