Formation and significance of yeast P-bodies on A. thaliana leaves.

(A) P-body formation in the WT strain on growing Arabidopsis leaves. Edc3-Venus fusion protein was expressed under the control of the native EDC3 promoter as a marker. The strain was spotted on leaves, collected from the leaves at the indicated day/time post-inoculation and observed by a fluorescence microscope. DIC: differential interference contrast. Bar, 2 µm. dpi, days post-inoculation. (B) Quantification of Edc3-Venus dot formation detected in (A). Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). The number of dots is presented as mean ± standard error (SE) of 90 cells. Asterisks indicate statistical significance: * p < 0.05; ** p < 0.01; n.s. means not significant. (C) P-body formation in the WT and edc3Δ strains on SM media. Dcp2-Venus fusion protein was expressed under the control of the native DCP2 promoter as a marker. The strains grown on SM medium were collected for microscopic observation. Bar, 2 µm. (D) Quantification of Dcp2-Venus dot formation detected in (C). Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). The number of dots is presented as mean ± standard error (SE) of 90 cells. Asterisks indicate statistical significance: ** p < 0.01. (E) P-body formation in the WT and edc3Δ strains on growing Arabidopsis leaves. The strains expressing Dcp2-Venus were spotted on leaves and collected 4 hours after inoculation for microscopic observation. Bar, 2 µm. (F) Quantification of Dcp2-Venus dot formation detected in (E). Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). The number of dots is presented as mean ± SE of 90 cells. Asterisks indicate statistical significance: ** p < 0.01. (G) Confocal microscope images of the Venus-labeled WT and edc3Δ strains on growing Arabidopsis leaves. Venus fluorescent protein was expressed under the control of the constitutive ACT1 promoter. The strains were spotted on leaves and observed at the indicated days post-inoculation (dpi). Merged images of differential interference contrast (DIC) and Venus are depicted. Bar, 20 µm. (H) Quantification of C. boidinii cell populations on A. thaliana leaves by flowcytometry (FCM). The leaves onto which the strains were inoculated were retrieved at the indicated days post-inoculation (dpi) and were subjected to FCM for counting the cell population expressing Venus. Values are indicated as mean ± SE of four biological replicates.

Intracellular dynamics of P-bodies and mimRNAs on methanol growth conditions.

(A) Confocal microscopy images of the WT strain expressing Edc3-Cerulean and Venus-tagged DAS1 mRNA (strain Edc3-C/DAS1m-V) on growing Arabidopsis leaves. The strain was spotted on the leaves and observed 4 hours after inoculation. Squares I, II, III and VI were enlarged. Merged images were generated by combining Venus and Cerulean fluorescence images. White arrows indicate colocalization of Edc3-Cerulean and Venus-tagged DAS1 mRNA dots. The lengths of the bars are indicated in the images. DIC: differential interference contrast. (B) Fluorescence microscopy images of strain Edc3-C/DAS1m-V on growing Arabidopsis leaves. The strain, spotted on leaves, was collected for observation from the leaf surface 4 hours after inoculation during both the light and dark periods. Merged images were generated by combining Venus and Cerulean fluorescence images. White arrows indicate colocalization of Edc3-Cerulean and Venus-tagged DAS1 mRNA dots. Bar, 2 µm. (C) Quantification of dot formation of Edc3-Venus and Venus-tagged DAS1 mRNA and their colocalization detected in (B). Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). The number of dots is presented as the mean ± SE of 90 cells, and colocalization rates are shown as mean ± SE from three biological replicates (n = 30 cells per replicate). n.s. means not statistically significant. (D) Time-course analysis of the intracellular localization of P-bodies and mimRNAs in strain Edc3-C/DAS1m-V during methanol induction. The strain grown on an SD medium was transferred to SM medium and collected at the indicated time points for fluorescence microscopy. Merged images were generated by combining Venus and Celurean fluorescence images. Bar, 2 µm. (E) Quantification of dot formation of Edc3-Venus and Venus-tagged DAS1 mRNA and their colocalization detected in (D). Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). The number of dots is presented as mean ± SE of 90 cells and colocalization rates are shown as mean ± SE from three biological replicates (n = 30 cells per replicate). (F) Fluorescence microscopy images of the strains visualizing DAS1, AOD1, FLD1, FGH1, FDH1, PMP20 and CTA1 mRNAs. The strains grown on an SD medium were transferred to SM medium and incubated for 3 hours before fluorescence microscopy. Bar, 2 µm. (G) Quantification of mimRNA granule formation detected in (F). Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). The number of dots is presented as mean ± SE of 90 cells. (H) Intracellular localization of multiple mimRNAs during methanol induction. In strain DD, DAS1 mRNA was visualized by the Venus-tagged U1A-based RNA system and Cerulean-tagged MS2-based RNA system. In strain AD and strain FD, AOD1 or FLD1 mRNA was visualized by Venus-tagged U1A-based RNA system and DAS1 mRNA by Cerulean-tagged MS2-based RNA system. Merged images were generated by combining Venus and Cerulean fluorescence images. Bar, 2 µm. (I) Quantification of the colocalization of the multiple mimRNA granules detected in (H). Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). The number of dots is presented as mean ± SE of 90 cells. n.s. means not statistically significant.

