Pre-mRNA splicing is modulated by antifungal drugs in the filamentous fungus Neurospora crassa

Abstract

For this study, we sought to identify pre-mRNA processing events modulated by changes in extracellular pH, inorganic phosphate, and antifungal drugs. We examined genes with at least four putative introns whose transcriptional level responded to these effectors. We showed that the intron retention levels of genes encoding asparagine synthetase 2, C6-zinc finger regulator (fluffy), and a farnesyltransferase respond to amphotericin B, ketoconazole, and other effectors. In general, the assayed antifungals promoted the disruption of the structural domains of these proteins probably leading to their inactivation, which emphasize the complexity of the metabolic modulation exerted by antifungal signaling.

Keywords: Neurospora crassa; Pi regulation; alternative splicing; amphotericin B; asparagine synthetase; intron retention; ketoconazole.

Figure 1

In vitro susceptibility of two N. crassa strains exposed to antifungal drugs at pH 5.4. The inhibition zones, measured in mm, were plotted against antifungal concentrations. The St.L.74A and Δmak‐2 strains were incubated in the absence of antifungal (control) (green circles). Strains were also incubated with ketoconazole (A), amphotericin B (B), terbinafine (C), and nystatin (D). Blue squares and red triangles represent the inhibition zones observed for the St.L.74A and Δmak‐2 strains, respectively.

Figure 2

Intron retention evaluated by RNAseq in N. crassa. The Sequence Read Archive at the NCBI database (http://www.ncbi.nlm.nih.gov/sra) was used to quantify intron retention. Reads were then aligned to fasta files containing sequences of 50 nt representing each exon‐intron boundary for the genes of interest. The number of reads mapping at each boundary was used to estimate the prevalence of intron retention in each gene analyzed (5′‐ gray; 3′‐ white). (A) Asparagine synthetase 2 (asn‐2) (NCU04303); (B) C6‐zinc finger regulator (fluffy) (NCU08726); (C) farnesyltransferase (ram1) (NCU05999).

Figure 3

(I) Retention of intron‐3 visualized by RT‐PCR during pre‐mRNA processing of the asn‐2 gene in N. crassa. Strains Δmak‐2 mutant and St.L.74A were incubated for 5 h and 16 h in high‐ (10 mm) (↑) or low‐Pi (100 μm) (↓) at pH 5.4 and pH 8.0 in the absence of antifungal (control) (A, B), with amphotericin B (C, D), and with ketoconazole (E, F). (M) Molecular weight ladder. Sizes expected for the amplified fragments were 49 bp and 188 bp for nonretention or retention of the intron, respectively. (II) Schematic overview of intron‐3 retention, as compared to the genomic DNA and mRNA organization of the asn‐2 gene of N. crassa.

Figure 4

Real‐time PCR (qRT‐PCR) validation of intron‐3 transcript levels in the asn‐2 gene in N. crassa. Strains Δmak‐2 mutant and St.L.74A were incubated for 5 h and 16 h in high‐ (10 mm) (↑) or low‐Pi (100 μm) (↓), at pH 5.4 and pH 8.0, in the absence of antifungal (control) (A, B), with amphotericin B (C, D), and with ketoconazole (E, F). qRT‐PCR data are representative of the average values ± standard deviation (SD) obtained from three independent experiments. Statistically significant values are indicated by asterisks: Tukey's ad hoc test, ***P < 001.

Figure 5

(I) Retention of intron‐4 visualized by RT‐PCR during pre‐mRNA processing of the asn‐2 gene in N. crassa. Strains Δmak‐2 mutant and St.L.74A were incubated for 5 h and 16 h in high‐ (10 mm) (↑) or low‐Pi (100 μm) (↓) at pH 5.4 in the absence (A) and the presence of ketoconazole (B). (M) Molecular weight ladder. Sizes expected for the amplified fragments are 57 and 142 bp for nonretention or retention of the intron, respectively. (II) Schematic overview of intron‐4 retention, as compared to the genomic DNA and mRNA organization of the asn‐2 gene of N. crassa.

Figure 6

(I) Retention of intron‐5 visualized by RT‐PCR during pre‐mRNA processing of the asn‐2 gene in N. crassa. Strains Δmak‐2 mutant and St.L.74A were incubated for 5 h and 16 h in high‐ (10 mm) (↑) or low‐Pi (100 μm) (↓) at pH 5.4 and pH 8.0 in the absence of antifungal (A, B), with amphotericin B (C, D), and with ketoconazole (E, F). (M) Molecular weight ladder. Sizes expected for the amplified fragments are 112 and 182 bp for nonretention or retention of the intron, respectively. (II) Schematic overview of intron‐5 retention, as compared to the genomic DNA and mRNA organization of the asn‐2 gene of N. crassa.

Figure 7

(I) Retention of intron‐1 visualized by RT‐PCR during pre‐mRNA processing of the fluffy gene in N. crassa. Strains Δmak‐2 mutant and St.L.74A were incubated for 5 h and 16 h in high‐ (10 mm) (↑) or low‐Pi (100 μm) (↓) at pH 8.0 in the absence (A) and presence (B) of ketoconazole. (M) Molecular weight ladder. Sizes expected for the amplified fragments are 149 and 299 bp for nonretention or retention of the intron, respectively. (II) Schematic overview of intron‐1 retention, as compared to the genomic DNA and mRNA organization of the fluffy gene of N. crassa.

Figure 8

(I) Retention of intron‐3 visualized by RT‐PCR during pre‐mRNA processing of the gene coding for farnesyl transferase beta subunit Ram1 in N. crassa. Strains Δmak‐2 mutant and St.L.74A were incubated for 5 h and 16 h in high‐ (10 mm) (↑) or low‐Pi (100 μm) (↓) at pH 5.4 in the absence (A) and presence (B) of ketoconazole. (M) Molecular weight ladder. Sizes expected for the amplified fragments are 313 and 503 bp for nonretention or retention of the intron, respectively. (II) Schematic overview of intron‐3 retention, as compared to the genomic DNA and mRNA organization of the gene coding for a farnesyltransferase (beta subunit Ram1) of N. crassa.