Figure 8. mRNAs encoding heat shock proteins undergo enhanced multiplexing upon heat shock.

MATa yeast strains (BY4741) expressing MS2 aptamer-tagged HSP104, UGO1, or STE5 from their genomic loci or untagged control (WT) cells were grown to mid-log phase (O.D.₆₀₀ = 0.5) and subjected to RaPID followed by qRT-PCR (RaPID-qRTPCR; see 'Materials and methods'). RNA derived from the total cell extracts or biotin-eluated fractions was analyzed by qRT-PCR using primer pairs corresponding to mRNAs expected to multiplex (see listed genes) or not (e.g., GAPDH mRNA). (A) mRNAs encoding heat shock proteins (HSPs) multiplex upon heat shock. Cells expressing MS2 aptamer-tagged HSP104 were either exposed to heat shock (10 min; 40°C; HS) or maintained at 30°C (NHS) prior to fixation and RaPID-qRT-PCR. Three biological replicates were performed and an unpaired t-test was used to compare each under non-heat and heat shock conditions with WT; ****p<0.0001; **p<0.001; *p<0.01. (B) mRNAs of HSPs co-localize upon heat shock. Representative single-molecule fluorescence in situ hybridization (smFISH) images of BY4741 WT cells that underwent heat shock (HS) or did not (NHS). BY4741 were processed for smFISH labeling using designated FISH probes complementary to the HSP104 and SSA2 and HSP104 and STE2 mRNA pairs prior to labeling with DAPI. SSA2 – Cy3 labeling; HSP104/STE2 – Alexa488 labeling; merge – merged Cy3 and Alexa-488 windows with DAPI staining. Size bar = 2 μm. The representative image shown is from a single focal plane. (C) Scatter plot of data from (B) showing the proportion of co-localized smFISH foci. Black lines represent average ± SEM distribution. Each data point represents a single cell. *****p-value<0.00001.

Figure 8—figure supplement 1. Motifs in HSP mRNA coding regions and role of histone H4 in transperon formation.

(A) A conserved motif is common to all six heat shock proteins (HSPs) mRNAs. MEME analysis of the sequences of all six mRNAs was performed and revealed three conserved motifs in the coding regions, shown schematically as sequence logo based on nucleotide representation. (B) Distribution of the motifs over the HSP genes. Motif location is plotted, along with their p-values over the different genes. Motifs 1–3 correspond to those shown in (A), respectively. (C) Single gene histone deletions do not affect cell growth after heat shock. The doubling times (minutes) of WT, hhf1Δ, hhf2Δ, hta1∆, hta2∆, htb1∆, htb2∆, hht1∆, and hht2∆ strains were assessed for cells grown on a liquid-rich medium (YPD) exposed to heat shock (50°C; 60 min;+HS) or not (-HS) and then allowed to recover for 18 hr at 30°C. Error bars represent the avg. ± sd of four biological repeats. A one-way ANOVA with multiple comparisons with and without heat shock for each strain was performed to calculate the statistical significance; p<0.0001 for all comparisons. (D) Combined hhf2∆ and AID-HHF1 alleles result in increased doubling time upon auxin induction and heat shock. MATa wild-type (WT) and hhf2∆ AID-HHF1 cells were grown to mid-log phase, treated either with or without auxin (3-IAA; 4 mM) for 3.5 hr, and exposed to heat shock (50°C; 0, 15, 30, 60 min; HS). After removal to a fresh YPD medium lacking auxin, strains were grown for 18 hr at 30°C and the doubling times assessed. Three biological replicates were performed and a one-way ANOVA test with multiple comparisons was performed to calculate the statistical significance between time points; ****p-value<0.0001. ns – non-significant.

Figure 8—figure supplement 2. Histone H4 function is required for HSP mRNP assembly.

(A) Depletion of histone H4 inhibits heat shock protein (HSP) mRNA multiplexing. MATa hhf2∆ AID-HHF1 cells expressing MS2 aptamer-tagged HSP104 from its genomic locus were grown to mid-log phase, treated either with or without auxin (3-IAA; 4 mM) for 3.5 hr, and then heat shocked (50°C; 10 min) as shown in (B) or maintained at 30°C, as shown in (A). RaPID pulldown of HSP104 mRNA was performed, followed by qRT-PCR of the co-precipitated mRNAs. Three biological replicates were tested and unpaired t-tests were used to compare the levels of each mRNA either with or without auxin treatment; ****p<0.0001. (C) The co-localization of HSP104 and SSA2 mRNAs is reduced upon histone H4 depletion. Wild-type (WT) and AID-HHF1 hhf2∆ cells were grown to mid-log phase, treated either with or without auxin (3-IAA; 4 mM) for 3.5 hr, and fixed for single-molecule fluorescence in situ hybridization (smFISH). A scatter plot showing the level of co-localized smFISH puncta. Co-localization was assessed using FISHquant (see 'Materials and methods'). Black lines indicate the avg. ± SEM for each sample. Each data point represents a single cell. ****p-value<0.0001. (D) Neither auxin (3-IAA) treatment nor histone H4 depletion abolishes heat shock-induced gene expression. WT and HA-AID-HHF1 hhf2∆ cells were grown to mid-log phase prior to treating cells with or without auxin (3-IAA; 4 mM; 3.5 hr), with or without subsequent heat shock (50°C for 10 min), both auxin treatment and heat shock, or neither. After treatment, total cell lysates were prepared, RNA extracted, and analyzed by qRT-PCR using primers against HSP104 and SSA2. RNA levels derived from WT and HA-AID-HHF1 hhf2∆ cells under the different conditions were compared; ***p-value<0.0005; *p-value<0.01.