Elimination eIF2A has no effect on bulk protein synthesis in the presence or absence of amino acid starvation.

(A) Polysome profiles of WT strain (BY4741) and eIF2AD mutant (F2247) untreated (i)-(ii) or treated with SM (iii)-(iv). For (i)-(ii), cells were cultured in SC medium at 30°C to log-phase and treated with 50 μg/mL of cycloheximide 5 min prior to harvesting. For (iii)-(iv), cells were cultured in SC medium lacking Ile/Val and treated with 1 µg/mL of SM for 20 min before addition of cycloheximide. Cell extracts were resolved by sedimentation through sucrose density gradients and scanned continuously at 260 nm during fractionation. The plots show the A260 measured across the gradient with the top of the gradient on the left. (B) Schema of translational control of GCN4 mRNA, wherein translation of the main CDS is induced by phosphorylation of eIF2α through a specialized “delayed reinitiation” process mediated by four short upstream open reading frames (uORFs). (See text for details). (C) Genome browser view of ribosome profiling data for GCN4 mRNA. Tracks display RPF or mRNA reads mapped across the transcription unit, with the scales given in rpkm (Reads Per Kilobase of transcript per Million mapped reads). Data are presented for WT (blue) and eIF2Δ cells (purple) with or without SM treatment, as indicated. Each genotype/treatment includes two biological replicates, designated _a and _b. The main CDS is shown schematically in orange below the tracks; the four uORFs are in grey. The calculated values for log2ΔTEWT+SM/WT and log2ΔTEeIF2AΔ+SM/eIF2AΔ+SM and the respective FDRs are shown on the right.

eIF2A is not critical for translation of any individual mRNAs in non-starved cells and has little impact on the reprogramming of TEs conferred by amino acid starvation.

(A) Volcano plot depicting the log2 ratios of TEs in eIF2AD versus WT cells (log2ΔTEeIF2AΔ/WT values) for each mRNA (x-axis) versus negative log10 of the FDR (y-axis) determined by DESeq2 analysis of ribosome profiling data for the 5340 mRNAs with evidence of translation. Genes showing a significant increase in TE in eIF2AD versus WT cells at FDR < 0.25 (ΔTEeIF2AΔ+SM/WT_up) are plotted in orange circles. The dotted line marks the 25% FDR threshold, below which all other 5337 mRNAs are plotted in grey. (B) Volcano plot as in (A) showing the log2 ratios of TEs in WT+SM cells versus WT cells (log2ΔTEWT+SM/WT values) for the 5441 mRNAs with evidence of translation. The dotted line marks the 1% FDR threshold. Genes showing a significant increase (ΔTEeIF2AΔ+SM/WT_up) or decrease (ΔTEWT+SM/WT_down) in TE in WT+SM versus WT cells at FDR < 0.01, are plotted in magenta and pink circles, respectively. (C) Hierarchical clustering analysis of log2ΔTE values for the 1884 mRNAs (arrayed from top to bottom) that exhibit significant TE decreases or increases in SM-treated versus untreated WT cells at FDR < 0.01 (defined in (B)) conferred by SM treatment of WT cells (col. 1) or SM treatment of eIF2AD cells (col. 2), with the log2ΔTE values represented on a color scale ranging from 4 (dark blue) to -4 (dark red). The Pearson coefficient (r) and corresponding p-value for the correlation between log2ΔTE values in the two cols. are indicated below. (D) Notched box plots of log2ΔTE values for the indicated mutant/condition for all mRNAs (cols. 1-4) or for the indicated mRNA groups identified in (B). The y-axis scale was expanded by excluding a few outliers from the plots.

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.

eIF2A has little or no effect on the translation of three mRNAs reported to contain IRESs.

Genome browser views of RPF and RNA reads from ribosome profiling data for (A) URE2 mRNA (B) GIC1 mRNA and (C) PAB1 mRNA presented as in Figure 1C. The calculated values for log2ΔTEeIF2AΔ/WT and log2ΔTEeIF2AΔ+SM/WT+SM with the respective FDRs are shown on the right. The scale is shown on the left. The region containing the URE2 IRES is enclosed in a dotted box, with the AUG start codon highlighted in red. Locations of the GIC1 and PAB1 IRESs have not been defined.

eIF2A plays little or no role in uORF-mediated translational control of CPA1 or YAP2/CAD1 mRNA.

Genome browser views of RPF and RNA reads from ribosome profiling data for (A) CPA1 mRNA and (B) YAP2/CAD1 mRNA presented as in Figures 1C & 4. CDS and uORFs are represented in orange and grey rectangles, respectively.

Minimal effects of eliminating eIF2A on translation of mRNAs harboring translated uORFs.

(A) Notched box plots of log2ΔTE values for all mRNAs (for which TEs could be determined from our ribosome profiling data) containing annotated AUG- or NCC-uORFs (i), conserved AUG- or NCC-uORFs (ii), or single functional inhibitory AUG-uORFs (iii), conferred by SM treatment of WT cells (maroon), by the eIF2AD mutation in untreated cells (orange), or by the eIF2AD mutation in SM-treated cells (green). Statistical significance determined using the Mann-Whitney U test is indicated for selective comparisons of changes observed for the indicated groups in comparison to the changes for all mRNAs. A few outliers were omitted from the plots to expand the y-axis scale. (B) Notched box plots as in (A) for the subsets of the same mRNA groups analyzed there exhibiting > 1.41-fold increases in TE in SM-treated versus untreated WT cells. A few outliers were omitted from the plots to expand the y-axis scale. Statistical significance determined as in (A). (C) Notched box plots as in (A-B) for the subsets of the mRNA groups analyzed there exhibiting > 1.41-fold decreases in TE in SM-treated eIF2AΔ versus SM-treated WT cells. A few outliers were omitted from the plots to expand the y-axis scale. Statistical significance determined as in (A). (D) Proportional Venn diagram showing overlap between the 17 mRNAs identified in Figure 3A showing evidence for a conditional requirement for eIF2A when eIF2 function is reduced by SM (transcripts marked with “#”s) and the 514 mRNAs bearing functional AUG or NCC-uORFs.

eIF2A plays no major role in stimulating translation elongation for particular tripeptide motifs.

