Ribonucleoprotein (RNP) condensates modulate survival in response to Mars-like stress conditions

Yeast strains and growth conditions

The yeast strains used in this study are listed in Table S1. All strains were transformed with a plasmid expressing Dcp2-mCherry and Pab1-GFP (green fluorescent protein) (a list of plasmids used in the study is listed in Table S2). All strains were grown at 30 °C in synthetic dropout media without uracil supplemented with 2% glucose.

Shock processing

We utilized the High-Intensity Shock Tube for Astrochemistry (HISTA) housed in the Physical Research Laboratory (PRL), Ahmedabad, India. It is a 7 m long shock tube with a 2 m long driver section and a 5 m long driven section, and these two sections are separated by an Aluminum diaphragm. The driver sections are filled with high-pressure Helium gas, and the driven sections are usually filled with low-pressure Argon gas. The various factors that influence the shock intensity in the shock tube are the bursting pressure of the driver side, the diaphragm's groove thickness, and the driven gas pressure. By adjusting these parameters, we can generate different shock intensities according to our requirements. The details of HISTA's operation principles, procedure, and data collection can be found in the literature by (13, 14)

Before the shock processing, we drop casted different yeast samples on the end flange of the shock tube, followed by closing the shock tube's driven section using the metal end flange. The entire driven sections are then pumped up to 10⁻² mbar order, followed by purging using Argon (Ar) to reduce any chance of contamination from the residual atmospheric gas. The driven section is filled with very low-pressure Ar. The driven section pressure can be varied according to the desired shock Mach number. A shock response curve can be collected using two pressure sensors with a separation distance of 30 cm attached to HISTA. After the shock processing the internal pressure of the shock tube becomes very high (can be as high as 18 bar). The high-pressure He and Ar mixture is slowly released further with the help of a leak valve. Once the internal pressure of the shock tube equilibrates with the atmospheric pressure, the end flange can be opened, and shocked yeast samples were collected. For the unshocked control, the yeast samples were similarly drop casted on the metal flange as done for shock processing and for a similar duration of time before collecting them for growth curve and imaging experiments.

Growth curve

Growth curve was performed on yeast cells recovered from the shock tube following the exposure to shock waves, along with unshocked cells. The cells were then cultured in fresh media by dilution to an initial optical density of 0.05 OD_600nm. The cultures were then incubated at 30 °C (180 rpm) and monitored the growth periodically till 40 h.

The growth curve for the perchlorate-treated condition was obtained by growing yeast in fresh media with dilution to an initial optical density of 0.1 OD_600nm. The cultures were then split into two parts, one treated with 100 mM of sodium perchlorate and one with vehicle control (milli-Q water). The cultures were then incubated at 30 °C (180 rpm) and monitored the growth periodically for 30–40 h.

For combined stress, yeast cells were first exposed to 5.6 Mach intensity shock waves, followed by dilution to an initial optical density of 0.05 OD_600nm in fresh medium supplemented with 100 mM sodium perchlorate. The cultures were incubated at 30 °C (180 rpm) and monitored the growth periodically till 60 h. Synthetic dropout media (-uracil) supplemented with 2% glucose were used for all growth curve experiments. All absorbance measurements were performed at 600 nm. The data were analyzed, and the growth curve was then plotted using GraphPad Prism 9.0. Lag time and growth rate were derived from the first derivative of the growth curve, subjected to linear regression with Gaussian fit using GraphPad Prism 9.0. For Figs. 1C and S3B, C, lag time was calculated as the time taken to reach 0.25 OD_600nm.

Sample preparation for microscopy

Postshock wave treatment, samples were recovered from the shock tube and fixed with 2% paraformaldehyde in 1× PBS (phosphate buffer saline pH 7) at room temperature (RT). Fixed samples were then incubated with poly-L-lysine-coated coverslips at RT for 45 min, followed by mounting on a glass slide with Fluorochrome G.

For the sodium perchlorate-treated condition, cells were grown in the presence of 100 mM sodium perchlorate to reach 0.5 OD, followed by fixing with 2% paraformaldehyde as mentioned above.

