Modified Ribosomal RNA Molecules Resistant to Breakdown Are Made When Cell Growth Is Slowed

Saccharomyces cerevisiae S288C (ATCC), BY28996 (MATa ura3‐1 leu2‐3, 112trp1‐1 his3‐11, 15 ade2‐1 can1‐100 tor1Δ::KanMX tor2Δ::HIS3 pRS316[TOR2] (YGRC/NBRP Japan)), W303‐1a (MATa leu2‐3,112 trp1‐1 can1‐100 ura3‐1 ade2‐1 his3‐11,15) and FB229‐7A (MATalpha leu2‐3,112 trp1‐1 can1‐100 ura3‐1 ade2‐1 his3‐11,15, rrn3∆‐:: NAT‐MX4, rpa43∆‐::NAT‐MX4 + pGEN‐CARA (2micron TRP1 pPGK1‐CARA) were preserved in 50% glycerol in YPD broth (2% w/V tryptone, 1% w/v yeast extract, 2% w/v dextrose) at −80°C. For activation, cells were inoculated into YPD broth and incubated at 30°C. The yeast culture was maintained on Sabouraud dextrose agar at 4°C and subcultured every 4–6 weeks, with a maximum of five passages. Before experiments, yeast cells were harvested from the agar surface and cultured in YPD broth at 30°C. Cell concentrations were determined using a hemocytometer. Starting yeast concentrations for all experiments were 1 × 10^6 cells/mL. Cultures were incubated during 6 h at 30°C for the Mid log condition and 16 h for the diauxic phase.

Yeast cells (S. cerevisiae S288C, BY28996, W303‐1a and FB229‐7A) were collected at multiple time points, starting at 4 h and ending at 48 h. Cell concentration was calculated using a hemocytometer. The data were plotted using GraphPad Prism 9 software where each time point represents the average of three experiments. Standard deviation is also shown at each time point.

Cells were collected by centrifugation, washed with sterile phosphate‐buffered saline (PBS) and placed on ice until total RNA extraction. Cell disruption was performed using RNase‐free zirconia beads, and RNA was extracted using the RiboPure RNA Purification Kit for Yeast (Invitrogen) following the manufacturer's instructions. RNA concentration and quality were assessed using a Qubit 4 fluorometer and an Agilent 2100 Bioanalyzer.

Exonuclease (Terminator or Xrn1) treated and untreated RNA samples were loaded onto an RNA 6000 Nano chip and analyzed using an Agilent 2100 bioanalyzer system (Agilent Technologies, INC). Electropherograms were used to determine the percentage of exonuclease‐resistance RNA by calculating the areas under the peaks of treated (Glynn et al. 2012) RNA and dividing it by the area of untreated RNA (uncut).

Cap‐Clip acid pyrophosphatase (CellScript) and alkaline phosphatase calf intestinal (CIP) (New England Biolabs) were used according to the manufacturer's instructions. RNA samples were treated with CIP only, CIP followed by CapClip or CapClip alone. RNAse inhibitors were used to decrease endonuclease activity of CapClip. All samples were purified by ethanol precipitation before proceeding to the exonuclease (Terminator or Xrn1) digestion assay.

Total RNA was treated with either Terminator (Biosearch Technologies) or XrnI (New England Biolabs) using the supplied buffer following the manufacturer's protocol. To ensure efficient cleavage, enzymes were applied at a ratio of 1 unit per 1 µg of RNA. RNase inhibitors were included in all assays.

Yeast cells were incubated with 1 µg of rapamycin (Sigma‒Aldrich) per 1 × 10⁶ cells. The incubation time was 6 h at 30°C with constant shaking. After incubation, the cells were washed with PBS and used for the appropriate assay.

Yeast cells were cultured in YPD medium until they reached the desired growth phase, either mid‐log (5 h) or stationary (16 h). Subsequently, 100 uM Thiouracil was added to the culture, and the cells were incubated for an additional hour. Cells were washed in PBS and stored for RNA extraction. For the chase component of the experiment, the cells were spun down, and the YPD medium containing thiouracil was removed. The cells were then resuspended in YPD, which had previously supported the growth of an identical number of yeast cells for the same length of time. Those yeast cells were removed by spinning them down before resuspension.