(A) Scatterplot of average pause scores for 8006 tripeptide motifs, comparing the two biological replicates of ribosome profiling data for eIF2AΔ versus WT cells. Each dot on the plot represents a tripeptide motif. Pause scores were computed using a shift value of 18 nt from the 3′-end of the footprint, positioning the first codon of the tripeptide motif in the E site. (B) Scatterplot of average pause scores for 6267 tripeptide motifs, comparing the two biological replicates of SM-treated versus untreated WT cells. All 351 detected motifs with valine codons in the A site are highlighted in red. Pause scores were computed as in (A).

Yeast strains used in this study.

Plasmids used in this study.

Primers used in this study.

High reproducibility between biological replicates of ribosome footprint profiling and RNA-seq analyses.

(A-H) Scatterplots depict the RPF (A, C, E, G) or mRNA (B, D, F, H) read densities for all expressed mRNAs across biological replicates of the WT (A, B), the eIF2AD mutant (C, D) SM-treated WT (E, F) and SM-treated eIF2AD mutant (G, H). The read densities were calculated by mapping the reads to the CDS of each gene and expressed as reads per million mapped reads (RPM) in individual libraries of biological replicates. The Pearson’s coefficient (r) is indicated in each plot, quantifying the degree of correlation between the replicate datasets.

Relative TE changes evoked by increased eIF2α phosphorylation in cells lacking eIF2A are broadly similar to relative TE changes conferred by increased eIF2α phosphorylation in WT cells.

(A) Volcano plot as in Figure 2A showing the log2 ratios of TEs in SM-treated eIF2AΔ versus untreated eIF2AD cells (ΔTEeIF2AΔ+SM/eIF2AΔ values) for the 5426 mRNAs with evidence of translation. The dotted line marks the 1% FDR threshold. Genes showing a significant increase (ΔTEeIF2AΔ+SM/eIF2AΔ_up) or decrease (ΔTEeIF2AΔ+SM/eIF2AΔ_down) in TE in SM-treated eIF2AD versus eIF2AD mutant cells at FDR < 0.05, are plotted in dark and light blue circles, respectively. (B) Proportional Venn diagram showing overlap between the 1884 mRNAs identified in Figure 2B and the 786 mRNAs identified in Figure 2-figure supplement 1A.

Representative separation of polysomes by sedimentation through a sucrose density gradient in the experiment depicted in

Figure 3E.

The A260 values were determined continuously during fractionation of the gradient. Fractions pooled for isolation of RNA from 80S monosomes or the various polysomal species are indicated by boxes.

eIF2A plays a minimal in regulating uORF-mediated translation.

(A-B) Smoothed scatterplots displaying the relationship between log2RROWT (x-axis) and log2RROeIF2AΔ (y-axis) for all mRNAs containing annotated AUG- or NCC-uORFs (A) or evolutionarily conserved AUG- or NCC-uORFs (B) in WT versus eIF2AD cells without SM treatment. No mRNAs showed ≥2-fold changes in RRO in the eIF2AD mutant versus WT cells at FDR < 0.5. (C-D) Smoothed scatterplots displaying the relationship between log2RROWT+SM (x-axis) versus log2RROeIF2AΔ+SM (y-axis) for the same mRNAs analyzed in (A)-(B) but in the presence of SM. Again, no mRNAs showed ≥2-fold changes in RRO in the eIF2AD mutant versus WT cells at FDR < 0.5. (E-H) Notched box plot displaying log2RRO values for all mRNAs containing annotated AUG- or NCC-uORFs (E, G) or evolutionarily conserved AUG- or NCC-uORFs (F, H) in untreated WT and eIF2AD mutant (E-F) or SM-treated WT and eIF2AD mutant (G-H). The y-axis scale was expanded by omitting a few outliers. Statistical significance determined using the Mann-Whitney U test is shown for the bracketed comparisons in panels E & G.

Lack of genetic interaction between eIF2A and the purine salvage pathway.

(A-B) Cell spotting assays were performed on SC plates (A) or SD (B) plates to assess the growth of WT, eIF2AD, fcy2D and eIF2AD fcy2D strains. Ten-fold serial dilutions of saturated cultures were applied to SC or SD plates supplemented with the indicated concentrations of adenine and incubated at 30°C for 2 days. (C) WT and eIF2AD strains were transformed with the indicated lacZ reporter plasmids-IMD2 and IMD3. The transformants bearing the reporter plasmids-IMD2 containing IMD2 coding sequences along with 1186 bp upstream region while IMD3 contains IMD3 coding sequences containing 555 bp upstream region-were grown in SC-Ura to saturation. The cultures were then diluted in fresh SC-Ura containing 0.015 mM concentration of Adenine and grown for 6 h to A600 of ∼1.0. WCEs were prepared and assayed for β-galactosidase activities in units of nmol of ONPG cleaved per mg of protein per min. The results represent the fold change of means and ±SEMs of activities calculated from three independent transformants.