Role of P-bodies on post-transcriptional regulation of mimRNAs.

(A) Dot formation of Venus-tagged DAS1 mRNA in the WT and edc3Δ strains on SM medium. The strains grown on an SD medium were transferred to SM medium and incubated for 3 hours before fluorescence microscopy. Bar, 2 µm. (B) Quantification of Venus-tagged DAS1 mRNA dot formation detected in (A). Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). The number of dots is presented as mean ± SE of 90 cells. Asterisks indicate statistical significance: * p < 0.05. (C) Relative transcript levels of DAS1 mRNA in the WT and edc3Δ strains on SM medium. Values are indicated as mean ± SE of three biological replicates. Statistical significance between the WT and edc3Δ strains were assessed. Asterisks indicate statistical significance: * p < 0.05. (D) P-body formation under stress conditions. Strain Edc3-V was exposed to oxidative stress with 5 mM H2O2, heat shock at 37 degrees or salt stress with 3 M NaCl for 1 hour before microscopic observation. Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). The number of dots is presented as mean ± SE of 90 cells, and the proportion of cells containing at least one dot (dot containing rate) are shown as mean ± SE from three biological replicates (n = 30 cells per replicate). Bar, 2µm. (E) Fluorescence microscopy images of strain Edc3-C/DAS1m-V during oxidative stress and recovery from the stress. The strain incubated on an SM medium was treated with 5 mM H2O2 for 1 hour and then transferred to an SM medium. Merged images were generated by combining Venus and Cerulean fluorescence images. Bar, 2 µm. (F) Quantification of dot formation of Edc3-Venus and Venus-tagged DAS1 mRNA and their colocalization detected in (E). Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). The number of dots is presented as mean ± SE of 90 cells and colocalization rates are shown as mean ± SE from three biological replicates (n = 30 cells per replicate). (G) P-body formation in the WT and edc3Δ strains expressing Dcp2-Venus under oxidative stress. The strains grown on SM medium were treated with 5 mM H2O2 for 1 hour and collected for microscopic observation. (H) Quantification of Dcp2-Venus dot formation detected in (I). Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). The number of dots is presented as mean ± SE of 90 cells. Asterisks indicate statistical significance: ** p < 0.01. (I) Relative transcript level of DAS1 mRNA in the WT and edc3Δ strains on SM medium under oxidative stress. The strain was incubated on SM medium for 3 hours and then treated with 5 mM H₂O₂ for 1 hour. Transcript levels of all genes were first normalized to ACT1 and then expression levels were calculated relative to samples collected at 0 h of methanol cultivation. In the graph, the sample collected at 3 h is labeled as “Control” for comparison. Values are indicated as mean ± SE of three biological replicates. Statistical significance between the WT and edc3Δ strains was assessed. Asterisks indicate statistical significance: ** p < 0.01. (J) Growth assay of cells cultured under oxidative stress conditions. Cells were grown to early log phase, adjusted to OD610=1, and 3 μL of tenfold serial dilutions were dropped onto SM plates with or without 0.7 mM H2O2. Subsequently, the SM plates were incubated at 28°C. Cell growth was analyzed after 2 days. Growth of the wild-type (WT), edc3Δ, and edc3Δ expressing Edc3-Venus (edc3ΔCOMP) under the native promoter was compared.

