Growth curve and production of resistant 18S and 25S in wild-type Saccharomyces and in TOR-deleted mutants.
During the growth cycle of yeast, the rate of new yeast formation diminishes as nutrient availability decreases, eventually reaching a stationary phase. As shown in Fig. 1, the growth curve of the TOR-mutant S. cerevisiae (BY-28996) is similar to the wild-type Saccharomyces cerevisiae (S288C), containing a lag, logarithmic and stationary phase, but they differ on the timing of these phases. The production of resistant 18S and 25S is clearly phase dependent, as it increases as cells near the stationary phase of nutrient depletion (Fig. 2). The growth phase of TORC1-deleted BY-28996 was much longer (Fig. 1), but in contrast to that of S288C, the production of resistant 18S and 25S molecules remained constant and reached similar levels throughout all the phases.
5’-End analysis of the exonuclease resistance of the 18S and 25S wild-type strains at different growth phases
As mentioned, normally processed 18S and 25S molecules have a single phosphate at their 5’ end and are susceptible to these exonucleases. Thus, for them to become resistant, some changes need to occur. Several possibilities can make RNA molecules resistant to single 5’-phosphate-dependent RNA exonucleases (Fig. 3–5). One is the removal of the single phosphate at its 5’ end, resulting in an OH at the 5’ position of the first ribose. Another is that more phosphates are added at the 5’ end. A third possibility is the addition of a structure other than a phosphate such as a cap, for example, to the 5’ end phosphate. To determine which of these possibilities are most likely, we used digestion with alkaline phosphatase (AP) (Fig. 3A), the decapping enzyme Cap-Clip, which is a pyrophosphatase that cuts only between phosphates (Fig. 4A), and the combination of both enzymes (Fig. 5A).
As shown in Fig. 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 5’ end, as expected for transcribed and processed 18S and 25S molecules. Digestion with AP increases the resistance of the RNA to 100% by removing a single phosphate, leaving an OH group on the 5’ end, thus making the exonuclease unable to digest the RNA. As the cells transition to the stationary phase, 30–40% of them become resistant to exonucleases, indicating some change at the 5’ end of these molecules. When they are digested with AP, they also become 100% resistant.
Exonuclease resistance of RNA in stationary yeast can arrive from several ways as depicted in Fig. 3A. One possibility is that there might be more than one phosphate on the 5’ end, making the RNA exonuclease resistant. Digestion with AP removes all the phosphates, leaving the molecules resistant to exonuclease. Another possibility is that the resistant molecules underwent other structural changes that made them resistant to exonuclease and AP. When they are digested with AP, they maintain their exonuclease resistance, and the remaining resistance is due to the removal of the single phosphate from the usually processed molecules by AP.
When RNA from mid-log and stationary cells were treated with Cap-Clip (Fig. 4B), they were all digested by exonucleases. This indicates that, at minimum, the resistant molecules acquired additional phosphates to become resistant to exonucleases, and again became susceptible after Cap-Clip removed them, leaving a single phosphate (Fig. 4A).
The results of sequential digestion with AP and Cap-Clip are shown in Fig. 5. Molecules from mid-log yeast, which are fully susceptible to exonucleases due to their 5’-monophosphate, become approximately 90% resistant after AP digestion, and pyrophosphatase activity by Cap-Clip does not increase susceptibility. AP will eliminate the phosphates, resulting in an OH at the 5’ end, and Cap-Clip will have no effect. The observation that stationary organisms have approximately 40% exonuclease resistance also tells us that approximately 60% of 18S and 25S molecules still carry a single 5’-phosphate. We know that resistant molecules have more than one phosphate at their 5’ end since 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 digestion by some additional modification at the 5’ end. Cap-Clip will make them susceptible to exonucleases, even with the additional structure. The remaining 60% of molecules with 5’-monophosphates will become exonuclease resistant by the initial AP removal of the phosphates.
Effects of rapamycin inhibition and TORC1 deletion on resistant 18S and 25S
As shown in Fig. 6, both TORC1-deleted yeast cells and those treated with rapamycin produced resistant 18S and 25S in amounts similar to those produced by wild-type cells approaching growth inhibition. When treated with alkaline phosphatase or decapping pyrophosphatase (Cap-Clip) individually or in sequence, the results mirrored the molecules produced by wild-type Saccharomyces cerevisiae (Fig. 3–5 ) during the stationary growth phase. Thus, it appears that regardless of the mechanism by which these molecules are converted to an exonuclease-resistant state, they seem to function independently of TORC1.
Modification of the 18S and 25S molecules for exonuclease resistance
As our data regarding exonuclease resistance revealed a modification at the 5’ ends of resistant 18S and 25S that is independent of TORC1 regulation, we wanted to determine whether this process also functions independently of the usual polycistronic transcription by RNAPI and processing of rRNA. To this end, 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 (Fig. 7A) indicated that it reliably reflected the rate of rRNA synthesis, differentiating the mid-log phase from the stationary growth phase. This gave us the opportunity to label the rRNAs at a particular phase, and monitor them to determine if these molecules undergo post-synthesis modifications that confer resistance to exonuclease digestion. As shown in Fig. 7B Lane 1, the yeast that were labeled from hours 4 to 5 in fresh YEPD (their usual mid-log phase) were completely digested by a terminator (Lane 4). When yeast that were labeled for the same time period were allowed to rest for another two hours with the same degree of nutritionally depleted YEPD without thiouracil (approaching the stationary phase, Lane 2), they became resistant to Terminator 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 two hours, leading to exonuclease resistance. When the yeast cells were incubated overnight (stationary growth phase) and labeled with thiouracil for one hour (lane 6), they produced newly synthesized exonuclease-resistant 18S and 25S molecules. These data indicate that when this TOR-independent 5’-end modification system is active, both previously synthesized and newly formed ribosomal RNA molecules are targeted for these changes.