Microscopy

All images were acquired using a Deltavision RT microscope system running softWoRx 6.1.3 software (Applied Precision, LLC), using a 100X, oil-immersion objective. Exposure time and transmittance settings for the GFP channel were 0.3 s and 32%, and for the mCherry channel were 0.5 s and 50%, respectively. Images were acquired at a resolution of 512 × 512 pixels using a CoolSnapHQ camera (Photometrics) with 1 × 1 binning. All the images were deconvolved using standard softWoRx deconvolution algorithms. ImageJ was used to analyze the data. For each experiment, 100 cells were counted to determine the number of foci (Pab1-GFP, Edc3-mCherry, or Scd6-GFP) per cell. For Fig. 2F and G, live-cell images were acquired using an Apotome microscope (Zeiss Axio observer) and processed using ImageJ software, as there were technical issues with Deltavision RT microscope system. The images were taken at 63X, oil immersion objective. Exposure time and transmittance settings for the GFP channel were 0.15 s and 50%, and for the mCherry channel were 0.75 s and 100%, respectively.

Colony-forming unit assay

The yeast cells were diluted at an OD₆₀₀ of 0.1 from an overnight-grown primary culture and grown in synthetic dropout media without uracil (SD Ura-), supplemented with 2% glucose, with or without 100 mM sodium perchlorate, and allowed to reach 0.5 OD₆₀₀. After which the cultures were washed twice with perchlorate-free media. The cells were serially diluted and plated on SD Ura- agar plates. The plates were incubated at 30 °C for 48 h. The colony-forming unit (CFU) count was used to determine the survival.

mRNA stability assay

A thermo-sensitive mutant of RNA polymerase II, rpb1-1, was used to perform an mRNA stability assay (15, 16). The rpb1-1 strain was grown in YEPD with or without 100 mM sodium perchlorate at 30 °C (180 rpm) till 0.5 OD_600nm. For transcription shut off, prewarmed (55 °C) YEPD was added to cultures at 30 °C followed by incubating at 42 °C for heat shock. Cells were collected at time intervals of 0, 15, 30, 45, and 60 min of heat shock, followed by immediate snap freezing with liquid nitrogen. RNA isolation was performed by the hot-phenol method, followed by cDNA synthesis. Gene-specific primers were used for qRT-PCR and were normalized to PGK1. Statistical analysis was done by two-way ANOVA using Šídák's multiple comparisons test.

RNA isolation

RNA was isolated from biological replicates for each test sample using the hot acidic phenol protocol described in Current Protocols in Molecular Biology (1996), Unit 13.12 (17, 18). Briefly, the cell pellet was washed with DEPC-treated autoclaved Milli-Q water (DEPC-AMQ) once and resuspended thoroughly in TES (10 mM Tris-Cl pH 7.5, 10 mM ethylene-diamine-tetraacetic acid [EDTA], and 0.5% SDS). Four hundred microliters of acidic phenol pH 4.5 (0.1 M citrate buffer saturated) were added to the suspension and incubated at 65 °C for 60 min with occasional vortexing. Samples were spun at 14,000 rpm for 10 min at 4 °C. The top aqueous layer was transferred to a fresh tube. Subsequently, 400 μL chloroform was added, mixed, and spun as above. The aqueous layer was mixed with 1/10th volume 3 M sodium acetate, pH 5.2, and 2.5 volumes of 100% ethanol, followed by snap freezing in liquid N₂. The samples were allowed to thaw on ice, followed by pelleting down the precipitated RNA at 14,000 rpm at 4 °C for 30 min. The RNA pellets were washed with 70% ethanol (prepared in DEPC-AMQ), followed by air drying and resuspending the pellet in 50 μL DEPC-AMQ, and RNA quality was determined by 1% agarose formamide gel electrophoresis. The concentration and purity of RNA were quantified using a Nanodrop Spectrophotometer (Thermo Scientific; 2000). The integrity of RNA in the samples was assessed on TapeStation (Agilent). RNA concentration was quantified using a Qubit RNA HS assay kit (Q32855).