Thiouracil (Sigma) incorporation was measured by biotin detection using MTSA‐biotin‐XX (Biotium). Biotinylation reactions included RNA (1 µM), 10 mM HEPES (pH 7.5), 1 mM EDTA, and 25 µM MTS‐biotin (dissolved in DMSO at 250 µM).

RNA was separated on formaldehyde agarose gels (Lonza) and stained with SYBR Gold nucleic acid gel stain (Life Technologies) for 30 min. Gel images were captured with a digital camera (Canon Vixia HFS30). RNA was transferred by electroblotting (Bio‐Rad Trans‐Blot Turbo Transfer System) to a positively charged nylon membrane (Millipore) in 0.5x TBE (standard Tris/Borate/EDTA buffer). The RNA was cross‐linked to the membrane using UV (Stratagene UV Crosslinker). The Prosignal (Prometheus) chemiluminescence substrate was used to detect the HRP signal. The film was developed with an SRX‐101A Konica film processor.

All experiments were performed in triplicate, and data are presented as mean ± standard deviation (SD). Statistical significance was determined using two‐tailed, unpaired t‐test in GraphPad Prism (version 9.5, GraphPad Software, San Diego, CA, USA). A p < 0.05 was considered statistically significant.

During the growth cycle of yeast, the rate of new yeast formation diminishes as nutrient availability decreases, eventually reaching a diauxic phase. As shown in Figure 1, the growth curve of the TOR1‐mutant S. cerevisiae BY‐28996 (Figure 1C) is similar to the wild‐type Saccharomyces cerevisiae S288C (Figure 1A) and to the parental cell line W303‐1b (Figure 1B). They all present a lag, logarithmic and diauxic phase, but differ on the timing of these phases. In the wild type cells, production of resistant 18S and 25S is clearly phase dependent, as it increases when cells approach the diauxic phase (Figure 2A,B). TOR1‐deleted BY‐28996 took longer (Figure 1), but in contrast to S288C or W303‐1b cells, the production of resistant 18S and 25S molecules remained constant and reached similar levels throughout all the phases (Figure 1C).

As mentioned, normally processed 18S and 25S molecules have a single phosphate at their 5ʹ end and are susceptible to these exonucleases. Thus, for them to become resistant, some changes need to occur. Several factors can make RNA molecules resistant to single 5ʹ‐phosphate‐dependent RNA exonucleases (Figures 3, 4, 5). One possibility is the removal of the single phosphate at its 5ʹ end, leaving a hydroxyl group at the 5ʹ position of the first ribose. Another is the addition of extra phosphates at the 5ʹ end. A third option is the attachment of a nonphosphate structure, such as a cap, to the 5ʹ end phosphate. To identify which of these scenarios is most likely, we employed alkaline phosphatase (AP) (Figure 3), the decapping enzyme Cap‐Clip, which is a pyrophosphatase that cuts only between phosphates (Figure 4), and the combination of both enzymes (Figure 5).

Exonuclease resistance of RNA in the diauxic phase of yeast cells can arise through several mechanisms, as depicted in Figure 3A. One possibility is the presence of multiple phosphates at the 5ʹ end without a cap, which makes rRNA resistant to exonuclease digestion. AP removes these phosphates, yet the rRNA remains resistant to exonuclease. Another possibility is that the resistant molecules undergo structural modifications that confer resistance to both exonucleases and AP. In such cases, even after dephosphorylation, the rRNAs maintain their resistance, with the remaining resistance likely due to AP removing the single phosphate from normally processed molecules.

As shown in Figure 3B, in the mid‐log growth phase of the yeast, essentially all the 18S and 25S molecules were susceptible, indicating that they all contained a single phosphate at the 5ʹ end, as expected for transcribed and processed 18S and 25S molecules. Alkaline phosphatase treatment increases rRNA resistance to 100% by removing the single phosphate and leaving a hydroxyl group at the 5ʹ end. As the cells enter the stationary phase, 30%–40% of the rRNA molecules become resistant to exonucleases, suggesting a modification at their 5ʹ end. When these molecules, from the diauxic phase, are dephosphorylated (AP treatment), their resistance also reaches 100%. Total resistance increase is due to the removal of the single phosphate of previously susceptible rRNA molecules.

Figure 4A depicts the possibilities for Cap‐Clip activity. In the mid‐log phase Cap‐Clip does not modify rRNA since there is a single phosphate at the 5ʹ end. Therefore, exonuclease digestion is the same, with or without Cap‐Clip treatment. In the diauxic phase, the possibilities include single phosphate, triphosphate alone or with a modification, such as a cap. Cap‐Clip digestion leaves a single phosphate in all these possibilities.

