Yeast eIF2A has a minimal role in translation initiation and uORF-mediated translational control in vivo

Initiating translation of most eukaryotic mRNAs depends on recruitment of methionyl initiator tRNA (Met-tRNAi) in a ternary complex (TC) with GTP-bound eukaryotic initiation factor 2 (eIF2) to the small (40S) ribosomal subunit, forming a 43S preinitiation complex (PIC) that attaches to the mRNA and scans the 5’-untranslated region (5’ UTR) for an AUG start codon. Previous studies have implicated mammalian eIF2A in GTP-independent binding of Met-tRNAi to the 40S subunit and its recruitment to specialized mRNAs that do not require scanning, and in initiation at non-AUG start codons, when eIF2 function is attenuated by phosphorylation of its α-subunit during stress. The role of eIF2A in translation in vivo is poorly understood however, and it was unknown whether the conserved ortholog in budding yeast can functionally substitute for eIF2. We performed ribosome profiling of a yeast deletion mutant lacking eIF2A and isogenic wild-type (WT) cells in the presence or absence of eIF2α phosphorylation induced by starvation for amino acids isoleucine and valine. Whereas starvation of WT confers changes in translational efficiencies (TEs) of hundreds of mRNAs, the eIF2AΔ mutation conferred no significant TE reductions for any mRNAs in non-starved cells, and it reduced the TEs of only a small number of transcripts in starved cells containing phosphorylated eIF2α. We found no evidence that eliminating eIF2A altered the translation of mRNAs containing putative IRES elements, or harboring uORFs initiated by AUG or near-cognate start codons, in non-starved or starved cells. Thus, very few mRNAs (possibly only one) appear to employ eIF2A for Met-tRNAi recruitment in yeast cells, even when eIF2 function is attenuated by stress.


SUPPLEMENTARY FIGURE LEGENDS
The A 260 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.(A-B) Cell spotting assays were performed on SC plates (A) or SD (B) plates to assess the growth of WT, eIF2A∆, fcy2∆ and eIF2A∆ fcy2∆ 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 eIF2A∆ 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 βgalactosidase 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.Spreadsheet tabulates the changes in luciferase activity expressed from F.LUC reporters calculated for WT+SM versus WT and eIF2A∆+SM versus eIF2A∆ for three biological replicates (Figure 3D).Spreadsheet 1, "raw CT values for mRNA from qRT", tabulates the raw CT values for mRNA from qRT reactions for three biological replicates-a, b, and c.Spreadsheet 2, "Dilution factor exemplar WT_a", provides an example file for one of the biological replicate WT (wild type) samples, illustrating how to calculate the dilution factor.Spreadsheet 3, "% 18S rRNA", provides an example file illustrating how to calculate the dilution factor.Spreadsheet 4, "Area % & Polysome norm factor", tabulates following parameters for all the biological replicates: Area under peaks from UV trace; SUM 80S+Polysomes; Average SUM 80S+Polysomes; Polysome recovery normalisation factor; and % total Area under 80S + Polysomes from UV trace in each peak.Spreadsheet 5, "rRNA normalisation factor", illustrates the calculations required to determine the rRNA normalization factor.Spreadsheet 6, "SAG1", illustrates the calculations required to determine ∆Monosome-Polysome/Total RNA (See materials and methods section for details) (Figure 3E).Spreadsheet tabulates lists of the 514 mRNAs bearing functional AUG or NCC-uORFs, 17 mRNAs identified in Figure 3A showing evidence for a conditional requirement for eIF2A when eIF2 function is reduced by SM i.e., ∆TEeIF2A∆+SM/WT+SM_down* (N=17) group, and overlap of mRNAs with functional AUG-or NCC-uORFs (N=514) and ∆TEeIF2A∆+SM/WT+SM_down (N=17) (Figure 6D).105 and is also made available for use under a CC0 license.

FIGURE SOURCE DATA
(which was not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 U

Figure 1 -
Figure 1-figure supplement 1. 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

Figure 2 -
Figure 2-figure supplement 1. 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 Figure2Ashowing the log 2 ratios of TEs in SM-treated eIF2A∆ versus untreated

Figure 3 -
Figure 3-figure supplement 1. Representative separation of polysomes by sedimentation through a sucrose density gradient in the experiment depicted in Figure 3E.

Figure 6 -
Figure 6-figure supplement 2. Lack of genetic interaction between eIF2A and the purine salvage pathway.

Figure 2 -
Figure 2-source data 2. Spreadsheet tabulates the log2 ratios of TEs in WT+SM cells versus WT cells (log2∆TEWT+SM/WT values) and the corresponding FDR determined by DESeq2 analysis of ribosome profiling and parallel RNA Seq data for the 5441 mRNAs with evidence of translation (Figure 2B & Figure 2-figure supplement 1B).

Figure 2 -
Figure 2-source data 3.Spreadsheet tabulates the log2 ratios log2ΔTE values for the indicated mutant/condition for the indicated mRNA groups identified in Figure2B(Figure2D).
105 and is also made available for use under a CC0 license.(whichwas not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USCThe copyright holder for this preprint this version posted December 9, 2023.; https://doi.org/10.1101/2023.10.06.561292 doi: bioRxiv preprint Spreadsheet 1-3, "Fig6B(i-iii)", tabulates the lists and log2ΔTE values as in (Figure6-source data 1) for the subsets of the same mRNA groups analyzed there exhibiting > 1.41-fold increases in TE in SM-treated versus untreated WT cells (Figure6B).

Figure 6 -
Figure 6-source data 5. Spreadsheet tabulates the list of annotated AUG-or NCC-uORFs, chromosome coordinates, start codon of uORF, distances of the uORF AUG from the 5' end of the mRNA and the main CDS start codon, and the gene name.

Figure 6 -
Figure 6-source data 6.Spreadsheet tabulates the list of conserved AUG-or NCC-uORFs, chromosome coordinates, start codon of uORF, distances of the uORF AUG from the 5' end of the mRNA and the main CDS start codon, and the gene name.

Figure 6 -
Figure 6-source data 7. Spreadsheet tabulates the functional uORFs , chromosome coordinates, start codon of uORF, distances of the uORF AUG from the 5' end of the mRNA and the main CDS start codon, and the gene name (May et al. 2023).

Figure 6 -
Figure 6-figure supplement 1-source data 2.Spreadsheet 1 tabulates the log2 ratios of following parameters for all the evolutionarily conserved

Figure 6 -
Figure 6-figure supplement 1-source data 3.Spreadsheet 1 tabulates the log2 ratios of following parameters for all the expressed annotated uAUG or

Figure 6 -
Figure 6-figure supplement 1-source data 4.Spreadsheet 1 tabulates the log2 ratios of following parameters for all the evolutionarily conserved

Figure 6 -
Figure 6-figure supplement 1-source data 5.Spreadsheet 1 tabulates the β-galactosidase activities in units of nmol of ONPG cleaved per mg of protein Figure 1-figure supplement 1

Figure
Figure 1-figure supplement 1. High reproducibility between biological replicates of ribosome footprint

FigureFigure 2 -Figure 3 -Figure 6 -
Figure 2-figure supplement 1 105 and is also made available for use under a CC0 license.(whichwas not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC