FIG 4 .

Comparative transcriptional profiling shows major dysregulation of iron metabolism-associated genes in the aft1Δ mutant and reveals that Cth2 is a potential posttranscriptional regulator. (a) Relative mRNA levels of iron-associated genes in the WT strain compared to the aft1Δ, sef1Δ, and ftr1Δ mutants under iron starvation conditions. Microarray data are displayed in a normalized log₂ scale for downregulation (negative numbers, shaded red), upregulation (positive numbers, shaded green), or no detected expression (n.d.) of target genes. Asterisks (*) indicate target genes that carry one or multiple copies of the Aft1 consensus-binding site PyPuCACCCPu in the 5′ UTR. (b) qRT-PCR expression of the following genes affected by SEF1 deletion relative to the levels seen with the WT at 0 h: ACO1 (encoding mitochondrial aconitase), ISA1 (encoding an iron sulfur cluster assembly), and IDH2 (encoding isocitrate dehydrogenase). Data represent means of results of biological triplicate experiments ± SEM (nd, no expression detected). Data are from an unpaired Student’s t test and represent results of comparisons to WT results from the same time point (*, P value < 0.05; **, P value < 0.01). (c) The postulated target consensus motif of the Cth2 mRNA-degrading protein (5′-UUAUUUAUU-3′) is found in the downstream sequences of 36 (of 106) genes normally downregulated in the WT strain upon iron starvation (0 h to 4 h) (volcano plot; 2-fold downregulation; P value = 0.05; false-discovery-rate [FDR] corrected) but is artificially upregulated in the aft1Δ strain (4 h against WT) (volcano plot; 2-fold upregulation; P value = 0.05; FDR corrected). Motifs (red box, forward strand; blue box, reverse strand) are shown for the previously displayed striking microarray genes in the annotated 3′ intergenic region (lower gray line) or the predicted 3′ UTR of the target gene (upper gray line) in proportion to the actual length (1 dot represents 4 bp; // indicates a discontinued display). (d) Growth of the cth2Δ mutant under low-iron conditions (10 µM FeCl₃), intermediate-iron conditions (500 µM FeCl₃), and near-toxic iron conditions (40 mM FeCl₃) in Chelex-treated SD (pH 5.8) compared to the WT strain (zero, shaded white) determined by assessing the time of half-maximal growth (H) and the plateau height (P). Positive numbers indicate improved growth (shaded green), and negative numbers indicate growth defects (shaded red). Data represent means of results from triplicate experiments. (e) Growth of the cth2Δ mutant in the presence of the iron chelator BPS (50 µM) and under alkaline conditions (pH 6.4) on solid media.