When RNA from mid‐log and diauxic cells was treated with Cap‐Clip (Figure 4B), all RNA was digested by exonucleases. This suggests that resistant molecules in the diauxic phaser acquire additional phosphates to evade exonuclease degradation (Figure 4B).

Figure 5A shows the results of sequential treatment with AP and Cap‐Clip. RNA molecules from mid‐log yeast, which are fully susceptible to exonucleases due to their 5ʹ‐monophosphate, become resistant after AP plus Cap‐Clip digestion. The pyrophosphatase activity of Cap‐Clip does not restore susceptibility. AP removes the phosphates, leaving an OH group at the 5ʹ end, rendering the RNA resistant to exonucleases, while Cap‐Clip has no further effect. The observation that diauxic organisms have approximately 35% exonuclease resistance also indicates that approximately 65% of 18S and 25S molecules still carry a single 5ʹ‐phosphate. We observed a small amount of RNase activity when using Cap‐Clip (Figure S2) that accounts for the missing resistance percentage in both midlog and diauxic rRNA (Figure 5B). We know that resistant molecules have more than one phosphate at their 5ʹ end, as they become susceptible after decapping. Since AP was the first enzyme in the sequential digestion, and exonuclease resistance did not reach 100%, the extra phosphates must have been protected from AP by some additional modification at the 5ʹ end. Cap‐Clip makes them susceptible to exonucleases, even with the additional structure. The remaining molecules with 5ʹ‐monophosphates become exonuclease resistant by the initial AP removal of the phosphates.

As shown in Figure 6, both TOR1‐deleted yeast cells (BY‐28996) and wild type (WT) treated with rapamycin produced resistant 18S and 25S as in those produced by wild‐type cells nearing growth inhibition. When BY‐28996 and WT + Rap cells were treated with alkaline phosphatase or decapping pyrophosphatase (Cap‐Clip) individually or in sequence, the results resembled those of wild‐type S. cerevisiae (Figures 3, 4, 5) during the diauxic growth phase. Thus, it appears that regardless of the mechanism by which 18S and 25S are converted to an exonuclease‐resistant state, it seems to be suppressed by TOR1.

As our data regarding exonuclease resistance revealed a modification at the 5ʹ ends of resistant 18S and 25S when TOR1 is inactive, we wanted to determine whether this process functions independently of the usual polycistronic transcription by RNAPI and processing of rRNA. To this end, we used CARA cells, where RNAPI is constitutively active, regardless of the TOR pathway status. Figure 7A shows the growth curve of CARA cells indicating onset of exonuclease resistance production (red arrow). Figure 7B shows exonuclease resistance percentages of CARA cells during mid‐log and diauxic phases, and after rapamycin treatment. There is a significant difference (as indicated by the p values) between mid‐log CARA cells and diauxic or rapamycin treated cells.

We used timed thiouracil labeling of nascent RNA combined with exonuclease resistance measurements utilizing gel analysis and bioanalyzer measurements. A preliminary look at thiouracil labeling (Figure 8A) indicated that it reliably reflected the rate of rRNA synthesis, differentiating the mid‐log phase from the diauxic growth phase. This gave us the opportunity to label the rRNAs at a particular phase and monitor them to determine whether these molecules undergo post‐synthesis modifications that confer resistance to exonuclease digestion. As shown in Figure 8B Lane 1, the yeast that were labeled from hours 4–5 in fresh YEPD (their usual mid‐log phase) were completely digested by exonuclease (Lane 4). When yeast that were labeled for the same time period were allowed to rest for another 2 h with the same degree of nutritionally depleted YEPD without thiouracil (approaching the diauxic phase, Lane 2), they became resistant to exonuclease digestion (Lane 5). The fact that they contained thiouracil indicates that they were synthesized between hours 4 and 5, and clearly, some modification occurred to them over the next 2 h, leading to exonuclease resistance. When the yeast cells were incubated overnight (diauxic growth phase) and labeled with thiouracil for 1 h (lane 6), they produced newly synthesized exonuclease‐resistant 18S and 25S molecules. These data indicate that when the 5ʹ‐end modification system is active, both previously synthesized and newly formed ribosomal RNA molecules are targeted for these changes.