Examination of a small group mRNAs showing evidence of a conditional requirement for eIF2A when eIF2 is impaired.
(A) Volcano plot as in Figure 2A showing the log2 ratios of TEs in eIF2AD cells treated with SM versus WT cells treated with SM (log2ΔTEeIF2AΔ+SM/WT+SM values) for the 5482 mRNAs with evidence of translation. The dotted line marks the 25% FDR threshold. Genes exhibiting a significant increase (ΔTEeIF2AΔ+SM/WT+SM_up) or decrease (ΔTEeIF2AΔ+SM/WT+SM_down) at FDR < 0.25 are plotted in dark or light green circles, respectively. (B) Notched box plots of log2ΔTE values for the indicated mutant/condition for the 32 mRNAs in the group ΔTEeIF2AΔ+SM/WT+SM_down defined in (A). The y-axis scale was expanded by excluding a few outliers from the plots. Statistical significance determined using the Mann-Whitney U test is indicated for the changes in col. 4 compared to the changes observed for all mRNAs. (C) Hierarchical clustering analysis of log2ΔTE values for the 32 mRNAs (arrayed from top to bottom) in the group defined in (A) for the four comparisons listed across the top, with log2ΔTE values represented on a color scale ranging from 4 (dark blue) to -4 (dark red). The systematic gene names are listed for all 32 mRNAs, and the common name is indicated for those genes subjected to LUC reporter analysis below. Genes marked with “#”s display the pattern of TE changes consistent with conditional stimulation by eIF2A when eIF2 function is reduced by phosphorylation, showing Only 17 of the 32 transcripts (marked with “#”) displayed the diagnostic pattern of an appreciable reduction in TE both on elimination of eIF2A in SM-treated cells and on SM-treatment of cells lacking eIF2A (red or pink hues in cols. 1-2) but either a lesser reduction, no change, or increase in TE on SM-treatment of WT cells and on elimination of eIF2A from untreated cells (light pink, white or blue hues in cols. 3-4). (D) Expression of LUC reporters in different strains/conditions constructed for selected candidate genes analyzed in (C). The schematic depicts reporter construct design wherein the native gene promoter, 5’ UTR, and first 20 codons of the CDS are fused to firefly luciferase coding sequences (F.LUC), followed by a modified RPL41A 3’ UTR. Plasmid-borne reporter constructs were introduced into the WT and eIF2AD strains and three independent transformants were cultured in SC-Ura medium at 30°C to log phase (-SM) or treated with SM at 1 μg/mL after log-phase growth in SC-Ura/Ile/Val and cultured for an additional 6 h before harvesting. Luciferase activities were quantified in WCEs, normalized to total protein, and reported as fold change in relative light units (RLUs) per mg of protein, as means (±SEM) determined from the replicate transformants. The changes in luciferase activity plotted for each of the two comparisons depicted in the histogram were calculated as ratios of the appropriate mean activities. Results of student’s t-tests of the differences in fold changes between the indicated mutations/conditions are indicated. (E) Determination of relative TEs for the native mRNAs of selected candidate genes analyzed in (C & D. Cells were cultured in the four conditions described in (D) and WCEs were resolved by sedimentation through 10%-50% sucrose gradients and fractions were collected while scanning at 260 nm. Total RNA was extracted from 80S and polysome fractions, and the abundance of each target mRNA was quantified in each fraction by qRT-PCR, and normalized for (i) the amounts of 18S rRNA quantified for the same fractions and (ii) for the total amounts of monosomes/polysomes recovered in the gradient. The resulting normalized amounts of mRNA in each fraction were multiplied by the number of ribosomes per mRNA in that fraction, summed across all fractions, and divided by the input amount of mRNA in the WCEs, normalized to ACT1 mRNA, to yield the TEs for that mRNAs in each condition. (See Methods for further details.) The changes in TE conferred by SM treatment of WT or eIF2AD cells were calculated for each replicate culture, untreated of SM-treated, and the mean TE changes with SEMs were plotted for the indicated comparisons. The results of student’s t-tests of the differences in mean TE changes are indicated.