Regulation of mimRNA granule formation.

(A) Time-course analysis of the intracellular localization of Venus-tagged DAS1 mRNA during methanol induction. The strain grown on an SD medium was transferred to SM medium and incubated for up to 5 hours and collected at indicated time points. Bar, 2 µm. (B) Nuclear localization of Venus-tagged DAS1 mRNA. The strain grown on an SD medium was collected for fluorescence microscopy. A merged image was generated by combining Venus and DAPI fluorescence images. Bar, 2 µm. (C) Quantification of dot formation of Venus-tagged DAS1 mRNA detected in (A). Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). The number of dots is presented as mean ± SE of 90 cells. (D) Relative transcript level of DAS1 mRNA in the WT strain during methanol induction. Values are indicated as the mean ± SE of three biological replicates. (E) Immunoblot analysis of DAS in the WT strain during methanol induction. The expected molecular weight of DAS is 78.2 kDa. (F) Fluorescence microscopy images of the strains visualizing DAS1, TDH3, ADH1 and ACT1 mRNAs during cultivation on various carbon sources. The strains grown on an SD medium were transferred to either SM, SD or SE medium and incubated for 3 hours. Bar, 2 µm. (G-J) Quantification of mRNA dot formation detected in (F). Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). The number of dots is presented as mean ± SE of 90 cells. (G) DAS1 mRNA, (H) TDH3 mRNA, (I) ADH1 mRNA and (J) ACT1 mRNA.

Properties of mimRNA granules.

(A) Time-lapse images of strain Edc3-C/DAS1m-V under methanol-grown conditions. Twelve images extracted at the indicated time points are shown as a video (see also Figure 5 – video 1). Merged images were generated by combining Venus and Cerulean fluorescence images. White arrows indicate the dynamic properties of Edc3-Cerulean and Venus-tagged DAS1 mRNA dots. A new Venus-tagged DAS1 mRNA dot appeared at 56 seconds in the video, which colocalized with an Edc3-Cerulean dot from 1 min 24 s to 2 min 48 s, and disappeared at 3 min 16 s, while the Edc3-Cerulean dots remained. At 7 min 56 s, a Venus-tagged DAS1 mRNA dot appeared without colocalizing with a P-body; this granule remained at the same site at 8 min 24 s but disappeared at 8 min 52 s. Bar, 2 μm. (B) Fluorescence microscopy images of strain Edc3-C/DAS1m-V after treatment with protein synthesis inhibitors. The strain incubated on an SM medium was treated with either 0.1 mg/mL cycloheximide (CHX) or 0.1 mg/mL puromycin (PMC) before microscopic observation. Merged images were generated by combining Venus and Cerulean fluorescence images. Bar, 2 µm. (C) Quantification of dot formation of Edc3-Venus and Venus-tagged DAS1 mRNA and their colocalization detected in (B). Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). The number of dots is presented as mean ± SE of 90 cells and colocalization rates are shown as mean ± SE from three biological replicates (n = 30 cells per replicate). Asterisks indicate statistical significance. ** p < 0.01. n.s. means not significant. (D) Fluorescence microscopy images of strain Edc3-C/DAS1m-V after treatment with 1,6-hexandiol (HXD). The strain incubated on an SM medium was treated with 10% HXD before microscopic observation. Merged images were generated by combining Venus and Cerulean fluorescence images. Bar, 2 µm. (E) Quantification of dot formation of Edc3-Venus and Venus-tagged DAS1 mRNA and their colocalization detected in (D). Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). The number of dots is presented as mean ± SE of 90 cells and colocalization rates are shown as mean ± SE from three biological replicates (n = 30 cells per replicate). Asterisks indicate statistical significance. ** p < 0.01.

