18S and 25S ribosomal RNA molecules resistant to a 5'-monophosphate dependent exonuclease are produced by a mechanism independent of TOR

18S and 25S ribosomal RNA molecules resistant to a 5'-monophosphate dependent exonuclease are produced by a mechanism independent of TOR | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article 18S and 25S ribosomal RNA molecules resistant to a 5'-monophosphate dependent exonuclease are produced by a mechanism independent of TOR Miguel Rocha, Bhavani Gowda, Jacob Fleischmann This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4547749/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract It has been previously shown that Saccharomyces cerevisiae yeast cells produce 18S and 25S ribosomal RNA components that are resistant to exonucleases and require a single phosphate at the 5’- end of the RNA. These molecules are produced during the stationary growth phase when TOR activity decreases. We wanted to further define the relationship between TOR and these resistant RNA molecules. Active suppression of TOR activity by rapamycin results in the production of these molecules. Similarly, a TORC1-deleted mutant Saccharomyces cerevisiae produces resistant 18S and 25S in a steady fashion. Thiouracil labeling of these molecules showed that molecules previously produced during the logarithmic growth phase can be converted to this resistant state. Thiouracil uptake assays also revealed that fewer 18S and 25S genes are produced during the stationary phase. The decapping of these molecules converts them back to an exonuclease-sensitive state. These data indicate that the production of exonuclease resistance of 18S and 25S is independent of TOR activity and is perhaps suppressed when TOR is active. Decapping converts them back to an exonuclease-sensitive state, indicating that at the minimum, there is an additional phosphate at their 5’-end. These molecules likely allow the presence of some ribosomes in the nutritional decline phase to maintain protein production. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction In robustly growing cells, ribosome production consumes the greatest portion of the energy derived from cellular metabolism [ 1 ]. This process is highly sensitive to the availability of nutritional resources and is primarily regulated at the transcriptional level. Three RNA polymerases are utilized in this process [ 2 ]. RNA polymerase I (RNAP I) transcribes multiple repeating ribosomal RNA genes (rDNAs) as 35S precursors, which are processed into 18S, 25S and 5.8S components. RNA polymerase II (RNAP II) transcribes ribosomal protein genes, and RNA polymerase III (RNAP III) copies 5.0S from the opposite strand of the intergenic sequences of rDNA. Typically, the 18S and 25S molecules emerging from the endonucleolytic activity of the processome have a single phosphate at their 5’end, represented by the phosphate group connecting the 3’ position of one ribose to the 5’ position of the next ribose [ 3 ]. This makes them vulnerable both in vivo and in vitro to attack by 5′ → 3 exonucleases that require 5′-monophosphates [ 4 ]. Examples of such nucleases are Terminator (Lucigen) and XrnI (New England Biolabs), which are commercially available for the purpose of degrading rRNA from total RNA extracted from cells. In studies with the pathogenic yeast Candida albicans , we unexpectedly found that the cells produced exonuclease-resistant 18S and 25S molecules [ 5 ]. These molecules appeared as the cells approached the stationary growth phase with decreasing nutrient availability. We further detected these genes in Saccharomyces cerevisiae , including in polymerase-switched cells [ 6 ], which lack RNAPI activity. These molecule have at least one extra phosphate at their 5’ end, making them susceptible to exonuclease digestion after being treated with decapping enzymes like tobacco acid pyrophosphatase(TAP) [ 7 ]. TAP cuts between two phosphates exclusively, thus leaving a single phosphate at the 5’ end of these molecules, making them vulnerable to exonucleases. Having more than one phosphate at the 5’-end of an RNA is observed in newly transcribed molecules, as RNA