RrmA regulates the stability of specific transcripts in response to both nitrogen source and oxidative stress

Abstract

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.

Figure 1

Identification of AN9090 (rrmA) among proteins bound to areA mRNA 3′ UTR.A. Electrophoretic mobility shift assay (EMSA) of the conserved 3′ sequence of areA mRNA incubated in the presence of protein extracts from mycelia grown in the presence of Gln (Lane 1) or −N (Lane 2). As controls a 100× excess of unlabelled RNA was included in the incubation mixture (Lane 3) or the radiolabelled RNA sequence without protein (Lane 4).B. 2D gel electrophoretic separation of proteins bound to areA mRNA 3′ UTR after isolation using oligo-dT immobilized on magnetic beads. The three spots in the 50 kDa range were extracted, subjected to trypsin digestion and analysed using tandem mass spectrometry. MASCOT analysis of peptides from the middle spot identified this as AN9090 from five peptides together giving 16% sequence coverage.

Figure 2

RrmA is required to destabilize specific transcripts in response to Gln and nitrate stabilization of niaD mRNA.A. Northern analysis of areA (encoding a global transcription factor mediating nitrogen regulation) and meaA (encoding a high-affinity ammonium transporter) in both wild type and ΔrrmA strains is shown. In order to determine RNA degradation rates under conditions of nitrogen sufficiency (Gln) or nitrogen starvation (−N), transcript levels were monitored over a 30 min time-course after the inhibition of transcription. These two nitrogen regimes were chosen since both areA and meaA transcript stability is differentially regulated under these conditions.B. Northern analysis of niaD (encoding nitrate reductase) transcript levels in rrmA⁺ and ΔrrmA strains. Both strains include the gpd::crnA construct which constitutively expresses CrnA, a nitrate permease, thus facilitating nitrate uptake in the presence of repressing nitrogen sources such as Gln (Caddick et al., 2006b). Transcript levels were monitored under four nitrogen regimes; glutamine sufficiency (Gln), nitrogen starvation (−N), nitrate (NO₃⁻) or both glutamine and nitrate (Gln + NO₃⁻).Quantitative data from multiple (≥ 3) experiments was utilized to estimate the transcript median half-life (min) under each growth condition for areA (C), meaA (D) and niaD (E). Degradation rates were monitored over a 30 min time-course after transcription was inhibited. 18S rRNA was used as a loading control. Full statistical analysis of these data is presented in Tables S1, S2 and S3. *P < 0.05; **P < 0.01; ***P < 0.001; NS non-significant (P > 0.05).

Figure 3

Analysis of deadenylation of areA mRNA using RNase H assay. Total RNA was subjected to RNase H analysis using oligonucleotides specific to the areA 3′ UTR (Morozov et al., 2001). Total RNA was extracted from both wild type and ΔrrmA strains over a 30 min. time-course after the inhibition of transcription. Cultures were incubated either in the absence of a nitrogen source (−N) or in the presence of Gln, as indicted. A deadenylated control was included [-A], providing a reference point (A₀). Cycloheximide was added to the culture before the beginning of the time-course to prevent decapping and 5′-3′ degradation of the mRNA. The deadenylation and 3′-5′ degradation observed for the wild type in the presence of Gln is lost in the ΔrrmA strain.

Figure 4

Loss-of-function mutations in rrmA result in hypersensitivity to oxidative stress.A. The rrmA insertional mutant (rrmA::impala), the deletion strain (ΔrrmA) and the wild type (WT) were grown on complete medium with H₂O₂or tert-butyl hydroperoxide (t-BHP). This demonstrates increased sensitivity of rrmA⁻ mutants to oxidative stress.B. The involvement of polyamines in the oxidative stress response was shown by comparing wild type and ΔrrmA on minimal medium with H₂O₂, putrescine (put) and/or the ornithine decarboxylase inhibitor 1,4-diamino-2-butanone (DAB). In order to compare the response of the wild type and ΔrrmA strains to DAB and putrescine, H₂O₂ was used at the maximum concentrations which allowed growth; 12 mM for the wild type and 4 mM for the ΔrrmA strain.

Figure 5

RrmA influences the stability of eifE and dhsA mRNAs under oxidative stress conditions. Stability of the eifE transcript was determined by quantitative northern analysis with 18S rRNA as a control (A). For dhsA, qPCR was utilized due to the low level of expression (data not shown). In this case tubC, which encodes β-tubulin (May and Morris, 1988), was used as the reference. Stability was monitored in both wild type (WT) and ΔrrmA strains over a 30 min time-course after transcription was inhibited. The estimated median half-life for each transcript is given (B). In both cases there is a significant difference between the two strains (P < 0.01; **). Full statistical analysis of these data is presented in Table S6.

Figure 6

RrmA localization and role in P-body formation.A. Functionality of an RrmA::GFP fusion was assessed utilizing growth tests, with a near wild type growth phenotype and sensitivity to oxidative stress.B. Fluorescence microscopy revealed that RrmA::GFP is evenly distributed throughout the cytoplasm.C and D. P-body formation, as observed using Dpc1::GFP (Morozov et al., 2010b) in rrmA⁺ (C) or ΔrrmA (D) strains. In the ΔrrmA strain the P-bodies appeared normal, being evenly distributed and highly dynamic, as can be seen by comparing the four images taken at 10 min intervals. The number of P-bodies in each image is indicated and a 10 μm scale included.E–I. Quantitative analysis of the number of the P-bodies, normalized by cell volume, based on a minimum of four independent experiments with five to seven cells in each. The numbers given (± SE) were those observed in the rrmA⁺ (solid line) and ΔrrmA mutant (broken line) after different growth shifts. As a control, P-body number was monitored without shifting conditions (NH₄⁺) for 30 min (E). Compared with wild type no shift in numbers was observed on transfer to carbon free media (F) or after the addition of H₂O₂ to the media (G) suggesting the loss of a regulatory response. On transfer from ammonium to nitrate (NH₄⁺ to NO₃⁻), the number of P-bodies did change but this was less dramatic than in the wild type background (H). Cycloheximide treatment which leads to loss of P-bodies, probably due to disruption of RNA decapping and degradation (Beelman and Parker, 1994), was observed and they were lost at a greatly reduced rate in the ΔrrmA strain compared with wild type (I). This would be consistent with delayed degradation of mRNA associated with P-bodies.