Schematic image illustrating the role of yeast P-bodies in the post-transcriptional regulation of methanol-induced genes in the phyllosphere.

On A. thaliana leaves, C. boidinii assimilates methanol as the carbon source while adapting to environmental stresses. In this leaf environment, P-bodies regulate methanol-induced genes post-transcriptionally through the following steps: 1) Methanol induces transcription of genes involved in methanol metabolism leading to the accumulation of methanol-induced mRNAs (mimRNAs) in the cytosol; 2) Multiple mimRNAs accumulate into cytosolic mimRNA granules that contain both ribosome-free and ribosome-associated mimRNAs; 3) mimRNA granules dynamically interact with P-bodies where ribosome-free mimRNAs partially accumulate; 4) In response to environmental stimuli such as oxidative stress, P-bodies actively sequester mimRNAs, protecting them from degradation; 5) Upon stress removal, P-bodies release mimRNAs to the cytosol for translation.

Intracellular localization of Edc3-Venus and Pbp1-Venus on A. thaliana leaves.

(A-B) Investigation of the P-body and SG formation in the WT strains on Arabidopsis leaves. Edc3-Venus fusion protein was expressed under the control of the native EDC3 promoter as a P-body marker (A), whereas Pbp1-Venus fusion protein expressed under the control of the native PBP1 promoter was used as an SG marker (B). The strains were spotted on growing, matured, wilting or dead leaves, collected from the leaves 4 hours after inoculation and observed by a fluorescent microscope. Bar, 2 µm. DIC: differential interference contrast.

Diagram of methanol metabolism in yeast.

Enzymes: AOD, alcohol oxidase; DAS, dihydroxyacetone synthase; FDH, formate dehydrogenase; FGH, S-formylglutathione hydrolase; FLD, formaldehyde dehydrogenase; CTA, catalase; PMP20, peroxisome membrane protein with glutathione peroxidase activity. Abbreviations: DHA, dihydroxyacetone; GAP, glyceraldehyde 3-phosphate; GS-CH2OH, S-hydroxymethyl glutathione; GS-CHO, S-formylglutathione; GSH, reduced form of glutathione; GSSG, oxidized form of glutathione; RCOOOH, alkyl hydroperoxide.

Integration of the U1Abs did not alter protein function.

(A) Schematic overview for visualizing methanol-induced mRNAs (mimRNAs) in vivo. GENE, DNA sequence encoding a target mRNA; White box, UTR; 4xU1Abs, 4 x U1A binding sequence; NLS, nuclear localization sequence; U1A, U1A coding sequence; FP, fluorescent protein coding sequence. Yeast cells were transformed with vectors expressing a fusion of U1Abs with a gene sequence encoding the target mRNA sequence (GENE-4xU1Abs) and a fluorescent protein combined with NLS-U1A (NLS-U1A-FP). GENE-4xU1Abs was expressed under its own promoter, whereas NLS-U1A-FP was expressed under the TDH3 promoter. (B) Fluorescent microscopic images of DAS1 mRNA visualized by the U1A-based RNA system and fluorescence in situ hybridization (FISH) method. FISH was performed for visualizing the canonical DAS1 gene (methodological details are described in Materials and Methods section). Strain DAS1m-V grown on an SD medium was transferred to an SM medium and incubated for 3 hours. Merged images were generated by combining Venus and cy5 fluorescence images. Bar: 2 µm. (C) Colocalization of the DAS1 mRNA detected by the U1A-based RNA system and FISH method in (B). mRNA localization was visualized by FISH using Cy5-labeled probes and by the U1A-based RNA system using Venus fluorescence. Fluorescence intensity was measured along a defined line across the cells. Fluorescence intensity (Y-axis) was plotted against distance (X-axis) to show the spatial distribution of mRNA signals. (D) Growth assay of C. boidinii strains on SD or SM agar plates. The yeast strains were grown to early log phase, adjusted to OD610=1, and 3μL of tenfold serial dilutions were streaked onto SD or SM plates. The das1ΔINT strain expresses 4xU1Abs-tagged native DAS1 gene under its native promoter and the fld1ΔINT strain expresses 4xU1Abs-tagged native FLD1 gene under its native promoter.

