Figure 1.
Affinity Purification of Tagged Ribosomes
(A) Polysomal association of tagged Rpl16a protein in cells grown in YPD medium. The absorbance profile at 254 nm of a sucrose gradient is shown at the top, and the positions of the 40S and 60S ribosomal subunits, 80S monosomes, and polyribosomes are indicated. Fractions were separated by SDS-PAGE, blotted, and probed with Peroxidase-Anti-Peroxidase Soluble Complex (PAP) to detect tagged Rpl16p (Rpl16a-ZZ), and with specific antibodies to detect Rpl35p (a ribosomal protein) and Zwf1p (a cytoplasmic protein not associated with polysomes). Molecular masses are indicated in kilodaltons on the left.
(B) Immunoblot analysis following the affinity purification. Lanes: 1, cell-free extract (E); 2, supernatant after incubation of extracts with IgG beads (S); 3, captured beads (Bb); and 4, beads after elution with TEV protease (Bc).
(C) Polysomal profile of affinity-purified ribosomes isolated from cells grown in SC medium.
(D) Affinity-purified ribosomes (Rpl16a) were separated by SDS-PAGE, and proteins were visualized with silver (L16a). A sample from a control purification with untagged wild-type cells is also shown (Mock). Bands representing BSA and TEV protease are marked, and the protein marker (M) with molecular masses is depicted to the left.
(E) Total RNA isolated from extracts (T), and from affinity-purified ribosomes (RA) was separated on a 1% denaturing agarose gel, and RNA was visualized with ethidium bromide. The positions of 26S, 18S ribosomal RNAs, and tRNAs are indicated to the right. Lengths are indicated in kilobases (kb) on the left.
Figure 2.
Stress-Specific Transcriptome and Translatome Profiles
(A) Venn diagrams showing the overlap of genes that significantly changed steady-state mRNA levels (Total, black circle) and ribosome associations (RA, red circle) after application of the indicated stress. C: set of genes that was significantly changed in both groups; R: set of genes with preferentially altered ribosome associations (translatome); T: set of genes with significantly altered mRNA levels (more than 2-fold, p ≤ 0.05) but no significant change of ribosome association. The bars to the right refer to the number of up- or down-regulated genes.
(B) A hierarchically clustered heat map of transcriptome and translatome profiles of stress-treated cells. The cluster contains 1,422 genes that changed more than 2-fold (p ≤ 0.05) in at least one of the stress conditions as specified in (A). Each row represents an experiment set (averaged from independent replicates), and each column represents one gene. Pearson correlations of experiments are indicated and visualized with a tree. The relative expression of genes is represented with a blue–yellow color bar, where blue means lower and yellow means higher expression compared to untreated cells. Subclusters are numbered with roman letters, and the number of genes and significantly enriched GO terms are indicated.
Figure 3.
Enrichment of Functional Themes among Stress-Regulated Messages
Enrichment of GO terms among significantly changed gene sets (T, C, and R) specified in the upper-left Venn diagram shown in Figure 2A. The significance of enrichment of the GO term is represented as a heat map in which the color code corresponds to p-values (scale is indicated at the bottom). Themes among up-regulated genes are shown in yellow–red scale, themes among down-regulation genes are in blue scale. GO terms are colored according to their classification: “biological process” in blue, “biological function” in black, and “cellular component” in red; GO identification numbers are added in italics. Enrichment of respective GO terms among the ESR genes [22] is shown to the left.
Figure 4.
Enhanced Ribosome Association and Activity of the Mitochondrial F1F0-ATPase by Low Doses of CFW
(A) Heat map representing relative changes of expression of 17 mitochondrial F1F0-ATPase components after mild stress treatments (blue–yellow color scale). Total: changes of steady-state mRNA levels, RA: change of ribosome associations. The gene names are indicated to the left, with the name of the corresponding subunit in parentheses.
(B) Structure of the mitochondrial F1F0-ATPase. Each component is colored according to changes in ribosome associations depicted in (A). The membrane-associated part (F0) uses a proton motive force to mechanically drive the soluble part (F1) that exhibits ATPase activity [78,79].
(C) Distribution of TIM11, ATP4, and ACT1 mRNAs in polysomal gradients obtained from untreated cells (upper panel), and from cells treated with CFW (lower panel). RNA was isolated from each fraction of the polysomal profile and quantified by RT-qPCR (see Materials and Methods).The mRNA level in each fraction was calculated as a percentage of the total; data and absorbance (254 nm) profiles from representative experiments are plotted. ACT1 is as a negative control mRNA that was not expected to alter ribosome association.
(D) Relative mitochondrial ATPase activity of drug-treated versus untreated control cells. Cells were treated with CFW (CFW) for 20 min; preincubated for 10 min with CHX prior to addition of CFW (CHX + CFW); or treated with CHX or menadione. The activity of purified mitochondria was normalized to the untreated control sample. Average activities are indicated with a dashed line, standard errors of the mean (SEM) with continuous lines. Single dots represent biologically independent experiments (double asterisks [**] indicate p < 0.01; a single asterisk [*] indicates p < 0.05).