Article Figures & Data
Figures
- FIG. 1.
Phenotypic and transcriptional analysis of the A. niger ΔhacA strain. (A) Growth of the ΔhacA strain compared to that of wild-type strain AB4.1 on different agar media. MM, minimal medium; CM, MM supplemented with 0.5% yeast extract; PDA, potato dextrose agar; tm, 10 μg/ml tunicamycin. (B) Northern blot analysis showing the lack of a functional UPR in the ΔhacA strain and the restoration of the response by transforming the ΔhacA strain with plasmid pHacA-pyrG*.
- FIG. 2.
Mutational analysis of the uORF and the putative 5′ splice site, two elements present in the 5′UTR of hacAu mRNA. (A) Schematic overview of the 5′UTR of the hacA gene. ATG indicates the start codon of hacA, UPREs are indicated by open boxes, the starts of hacAu and hacAi mRNAs are indicated by black boxes, and the 44-codon uORF is indicated by an open arrow. The mutations introduced to disrupt the uORF and the putative 5′ splice site are indicated below. The putative IreA cutting site is indicated by an arrow. (B) Northern blot analysis showing the effect of ER stress imposed by tunicamycin (tm) on the transcription of hacA, bipA, and pdiA. 18S rRNA was used as a loading control. (C) Relative bipA levels in the wild type (wt), the ΔuORF strain, and the mut-5′ splice-site strain in the absence (black bars) and presence (gray bars) of 20 μg/ml tunicamycin.
- FIG. 3.
Role of the UPREs in regulation of hacA. (A) Schematic overview of the promoter region of hacA. The transcription start points of hacAu and hacAi are indicated by black boxes. The UPREs are represented by open boxes. The binding constant of each UPRE is shown at the bottom. (B) EMSAs showing the inability of HacA to bind to the mutated UPREs. The sequences of the 34-bp oligonucleotides representing the UPREs are shown, and mutations introduced into each UPRE to abolish binding by HacA are indicated in italics and are underlined. wt, wild type. (C) Northern analysis showing the effect of mutated UPREs on transcription levels of hacA, bipA, and pdiA. ER stress was imposed by the addition of 20 mM DTT to the medium. (D) Relative hacA mRNA levels in the different UPRE mutants under nonstress conditions.
- FIG. 4.
Phenotypes of the strains used in this study. (A) Growth of wild-type (wt) and mutant strains on CM plates in the absence (top row) or in the presence (bottom row) of 10 μg/ml tunicamycin (tm). (B) Microscope images of the wild-type strain, the hacA deletion strain (ΔhacA), the strain bearing the intronless hacA gene (Δintron), and the strain bearing the unspliceable hacA variant (mut-intron).
- FIG. 5.
Function of the unconventional intron and long-range base pairing in the regulation of the UPR. (A) Schematic overview indicating the unconventional intron and the location of the inverted repeats in relation to other elements present in hacA. The hacA ORF is indicated by a solid black arrow, and the 5′UTR is indicated by a gray box. The inverted repeats (IR) are depicted by open boxes, and the base pairing of the inverted repeats is shown at the bottom. The theoretically remaining base pairings after the introduction of silent mutations are also indicated. The structure of the unconventional intron is depicted by the stem-loop structure, and mutations introduced to prevent splicing by disrupting the structure are indicated. The intron sequence is shown in lowercase type, and cleavage sites are indicated by arrows. (B) Northern blot analysis showing the effect of the different mutations or deletions on the expression and truncation of hacA and on the transcript levels of bipA and pdiA. ER stress was imposed by treatment with tunicamycin (tm), and the gene coding for 18S rRNA was used as a control probe. (C) RT-PCR showing the transcript levels of bipA and pdiA in the wild-type (wt) and mut-intron strains. The four lanes under each condition correspond to 10, 15, 20, and 25 PCR cycles.
- FIG. 6.
Schematic overview of hacA regulation. The model depicts proteins and mRNAs between brackets indicating that their (active) concentration can vary. Elements present in the hacA promoter are in gray boxes, and elements present in the hacA mRNA are in white boxes. The splicing of full-length hacA (fl-hacAu) and truncated hacA (tr-hacAu) by IreA is depicted by the on/off switches. Translational attenuation is indicated by the thin arrow, compared to the arrow indicating nonattenuated translation. IR, inverted repeat.
Tables
- TABLE 1.
PCR oligonucleotide primers used in this studya
Oligonucleotide Sequence Description P1 5′-GGAGAACCAAGGTGCTATTG −2,071-bp hacA start codon P2 5′-GTTTAGGAGCCTTTACCTCC +1,152-bp hacA stop codon P3 5′-ATTTACCGTACGGTCAATTGGGGC −503-bp hacA start codon P4 5′-CCCTGCCTGTACTGACGAGTCATC +120-bp hacA start codon P5 5′-GGCCTGATCTGAACACGGACGCCTTTTAAAGAGT UPRE1 P6 5′-TCCCCCGTTATGACACGGACGCCTGTGTTCCTGT UPRE2 P7 5′-ATGGTTCTTAAGGACACCACTCCTTCTTGGCCCT UPRE3 P8 5′-TCTTTTTATTGTTCTCTGGTTCTTAAGGACACC ATG uORF P9 5′-CGACCTACATCACCGTCCTCCCAACGTCAGCGGTTAAGATAAGGCTCATAGTAAATCGATTG 5′ splice site P10 5′-TAGTGCCGTCCTCTGCGATCTTCA Unspliceable intron us P11 5′-TGAAGATCGCAGAGGACGGCACTA Unspliceable intron ls P12 5′-GCCTGGGTTAGCGCCCCCTGCAAGCCCCGTTATGACACGGTGGCCTGTGTTC Inverted repeat 5′UTR P13 5′-GACAGGTAATTCCTGCCCCCATGACTTTCTCTTCTTCACAGG Inverted repeat hacA gene P14 5′-TCCCCCGTTATGACACGGTGGCCTGTGTTCCTGT −168-bp hacA start codon P15 5′-TCAAACCGCTCAAGATTCGTTT +1,253-bp hacA start codon P16 5′-CGTCGAGAACGTCAAAGGCGAACCCGTC For 5′RACE hacA P17 5′-TTCGATGACAAGGATGTCCAGA For RT-PCR bipA P18 5′-GGGGATGAGCTTGGTCATGA For RT-PCR bipA P19 5′-ATTTAAATACCCTCTCCCATCGTCCTC For RT-PCR pdiA P20 5′-CCTCCTCGGCGGTGCAGTCAACCTTCAC For RT-PCR pdiA ↵ a Mutations are represented in boldface type.