polymerases initiate transcription with triphosphate nucleotides [ 8 ]. It is unlikely that these resistant 18S and 25S molecules are newly transcribed. It is more likely that a modification occurs as the cells experience nutritional deprivation, making them resistant to exonuclease degradation. This would be of some advantage to the cells in maintaining ribosome generation with dwindling nutritional sources. The main regulators of cell metabolism in yeast are the Ser/Thr kinases residing in the protein complexes TORC1 and TORC2 [ 9 ]. Complex 1 (TORC1) regulates ribosome biogenesis [ 10 ] and translation [ 11 ], including the transcriptional activity of RNAPI [ 12 ]. The current understanding is that TORC1 downregulates the transcriptional activity of RNAPI in response to dwindling nutrient sources [ 12 ]. Thus, it was of interest to explore any possible role that TORC1 may play in relation to these exonuclease-resistant 18S and 25S genes. We show here that the production of such exonuclease-resistant 18S and 25S molecules, is not TOR dependent, as they are produced in rapamycin-suppressed cells and in TORC1-deleted mutants. We also show that thiouracil-labeled nascent RNAs that are transcribed and processed from 18S and 25S can be modified to become exonuclease resistant via a mechanism that has yet to be described. Materials and Methods Organisms Saccharomyces cerevisiae S288C (ATCC) and BY28996 ( MAT a ura3-1 leu2-3, 112trp1-1 his3-11, 15 ade2-1 can1-100 tor1Δ::KanMX tor2Δ::HIS3 pRS316[TOR2] (YGRC/NBRP Japan)) were maintained in 50% glycerol in YPD broth (2% w/V tryptone, 1% w/v yeast extract, 2% w/v dextrose) at -80°C. The cells were activated in YPD broth at 30°C, maintained on Sabouraud dextrose agar at 4°C, and passaged every 4–6 weeks up to 4–5 times. Yeasts were lifted from the agar surface and grown in YPD broth for various lengths of time at 30°C. Yeast cell concentrations were established using a hemocytometer. Growth Curve Analysis Yeast cells ( S. cerevisiae S288C and BY28996) were collected at multiple time points, starting at 4 hours and ending at 48 hours. The cell concentration was calculated using a hemocytometer. The data were plotted using GraphPad Prism 9 software. RNA isolation The cells were collected by centrifugation, washed with sterile phosphate-buffered saline (PBS) and placed on ice pending total RNA extraction. The cells were disrupted with RNase-free zirconia beads, and RNA was isolated using a RiboPure™ RNA Purification Kit for Yeast (Invitrogen) according to the manufacturer’s instructions. RNA quantification and quality were assessed by using a Qubit 4 fluorometer and an Agilent 2100 Bioanalyzer. RNA analysis Exonuclease treated and nontreated RNA samples were loaded into an RNA 6000 Nano chip and analyzed with an Agilent 2100 bioanalyzer system (Agilent Technologies, INC). Electropherograms were used to calculate exonuclease resistance percentages by measuring the areas under the peaks of untreated (uncut) RNA and dividing it by the area of treated RNA (Supplementary Fig.) Alkaline Phosphatase and Decapping Assays 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. All samples were purified by ethanol precipitation before proceeding to the exonuclease (Terminator) digestion assay. 5’-Monophosphate-dependent Exonuclease Experiments and 5’-End Analysis Total RNA was treated with either Terminator (Biosearch Technologies) or XrnI (New England Biolabs) following the manufacturer’s protocol using the supplied Buffer A. The ratio of enzyme to substrate employed was 1 U per 1 µg of RNA to ensure adequate cleavage. RNase inhibitors were used in all the assays. Rapamycin Assays Yeast cells were incubated with 1 µg of rapamycin (Sigma‒Aldrich) per 1x10 6 cells. The incubation time was 6 hours at 30°C with constant shaking. After incubation, the cells were washed with PBS and used for the appropriate assay. Thiouracil Experiments Yeast cells were cultured in YPD medium until they reached the desired growth phase, either mid-log (5 hours) or stationary (16 hours). 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 prior to resuspension. Detection of Thiouracil by blotting 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 minutes. 