RNA library preparation and sequencing

RNA-sequencing (RNA-seq) libraries were prepared with Illumina-compatible NEBNext® Ultra II. Directional RNA Library Prep Kit (New England BioLabs, MA, USA) at Genotypic Technology Pvt. Ltd., Bangalore, India. 200 ng of total RNA was used for mRNA isolation, fragmentation, and priming. Fragmented and primed mRNA was then subjected to first-strand synthesis followed by second-strand synthesis. The double-stranded cDNA was purified using NEBNext sample purification beads. Purified cDNA was end-repaired, adenylated, and ligated to Illumina adapters as per NEBNext Ultra II Directional RNA Library Prep protocol, followed by second-strand excision using the USER enzyme at 37 °C for 15 min. Adapter-ligated cDNA was purified using NEBNext beads and was subjected to 12 cycles for Indexing-(98 °C for 30 s, cycling (98 °C for 10 s 65 °C for 75 s) and 65 °C for 5 min) and enriched the adapter-ligated fragments. Final PCR products (sequencing library) were purified with NEBNext beads, followed by a library quality control check. Illumina-compatible sequencing libraries were quantified by Qubit fluorometer (Thermo Fisher Scientific, MA, USA), and fragment size distribution was analyzed on Agilent 4150 TapeStation. The libraries were paired-end sequenced on an Illumina NovaSeqX Plus sequencer for 150 cycles (Illumina, San Diego, USA) following the manufacturer's instructions.

Transcriptome analysis

The QC-passed reads were mapped to the indexed S. cerevisiae reference genome (S288c) using a splice-aware aligner (19). The quantification of expressed transcripts was performed using the genome-aligned reads. This expression data was subsequently utilized for downstream analyses, including gene ontology (GO) and pathway annotations, to study the function of each expressed transcript. The Featurecounts4 tool was used to estimate and calculate transcript abundance (20). DESeq25 was used to calculate the differentially expressed transcripts (21). Transcripts were functionally annotated based on homology using the BLAST5 tool against Fungal protein sequences available from the UniProt database (22). Pathway analysis was done on the KAAS6 server using S. cerevisiae as the reference organism (23).

Reverse transcription and quantitative real-time PCR

The isolated RNA was subjected to DNase treatment to remove any DNA contamination. For this, 5 μg of total RNA, 0.75 units of DNase I (Thermo Fisher Scientific EN0525), and DNase I buffer (with MgCl₂ added to it), 0.2 units of RiboLock RNase Inhibitor (Thermo Fisher Scientific, EO0381), and DEPC-AMQ were added for a 20 μL reaction. The above reaction was incubated at 37 °C for 30 min, followed by adding 2 μL EDTA and incubating it at 65 °C for 10 min to inhibit the DNase I activity. The DNaseI-treated RNA concentration was measured in a spectrophotometer, followed by an RNA quality check by 1% agarose formamide gel electrophoresis. DNase I-treated RNA was used for cDNA preparation. One microgram of RNA was used to synthesize cDNA using the RevertAid RT Reverse Transcription Kit (Thermo K1691) according to the manufacturer's protocol. The synthesized cDNA was then diluted 1:10, followed by quantitative real-time PCR.

TB GreenTM Premix Ex TaqTM (TakaRa). The reactions were set up in technical duplicates for each biological replicate. Data points in all graphs represent biological replicates. Primers used for qRT-PCR are listed in Table S3. The PCR conditions were 95 °C for 10 min for initial denaturation, followed by 30 cycles of 95 °C for 15 s and 60 °C for 45 s. DNA was quantified in every cycle at the step of extension. Melt cure acquisition was carried out at 60 °C for 15 s. PGK1 was used as an internal control. To calculate the final log2 fold-change values, ΔΔCt method was used, where log2 Fold-change values were plotted using GraphPad Prism software, version 9.00. The significance of the data was calculated by a paired t test.

