Vanillin inhibits translation and induces messenger ribonucleoprotein (mRNP) granule formation in saccharomyces cerevisiae: application and validation of high-content, image-based profiling.

Vanillin, generated by acid hydrolysis of lignocellulose, acts as a potent inhibitor of the growth of the yeast Saccharomyces cerevisiae. Here, we investigated the cellular processes affected by vanillin using high-content, image-based profiling. Among 4,718 non-essential yeast deletion mutants, the morphology of those defective in the large ribosomal subunit showed significant similarity to that of vanillin-treated cells. The defects in these mutants were clustered in three domains of the ribosome: the mRNA tunnel entrance, exit and backbone required for small subunit attachment. To confirm that vanillin inhibited ribosomal function, we assessed polysome and messenger ribonucleoprotein granule formation after treatment with vanillin. Analysis of polysome profiles showed disassembly of the polysomes in the presence of vanillin. Processing bodies and stress granules, which are composed of non-translating mRNAs and various proteins, were formed after treatment with vanillin. These results suggest that vanillin represses translation in yeast cells

Distribution of morphological similarity between vanillin-treated cells and non-essential deletion mutants.

<p>Morphological data of wild-type cells incubated with various vanillin concentrations were used to infer the cellular processes affected by vanillin, as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061748#pone.0061748-Ohnuki1" target="_blank">[11]</a>. The X-axis shows the ORFs in alphabetical order. The Y-axis indicates –log₁₀ P of the correlation coefficient (R) between morphological profiles of vanillin-treated cells and that of each of the 4,718 non-essential gene deletion mutants (grey circles). The horizontal dashed line indicates a P value of 0.05 after Bonferroni correction. (A) Distribution of the cytosolic large ribosomal subunit (GO:0022625) mutants (black filled circles). (B) Photographs of Δ<i>his3</i> Δ<i>adh6</i> cells treated with 0.1% DMSO (MOCK), Δ<i>his3</i> Δ<i>adh6</i> cells treated with 1 mM vanillin (Vanillin), Δ<i>his3</i> (WT), and the significantly similar deletion mutants of the large ribosomal subunit. Cells were stained with FITC-ConA (green) for cell wall, Rh-ph (red) for actin and DAPI (blue) for DNA. (C) Distribution of the cytosolic small ribosomal subunit (GO:0022627) mutants (black filled circles). (D) Distribution of the ergosterol biosynthetic pathway (GO:0006696) mutants (black filled circles).</p

Polysome profile analysis of cells after treatment with vanillin.

<p>BY4741 cells at a concentration of 1×10⁶ cells/ml in SD medium were treated with 4 mM vanillin for 0–12 h (A) or 0–40 mM vanillin for 30 min (B). The polysome, 40S (small ribosomal subunit), 60S (large ribosomal subunit), and 80S (monosome) peaks are labeled.</p

Morphological changes of yeast (<i>Saccharomyces cerevisiae</i>) cells after treatment with vanillin.

<p>(A) Time-dependent change in the tPC1 score. Wild-type (Δ<i>his3</i> Δ<i>adh6</i>) cells were pre-cultured in YPD medium, inoculated into YPD medium containing 4 mM vanillin at 1×10⁶ cells/ml and incubated at 25°C. The cells were fixed at the indicated time points and stained with fluorescent dyes. Then, at least 200 cells were photographed under the fluorescence microscope and analyzed with CalMorph. For each parameter, the parameter values at each time point were summarized into one value from five replicate experiments with rank-order (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061748#s2" target="_blank">Materials and Methods</a>) and subjected to PCA. The contribution ratio of tPC1 was 0.559. (B) Venn diagram of dose-dependent and time-dependent parameters after vanillin treatment. For dose-dependency, wild-type cells were grown in YPD medium containing 0.00, 0.25, 0.50, 0.75 and 1.00 mM vanillin. For time dependency, time-course data were acquired as described in (A). Of the 501 parameters, 28 and 187 were dose- and time-dependent, respectively, at <i>P</i><0.01. The permutation tests determined that less than four and approximately four parameters were expected to be detected by chance at this <i>P</i> value, respectively. The parameters significantly changed in same direction (increase or decrease) were counted as the parameters changed similarly in both experiments. (C) Illustration of representative wild-type cells after vanillin treatment. In each dataset, the successive PCA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061748#pone.0061748-Ohnuki2" target="_blank">[16]</a> was performed to illustrate the representative cell shape. Blue filled ellipses and red circles indicate nuclei and actin patches, respectively. (D) Decrease in cell size after vanillin treatment. The parameter values of C11-1_A, C101_A1B and C101_C in each dataset were plotted for whole cell size in G1, S/G2 and M, respectively. For a full definition of the parameters, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061748#pone.0061748-Ohya1" target="_blank">[17]</a>.</p

Formation of cytoplasmic processing bodies (P-bodies) and stress granules (SGs) after treatment with vanillin.

<p>(A and B) BY4741 cells expressing indicated GFP-fusion constructs at a concentration of 1×10⁶ cells/ml in SD medium were treated with vanillin (0–50mM) for 30 min, deprived of glucose (- Glc) for 15 min, or administered heat shock at 46°C for 10 min. The assembly of P-bodies and SGs was examined with Dcp2-GFP (A) and Ngr1-GFP (B), respectively. (C and D) Cells were treated with cycloheximide (CHX) (100 µg/ml) for 1 min prior to treatment with vanillin (0–50 mM) for 30 min, glucose deprivation (- Glc) for 15 min, or robust heat shock at 46°C for 10 min. The assembly of P-bodies and SGs was examined using Dcp2-GFP (C) and Ngr1-GFP (D), respectively. (E and F) Percentages of cells containing cytoplasmic granule(s) of Dcp2-GFP (E) or Ngr1-GFP (F). The data are represented as means ± SD. Asterisk indicates P<0.05.</p