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 TM (Prometheus) chemiluminescence substrate was used to detect the HRP signal. The film was developed with an SRX-101A Konica film processor. Results 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. 3 A), the decapping enzyme Cap-Clip, which is a pyrophosphatase that cuts only between phosphates (Fig. 4 A), and the combination of both enzymes (Fig. 5 A). As shown in Fig. 3 B, 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. 3 A. 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. 4 B), 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. 4 A). 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. 7 A) 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. 7 B 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. Discussion We have established several reports on the existence of exonuclease-resistant ribosomal 18S and 25S RNA molecules in yeast. Consistently, these molecules are produced as the nutritional support for cell growth diminishes, a phase in which TOR downregulates RNAPI activity. Our data showing the production of these molecules with TOR inhibition by rapamycin indicate that this process is independent of TOR or possibly suppressed by TOR, under conditions of adequate nutritional status. The consistent production of resistant 18S and 25S by TOR-deleted yeast cells adds additional support for TOR independence. Thiouracil labeling of nascent ribosomal RNAs showed that this conversion to exonuclease resistance involves both previously produced and processed molecules, as well as newly transcribed molecules. Thus, once this process is activated, RNA molecules with a single 5’-phosphate end become targets for modification. The mechanism by which these molecules become exonuclease resistant is unknown. The fact that these processed molecules can be made susceptible to a 5’-dependent exonuclease by a decapping reaction, indicates that any modification occurring at the 5’-end that initially makes them resistant to exonuclease digestion, at minimum, includes an added phosphate by a kinase reaction. Certainly, it is well established for recycling of mRNAs in the cytoplasm that molecules whose triphosphate 5’-end is lost to a monophosphate state become targets for 5’-kinases and guanylyltransferases for recovery [ 13 ]. It is also well established that the polycistronic ribosomal RNA products of RNAPI transcription are processed to small and large ribosomal RNA components with a single phosphate at their 5’ end, making them targets for such kinases [ 14 ]. However, this alone does not explain why this resistance is detected exclusively as the cell approaches the stationary growth phase, and is not detected in the robust mid-log phase. We have also reported that this exonuclease resistance develops in the nucleus; thus, whatever this mechanism is, it is present there. This would be different from the capping of mRNA in the nucleus, which results from the removal of the terminal phosphate of the 5’-triphosphated RNA by RNA triphosphatase, resulting in biphosphate 5’ ends, to which a guanylyl triphosphate is added by a transferase with the release of two phosphate molecules. There are genes in Saccharomyces cerevisiae whose transcription is specifically activated in the stationary phase, and these genes need to be translated [ 15 ]. Thus, maintaining an adequate number of ribosomes is advantageous for the cell, and protecting 18S and 25S molecules from exonuclease digestion helps with this process. Indeed, such exonuclease-resistant ribosomal RNAs have been found in ribosomes isolated from Candida albicans [ 16 ]. It has also been proposed that yeast that approaches the stationary growth phase and the phase itself could be a possible model for the study of aging [ 17 ]. The conversion of otherwise susceptible to exonuclease-resistant ribosomal RNA components that we are describing is a phenomenon specific for the later stage of the yeast cycle, and defining the details of this process could be beneficial in this area of investigation. Declarations Ethical Approval and consent to