Saccharomyces cerevisiae survives 5.6 Mach intensity shock waves

Using a unicellular eukaryote, S. cerevisiae (budding yeast) as a model system, our study aims to understand the impact of Mars-like stress conditions on survival and growth. To address this question, we employed a multidisciplinary approach to simulate conditions similar to those on Mars, specifically shock waves. Using a HISTA, the S. cerevisiae wild-type BY4741 strain was subjected to varying intensities of shock waves such as 3.5, 4.8, and 5.6 Mach (M) (detailed in the Methods section). The various operational parameters for shock processing used in this study are outlined in Table 1. The shock response curve (5.6 M) of HISTA (Fig. 1A) was recorded on a digital oscilloscope using two piezoelectric pressure sensors, PS1 and PS2, positioned at the end of HISTA. The inset highlights the time delay between the pressure signals from PS1 and PS2 during the arrival of the incident shock. It is notable that shock wave treatment also exposes the cells to very high estimated temperatures, albeit for a very short (2 ms) period of time (Table 1; 24). This indicates that shockwaves could expose lifeforms to transient heat shock stress. To assess the effect of shock waves on S. cerevisiae survival, growth curve analysis was performed on wild-type BY4741 yeast cells collected postshock from the shock tube. Interestingly, as shown in Fig. 1B and C, the S. cerevisiae wild-type BY4741 strain survives 5.6 Mach intensity shock waves with an extended lag phase compared to the unshocked controls. 5.6 M shocked wild-type strain began to grow after ∼20 h of incubation at 30 °C, indicating a prolonged recovery period from the stress induced by shock waves. Cells exposed to 3.5 and 4.8 M intensity shockwaves also underwent an extended lag phase, but to a lesser extent than 5.6 M intensity shockwaves (Fig. 1B and C). This indicates a graded response by yeast cells to increasing intensity of shock waves.

Saccharomyces cerevisiae survives sodium perchlorate concentrations equivalent to Martian conditions

To further study the impact of Martian stress conditions on S. cerevisiae, we investigated the effect of perchlorate salts at concentrations equivalent to those found on the Martian surface. The Martian soil is reported to contain perchlorates in the range of 0.5–1% by weight (4). To test this, we used sodium salt of perchlorate (NaClO₄) as it is highly soluble in water among all other perchlorates. We then analyzed the growth of wild-type BY4741 yeast strain in the continuous presence of sodium perchlorate at concentrations of 50 mM, 100 mM, 200 mM, 500 mM, 1, and 2 M. As shown in Fig. 1D and E, we observed a concentration-dependent increase in growth defect, indicating the negative effect of sodium perchlorate on yeast growth. While 50 mM NaClO₄ impacts yeast growth slightly, concentrations of 100 and 200 mM show significant growth defects, and concentrations of 500 mM or above result in little or no growth till 40 h of incubation at 30 °C. Since perchlorates are present on Martian soil in the range of 0.5–1%, we have therefore used 100 mM (1.22% by weight) of sodium perchlorate for all subsequent experiments.

To check whether the observed growth defect is specific to sodium perchlorate, we looked at yeast growth in the presence of 100 mM sodium chloride. We observed no growth defect for wildtype at 100 mM NaCl compared to untreated control, unlike sodium perchlorate (Fig. 1F and G). This highlights the specific effect of perchlorate ions on growth.

5.6 M intensity shock induces RNP condensates in yeast

Yeast cells survive 5.6 M shock waves and grow after an 18–20 h lag, indicating a stress response. We, therefore, investigated the assembly of two conserved, classical RNP condensates: stress granules (Pab1-GFP) and P-bodies (Dcp2-mCherry) in response to shock waves. Exposure to 5.6 M intensity shock waves induced the assembly of both P-bodies as well as stress granules (Fig. 2A). This observation indicates that exposure to shock waves induces the assembly of ribonucleoprotein condensates as a cellular response to this stress. Moreover, the number of stress granules and P-bodies decreased in cells exposed to 4.8 M compared to 5.6 M (Fig. 2B and C). Interestingly, no condensates were induced in response to a 3.5 M shockwave, suggesting that the induction of the condensates occurs in a graded manner depending on the shockwave intensity. Since we observed the assembly of P-bodies and stress granules in wild-type strain upon exposure to 5.6 M shock waves, we wanted to confirm the assembly of these condensates with another condensate resident marker protein, Scd6, which is reported to localize to both P-bodies and stress granules. As shown in Fig. S1, the assembly of Scd6-GFP granules was also observed in wild-type yeast strains upon exposure to 5.6 M intensity shock. This again confirms our observation that exposure to shock waves induces the assembly of ribonucleoprotein condensates as a cellular response to the stress condition.