Intracellular dynamics of P-bodies and mimRNAs on wilting A. thaliana leaves.

(A) Fluorescence microscopy images of strain Edc3-C/DAS1m-V on wilting Arabidopsis leaves. The strain, spotted on leaves, was collected from the leaf surface 4 hours after inoculation for observation. Merged images were generated by combining Venus and Cerulean fluorescence images. White arrows indicate colocalization of Edc3-Cerulean and Venus-tagged DAS1 mRNA dots. Bar, 2 µm. (B) Quantification of dot formation of Edc3-Venus and Venus-tagged DAS1 mRNA and their colocalization detected in (A). Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). The number of dots is presented as mean ± SE of 90 cells and colocalization rates are shown as mean ± SE from three biological replicates (n = 30 cells per replicate).

Effect of the EDC3 gene deletion on methanol metabolism.

(A) Relative transcript level of AOD1 mRNA in the WT and edc3Δ strains on SM medium. Values are indicated as mean ± SE of three biological replicates. Statistical significances between the WT and edc3Δ strains were assessed. Asterisks indicate statistical significance: * p < 0.05. (B-C) Immunoblot analysis of AOD (B) and DAS (C) in the WT and edc3Δ strains during methanol induction. The expected molecular weights of AOD and DAS are 74.1 kDa and 78.2 kDa, respectively. (D) Growth assay of cells cultured on methanol. Cells were grown to early log phase, adjusted to OD610=1, and 3 μL of tenfold serial dilutions were dropped onto SM plates. Subsequently, the SM plates were incubated at 28°C. Cell growth was analyzed after 2 days. Growth of the wild-type (WT) and edc3Δ was compared.

Intracellular dynamics of P-bodies and DAS1 mRNA under stress conditions.

(A) Fluorescence microscope images of strain Edc3-C/DAS1m-V during heat and salt stress. The strain was exposed to heat shock at 37 degrees or salt stress with 3 M NaCl for 1 hour before microscopic observation. Merged images were generated by combining Venus and Cerulean fluorescence images. Bar, 2 µm. (B) Quantification of dot formation of Edc3-Venus and Venus-tagged DAS1 mRNA and their colocalization detected in (A). Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). The number of dots is presented as mean ± SE of 90 cells and colocalization rates are shown as mean ± SE from three biological replicates (n = 30 cells per replicate).

Granule formation and gene expression of various mimRNAs during methanol induction.

(A) Time-course analysis of the intracellular localization of mimRNAs during methanol induction. AOD1, FLD1, FGH1, FDH1, PMP20 and CTA1 mRNA were visualized. The strains grown on an SD medium were transferred to SM medium and incubated for up to 5 hours and collected at indicated time points. Bar, 2 µm. (B-G) Quantification of dot formation of mimRNAs detected in (A). (B) AOD1, (C) FLD1, (D) FGH1, (E) FDH1, (F) PMP20 and (G) CTA1 mRNA. At least 30 cells were analyzed per experiment. Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). The number of dots is presented as the mean ± SE of 90 cells. (H-M) Relative transcript level of mimRNAs in the WT strain during methanol induction. (H) AOD1, (I) FLD1, (J) FGH1, (K) FDH1, (L) PMP20 and (M) CTA1 mRNA. Values are indicated as mean ± SE of three independent experiments.

