Effects of single amino acid deficiency on mRNA translation are markedly different for methionine versus leucine

Abstract

Although amino acids are known regulators of translation, the unique contributions of specific amino acids are not well understood. We compared effects of culturing HEK293T cells in medium lacking either leucine, methionine, histidine, or arginine on eIF2 and 4EBP1 phosphorylation and measures of mRNA translation. Methionine starvation caused the most drastic decrease in translation as assessed by polysome formation, ribosome profiling, and a measure of protein synthesis (puromycin-labeled polypeptides) but had no significant effect on eIF2 phosphorylation, 4EBP1 hyperphosphorylation or 4EBP1 binding to eIF4E. Leucine starvation suppressed polysome formation and was the only tested condition that caused a significant decrease in 4EBP1 phosphorylation or increase in 4EBP1 binding to eIF4E, but effects of leucine starvation were not replicated by overexpressing nonphosphorylatable 4EBP1. This suggests the binding of 4EBP1 to eIF4E may not by itself explain the suppression of mRNA translation under conditions of leucine starvation. Ribosome profiling suggested that leucine deprivation may primarily inhibit ribosome loading, whereas methionine deprivation may primarily impair start site recognition. These data underscore our lack of a full understanding of how mRNA translation is regulated and point to a unique regulatory role of methionine status on translation initiation that is not dependent upon eIF2 phosphorylation.

Figure 1

Changes in the growth, peptide translation and polysome profile of HEK293T cells in response to deficiency of a single essential amino acid. Cells were grown in complete medium (Suff) or in medium deficient in histidine (His‒), arginine (Arg‒), leucine (Leu‒) or methionine (Met‒) for 12 h. Values are means ± SD for 3 separate experiments. Results in (a and c‒g) are from one set of experiments, and those in b are from a separate set of experiments. Bars labeled with different letters are significantly different by least squares analysis with Tukey’s post hoc test at p ≤ 0.05. Values labeled with an asterisk (*) had a 95% confidence interval that did not include 1.0, making values significantly different from the Suff control at p ≤ 0.05; when relative values for amino acid-deficient conditions were analyzed, the replicate number had no significant effect in the overall model. (a) Final protein content of cells grown in amino acid sufficient or deficient medium for 12 h, expressed as a fraction of the final protein content of cells grown in sufficient medium. (b) Representative western blot showing rate of mRNA translation as assessed by puromycin labeling of newly synthesized proteins. (c) Relative densities of puromycin-labeled peptides. The density for the Suff condition was set at 1.0 for each blot (i.e., each separate experiment). (d) Image of overlaid polysome profiles from a representative experiment, illustrating differences in polysome profiles. (e) Ratios of polysome area to monosome area of polysome profiles. (f) Number of peaks in the polysome fraction of the polysome profiles. (g) Cleaved caspase 3 assay on 293 T cells. A representative western blot for the cleaved caspase 3 assay; the negative (−) and positive (+) control extracts are Jurkat cells treated without (−) or with (+) cytochrome c.

Figure 2

Differences in 4EBP1 and eIF2 phosphorylation in HEK293T cells in response to deficiency of a single essential amino acid. Cells were grown in complete medium (Suff) or in medium deficient in histidine (His–), arginine (Arg–), leucine (Leu–) or methionine (Met–) for 12 h. Values are means ± SD for 3 separate experiments. All results (a‒f) are from the same set of experiments. Bars labeled with different letters are significantly different by least squares analysis with Tukey’s post hoc test at p ≤ 0.05. Values labeled with an asterisk (*) had a 95% confidence interval that did not include 1.0, making values significantly different from the Suff control at p ≤ 0.05; when relative values for amino acid-deficient conditions were analyzed. (a) A representative western blot of 4EBP1 in the lysis solution used for the eIF4E pulldown assays. (b) Relative abundance of total 4EBP1 expressed as fold the value for cells cultured in complete medium in the same replicate experiment after normalization for abundance of β-actin. (c) A representative western blot for the amount of 4EBP1 pulled down with eIF4E. (d) Amount of 4EBP1 associated with eIF4E expressed as fold the mean value for cells cultured in complete medium after normalization to eIF4E. (e) A representative western blot for phosphorylated and total eIF2α. (f) Ratio of phosphorylated eIF2α to total eIF2α expressed as fold the mean value for cells cultured in complete medium.

Figure 3

Effect of transfection of HEK293T cells with wild-type versus constitutively active 4EBP1. Cells were transfected with empty vector, wildtype 4EBP1, or constitutively active mutant 4EBP1(T37A/T46A) and grown in sufficient control medium for 24 h. Values are means ± SD for 3 separate experiments. All results (a‒h) are from the same set of experiments. Values for separate experiments were normalized by setting the vector control value at 1.0. Bars labeled with different letters are significantly different by least squares analysis with Student’s post hoc test at p ≤ 0.05. Values labeled with an asterisk (*) had a 95% confidence interval that did not include 1.0, making values significantly different from the vector control at p ≤ 0.05. (a) Representative western blot for abundance of total and Thr 37,46-phosphorylated 4EBP1 in transfected cells. (b) Abundance of total and Thr 37,46-phosphorylated 4EBP1, normalized for actin and expressed as fold the mean values for cells transfected with empty vector. (c) Representative western blot for m7 cap-analog pulldown of eIF4E and associated 4EBP1 from transfected cells. (d) Relative amount of 4EBP1 associated with eIF4E, expressed as fold the mean value for cells transfected with empty vector after normalization to eIF4E. (e) Cell growth assessed by total protein content at 24 h post-transfection, expressed as a percentage of total protein in cells transfected with the empty vector. (f) Representative image of polysome profiles for transfected cells. (g) Ratios of polysome to monosome areas of polysome profiles. (h) Number of peaks in the polysome fraction of the polysome profiles.

Figure 4

Ribosome profiling in response to deficiency of single essential amino acids. (a) Metagene analysis of ribosome density in HEK293T cells grown in complete medium or in medium deficient in leucine or methionine for 12 h. The density of ribosome footprints mapped to the human transcriptome are determined at single nucleotide positions and averaged across transcripts aligned at start and stop codons. The zoomed in region shows the read distribution by reading frame. (b) Fractional distribution of ribosome footprints mapped to the coding regions by reading frame using Ribo-seq data obtained in the presence of cycloheximide. (c) Metagene analysis of ribosome density in HEK293T cells prepared in the absence of cycloheximide. (d) Fractional distribution of ribosome footprints mapped to the coding regions by reading frame, using Ribo-seq data obtained in the absence of cycloheximide.