Sodium perchlorate induces P-body assembly

Saccharomyces cerevisiae survived 100 mM sodium perchlorate with a significant growth defect. Therefore, we investigated the assembly of RNP condensates in response to sodium perchlorate stress. As depicted in Fig. 2D and E, P-bodies but not stress granules were induced in wild-type strain grown in the presence of continuous 100 mM NaClO₄. This observation indicates that signaling pathways for the assembly of PBs and SGs are distinct in response to sodium perchlorate stress. To determine whether this response is specific to perchlorate ions or a general effect of sodium salts, we assessed assembly of RNP condensates in the presence of sodium chloride. Notably, neither stress granules nor P-bodies were induced in response to sodium chloride, indicating the effect is specific to perchlorate ions (Fig. 2F and G).

edc3Δlsm4ΔC mutant fails to assemble RNP condensates in response to shock waves or sodium perchlorate stress

We wanted to investigate the importance of these condensates in the recovery of yeast cells from the two Martian stress conditions mentioned above. To address this, we examined the mutant strain edc3Δlsm4ΔC, which is known to be defective in the assembly of P-bodies and stress granules under stresses such as glucose starvation (25). This strain is deleted for EDC3, the gene encoding a conserved P-body marker, and a part of the LSM4 gene encoding the prion-like C-terminal of the Lsm4 protein. We assessed whether this mutant could assemble RNP condensates in response to shock waves and/or perchlorate stress. As shown in Fig. 3A and B and Fig. S2A–C, P-body and stress granule assembly were hampered in the edc3Δlsm4ΔC mutant upon exposure to 5.6 M intensity shock wave and sodium perchlorate. This finding suggests that the formation of Dcp2-mCherry marked P-bodies and Pab1-GFP marked stress granules depends on the presence of Edc3 and Lsm4, highlighting similarities between the condensate assembly in various stress conditions.

The edc3Δlsm4ΔC mutant demonstrates an extended lag phase in sodium perchlorate stress. We hypothesized that if the formation of RNP condensates played a crucial role in survival under these stresses, then the edc3Δlsm4ΔC mutant, which fails to assemble these condensates, could exhibit altered growth under these stresses compared to wild-type. Therefore, we assessed the growth of the edc3Δlsm4ΔC mutant upon exposure to these stresses. Interestingly, the edc3Δlsm4ΔC mutant exhibited significantly slower growth than the wild-type strain (Fig. 3C and D) in continuous perchlorate stress. This suggests that RNP condensate assembly could facilitate better cellular adaptation and growth under perchlorate stress. In contrast, there was no significant difference in growth between wild-type and mutant strains during recovery after exposure to shock waves (Fig. 3E and F). Next, we examined the effect of sodium perchlorate on the survival of edc3Δlsm4ΔC. To test this, we performed a CFU assay. Our results revealed a significant decrease in CFU count in edc3Δlsm4ΔC compared to the wild-type following sodium perchlorate treatment, as shown in Fig. 3G and H, indicating decreased survival of edc3Δlsm4ΔC under these conditions. This is consistent with our earlier observation of slower growth in edc3Δlsm4ΔC compared to wild-type during continuous perchlorate treatment.

Transcriptome analysis reveals that granule assembly affects mRNA abundance in wildtype and edc3Δlsm4ΔC in response to sodium perchlorate RNP condensates regulate stress response by altering the mRNA abundance and translation. To understand the transcriptome-wide changes induced by the perchlorate stress and the possible contribution of RNP condensates to it, we performed sequencing of total RNA samples from wildtype and the edc3Δlsm4ΔC mutant (Fig. 4). For differential gene expression analysis, the category listed prior in all comparisons serves as a control. Genes with P-value ≤ 0.05 were considered significantly differentially expressed. Figure 4A–C and Figs. S4 and S5 show a summary of differentially expressed genes (DEGs) and GO enrichment.