Regulation of mimRNA granule formation at low methanol concentration.

(A) Fluorescence microscopy images of the strain visualizing Venus-tagged DAS1 mRNA on SM medium. The strain grown on an SD medium was transferred to SM medium and incubated for 3 hours and collected at indicated time points. Methanol concentration was either 0.0005% or 0.5%. Bar, 2 µm. (B) Quantification of mimRNA granule formation detected in (A). Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). The number of dots is presented as mean ± SE of of 90 cells. Asterisks indicate statistical significance: ** p < 0.01. (C) Relative transcript level of DAS1 mRNA in the WT strain during methanol induction. Methanol concentration was either 0.0005% or 0.5%. Values are indicated as mean ± SE of three biological replicates. Asterisks indicate statistical significance: * p < 0.05.

Visualization of methanol-induced KpAOX1 mRNA in K. phaffii.

Fluorescent microscopic images of the K. phaffii strain expressing U1Abs-tagged KpAOX1 mRNA, and the NLS- and Cerulean-tagged U1A protein. U1Abs; U1A protein binding sequence, NLS; nuclear localization sequence. The K. phaffii strain expressing only U1A-Cerulean (referred to as U1A-Cerulean only) was used as a control in which the Cerulean fluorescence remained in the nucleus. White arrows indicate the dots of KpAOX1 mRNA. Bar, 2 µm. DIC: differential interference contrast.

Regulation of dot formation of TDH3 mRNA.

(A) Time-course analysis of the intracellular localization of Venus-tagged TDH3 mRNA during methanol induction. The strain grown on an SD medium was transferred to SM medium and incubated for up to 5 hours. Bar, 2 µm. (B) Quantification of dot formation of Venus-tagged TDH3 mRNA detected in (A). Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). The number of dots is presented as mean ± SE of 90 cells. (C) Relative transcript level of TDH3 mRNA in the WT strain during methanol induction. Values are indicated as mean ± SE of three biological replicates. (D) Immunoblot analysis of GAPDH in the WT strain during methanol induction. The expected molecular weight of GAPDH is 35.5 kDa.

Intracellular dynamics of P-bodies and TDH3 mRNA under stress conditions.

(A) Fluorescence microscopy images of strain Edc3-C/TDH3m-V during oxidative stress. The strain incubated on an SM medium was treated with 5 mM H2O2 for 1 hour before microscopic observation. Merged images were generated by combining Venus and Cerulean fluorescence images. Bar, 2 µm. (B) Quantification of dot formation of Edc3-Venus and Venus-tagged TDH3 mRNA and their colocalization detected in (A). Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). The number of dots is presented as mean ± SE of 90 cells and colocalization rates are shown as mean ± SE from three biological replicates (n = 30 cells per replicate).

Association of mimRNA granules with other organelles and structures.

(A) Schematic image of the association or non-association of DAS1 mRNA with cellular organelles. (B-D) Association of DAS1 mRNA granules with the endoplasmic reticulum (ER) (B), peroxisomes (C) and actin patches (D). ER was visualized by mCherry tagged with KAR2 sequence signal at its N terminus and HDEL amino acid sequence at its C terminus. Peroxisomes were visualized by mCherry tagged with AKL amino acid sequence at its C terminus for peroxisome targeting signal. Actin patches were stained with rhodamine phalloidin. Cells grown on an SD medium were transferred to an SM medium and incubated for 3 hours. The association rate indicates the percentage of Venus-tagged DAS1 mRNA granules that are associated with the ER (B), peroxisomes (C) or actin patches (D). Cell count analysis was performed on fluorescence microscopy images (n = 30, n: number of cells analyzed; b = 3, b: biological replicates; total = 90 cells). Association rates are shown as mean ± SE from three biological replicates (n = 30 cells per replicate). Bar, 2 µm.