RNA-seq analysis of wild-type yeast cells treated with 100 mM sodium perchlorate identified 1,321 DEGs, of which 773 were upregulated and 548 were significantly downregulated. GO enrichment analysis of the upregulated genes revealed significant enrichment in several biological processes. Notably, genes associated with cellular respiration represented the largest category of upregulated genes in sodium perchlorate-treated wild-type cells (Fig. 4D and E). Other enriched categories among the upregulated genes included those involved in oxidative stress response, cell wall organization, carbohydrate metabolism, ion transport, and mitochondrial autophagy. Additionally, genes involved in ascospore formation and ubiquitin-dependent protein degradation processes were also significantly upregulated. Conversely, the downregulated genes were enriched in processes related to cytoplasmic translation, methylation, nucleocytoplasmic transport, and ribosome biogenesis (Fig. 4D). These findings suggest that sodium perchlorate might induce a metabolic shift, promoting stress adaptation while suppressing translation-related processes that are energy-intensive.

In the edc3Δlsm4ΔC mutant, sodium perchlorate treatment altered gene expression, with some DEGs overlapping with those in perchlorate-treated wild-type cells. Four hundred sixty-two upregulated genes and 154 downregulated genes were common between wild-type and mutant cells treated with sodium perchlorate, highlighting a perchlorate-specific stress response (Fig. 4F).

Comparison of the edc3Δlsm4ΔC mutant to wild-type under sodium perchlorate stress revealed 1,099 DEGs, with 610 genes upregulated and 489 genes significantly downregulated. GO enrichment analysis of the downregulated genes in the mutant highlighted significant enrichment of mRNAs encoding proteins involved in processes such as cell wall organization, protein folding, detoxification, transmembrane transport, and ascospore formation (Fig. 4G and H). This suggests that the edc3Δlsm4ΔC mutant likely exhibits an impaired response, particularly in pathways critical for maintaining cellular integrity and stress adaptation under perchlorate stress. This is consistent with the observed growth defects in the edc3Δlsm4ΔC mutant under continuous sodium perchlorate stress (Fig. 3C and D).

To further elucidate the alteration of the global transcriptome during recovery from the shock wave, transcriptomic analysis was performed on wild-type S. cerevisiae recovering from exposure to 5.6 M intensity shock waves. Direct analysis of shocked cells was not possible due to technical limitations of the shock tube, which yielded only a small sample amount. Since shocked cells demonstrated a marked lag phase, we were curious to identify the transcriptome changes just before the cells moved out of the extended lag phase induced by shock waves. Transcriptome analysis revealed differential expression of 1,322 genes. Among these, 686 genes were upregulated, and 636 genes were downregulated (Fig. 4A). GO enrichment analysis of the upregulated genes highlighted significant enrichment in processes such as methylation, translation, and nuclear export (Fig. S6). Conversely, the downregulated genes were enriched in pathways associated with glucose import and cellular respiration. Notably, genes involved in oxidative stress response and temperature stimulus were also downregulated, which is intriguing as these pathways are commonly associated with stress adaptation (Fig. S6). This suggests that the recovery from shock waves might involve a distinct regulatory mechanism prioritizing specific cellular processes over generalized stress responses. The edc3Δlsm4ΔC mutant was not subjected to transcriptome analysis upon shock wave exposure, as we did not observe a significant difference in growth rate compared to wild-type shocked cells (Fig. 3C and D).

Validation of the transcriptome data

To select an appropriate internal control for validating the RNA-seq results, we assessed the mRNA levels of three reference genes PGK1, ACT1, and SCR1 using qRT-PCR under the same conditions applied in the RNA-seq experiments. Among these, PGK1 and ACT1 exhibited no change in expression across all tested conditions, while SCR1 showed variable expression upon perchlorate treatment (Fig. S7A). Therefore, PGK1 was chosen as the internal control for mRNA target validation. To validate the differentially expressed transcripts identified through RNA-seq in wild-type yeast upon sodium perchlorate treatment, we focused on CTT1, GRE1, HXT5, and HSP31, which were upregulated, and TIF4631, TRM1, RPS0A, and RPL24A, which were downregulated. These transcripts were selected as representatives from each enriched GO category for qRT-PCR analysis. Notably, GRE1 was among the transcripts that exhibited the highest fold change in RNA-seq analysis. The qRT-PCR results validated the RNA-seq findings, with all tested transcripts exhibiting trends consistent with the sequencing data. The upregulated genes, including CTT1 and GRE1, which are associated with oxidative stress response, and HXT5, linked to carbohydrate metabolism, showed increased mRNA levels. Similarly, HSP31, which is involved in stress protection, was also significantly upregulated (Fig. 5A). Conversely, genes, such as TIF4631 and TRM1, associated with translation initiation and methylation, respectively, exhibited reduced mRNA levels. Ribosomal protein genes RPS0A and RPL24A also showed decreased expression, aligning with the observed downregulation of translation-related processes (Fig. 5B).

Among the transcripts downregulated in edc3Δlsm4ΔC perchlorate-treated condition compared to wild-type perchlorate-treated, we validated the six transcripts. ROM2, SSA1, PDR5, TPO3, ERO1, and ARN2 transcripts (Fig. 5C). These transcripts do not change in response to perchlorate in wild-type compared to untreated but are significantly downregulated in edc3Δlsm4ΔC perchlorate-treated condition compared to wild-type perchlorate-treated, suggesting that these may be destabilized in the absence of RNP condensates. We also validated four transcripts, such as DAK1, SRX1, SSE2, and SPI1, which were upregulated in wild-type treated compared to untreated, but significantly downregulated in edc3Δlsm4ΔC perchlorate-treated condition. Of all tested transcripts, except TPO3, all transcripts were validated (Fig. 5D and E). Furthermore, we validated three transcripts involved in ascospore wall assembly SPR3, SPO19, and SPS18—which were upregulated in wildtype upon sodium perchlorate treatment but are significantly downregulated in edc3Δlsm4ΔC mutant under perchlorate stress (Fig. 5F and G).

Similarly, to validate RNA-seq result for differentially expressed mRNAs in wild-type during recovery from exposure to 5.6 M intensity shock waves, four mRNAs such as TIF4631, NSR1, TRM1, and HMT1 were chosen as representatives from upregulated GO transcripts. TIF4631 was selected as it encodes a translation initiation factor crucial for translation regulation, while HMT1 was chosen as it is a major arginine methyltransferase. The following five transcripts CTT1, HXT5, SCO1, SDH1, and COX4 were chosen to validate downregulated mRNAs (Fig. S6E and F). CTT1 and HXT5 were among the transcripts that exhibited the lowest expression levels in RNA-seq analysis. We observed that all tested transcripts except COX4 validated the changes observed in RNA-seq analysis. Taken together, these observations validate the RNA-seq results by qRT-PCR and highlight the involvement of specific pathways that may facilitate adaptation in response to Martian stress conditions.

Stabilization of transcripts correlates with the induction of P-bodies upon sodium perchlorate treatment

We hypothesized that the reduced abundance of transcripts in the edc3Δlsm4ΔC mutant could be a result of post-transcriptional regulation due to the absence of P-bodies. To investigate this, we performed a transcription shut-off assay using a temperature-sensitive mutant of RNA polymerase II, rpb1-1. Upon heat shock, RNA polymerase II becomes inactive, halting mRNA synthesis, enabling us to assess mRNA stability (15, 16). We assessed the mRNA decay profile in untreated and NaClO₄-treated cells over a 60-min time course postheat shock to assess how transcript stability is affected by sodium perchlorate. When rpb1-1 untreated and sodium perchlorate-treated conditions were subjected to transcription shut-off by heat shock, we observed stabilization of specific transcripts such as SSA1 (involved in protein folding), TPO2 (involved in ion transport), ROM2 (involved in cell wall signaling) in sodium perchlorate-treated condition compared to untreated condition (Fig. 6B–D) whereas RPL24 which was used as a negative control shows a steady decrease in abundance over time in both untreated and treated condition. These results support the notion of stabilization of stress-responsive transcripts and correlation with induction of P-bodies under sodium perchlorate treatment.