participate Not applicable Consent to Publication Not applicable Conflict of interest The authors declare that they have no competing interests Funding Elias, Genevieve, Georgianna/ Atol Charitable Trust The Monica Lester Charitable Trust Acknowledgment Not applicable Author Contribution MR and JF contributed to the conception, design of the experiments, interpretation of the data and writing the manuscriptMR and BG contributed with the acquisition and analysis of the data References Warner JR. The economics of ribosome biosynthesis in yeast. Trends Biochem Sci. 1999;24(11):437–40. Gutierrez-Santiago F, Navarro F. Transcription by the Three RNA Polymerases under the Control of the TOR Signaling Pathway in Saccharomyces cerevisiae. Biomolecules 2023, 13(4). Henras AK, Plisson-Chastang C, O'Donohue MF, Chakraborty A, Gleizes PE. An overview of pre-ribosomal RNA processing in eukaryotes. Wiley Interdiscip Rev RNA. 2015;6(2):225–42. Jager D, Sharma CM, Thomsen J, Ehlers C, Vogel J, Schmitz RA. Deep sequencing analysis of the Methanosarcina mazei Go1 transcriptome in response to nitrogen availability. Proc Natl Acad Sci U S A. 2009;106(51):21878–82. Fleischmann J, Rocha MA. Nutrient depletion and TOR inhibition induce 18S and 25S ribosomal RNAs resistant to a 5'-phosphate-dependent exonuclease in Candida albicans and other yeasts. BMC Mol Biol. 2018;19(1):1. Rocha MA, Gowda BS, Fleischmann J. RNAP II produces capped 18S and 25S ribosomal RNAs resistant to 5'-monophosphate dependent processive 5' to 3' exonuclease in polymerase switched Saccharomyces cerevisiae. BMC Mol Cell Biol. 2022;23(1):17. Lockard RE, Rieser L, Vournakis JN. Labeling of eukaryotic messenger RNA 5' terminus with phosphorus – 32: use of tobacco acid pyrophosphatase for removal of cap structures. Gene Amplif Anal. 1981;2:229–51. Aprikian P, Moorefield B, Reeder RH. New model for the yeast RNA polymerase I transcription cycle. Mol Cell Biol. 2001;21(15):4847–55. Morozumi Y, Shiozaki K. Conserved and Divergent Mechanisms That Control TORC1 in Yeasts and Mammals. Genes (Basel) 2021, 12(1). Guerra P, Vuillemenot LPE, van Oppen YB, Been M, Milias-Argeitis A. TORC1 and PKA activity towards ribosome biogenesis oscillates in synchrony with the budding yeast cell cycle. J Cell Sci 2022, 135(18). Wang Y, Zheng X, Li G, Wang X. TORC1 Signaling in Fungi: From Yeasts to Filamentous Fungi. Microorganisms 2023, 11(1). Sengupta S, Peterson TR, Sabatini DM. Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress. Mol Cell. 2010;40(2):310–22. Trotman JB, Schoenberg DR. A recap of RNA recapping. Wiley Interdiscip Rev RNA. 2019;10(1):e1504. Woolford JL Jr., Baserga SJ. Ribosome biogenesis in the yeast Saccharomyces cerevisiae. Genetics. 2013;195(3):643–81. Riou C, Nicaud JM, Barre P, Gaillardin C. Stationary-phase gene expression in Saccharomyces cerevisiae during wine fermentation. Yeast. 1997;13(10):903–15. Fleischmann J, Rocha MA, Hauser PV, Gowda BS, Pilapil MGD. Exonuclease resistant 18S and 25S ribosomal RNA components in yeast are possibly newly transcribed by RNA polymerase II. BMC Mol Cell Biol. 2020;21(1):59. Werner-Washburne M, Roy S, Davidson GS. Aging and the survival of quiescent and non-quiescent cells in yeast stationary-phase cultures. Subcell Biochem. 2012;57:123–43. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4547749","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":315537592,"identity":"d9ea7650-57db-4b6b-b9ea-ab0c083324f3","order_by":0,"name":"Miguel Rocha","email":"","orcid":"","institution":"VA Greater Los Angeles Healthcare System","correspondingAuthor":false,"prefix":"","firstName":"Miguel","middleName":"","lastName":"Rocha","suffix":""},{"id":315537593,"identity":"0cc3e0fc-c95d-4230-85e1-f4644364230e","order_by":1,"name":"Bhavani Gowda","email":"","orcid":"","institution":"VA Greater Los Angeles Healthcare System","correspondingAuthor":false,"prefix":"","firstName":"Bhavani","middleName":"","lastName":"Gowda","suffix":""},{"id":315537594,"identity":"5cf37e0f-ffc6-4ac6-aa5c-fc315ef45ca7","order_by":2,"name":"Jacob Fleischmann","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAn0lEQVRIiWNgGAWjYBACxubDjQ8YGCxAbAMitbQlNgOVSpCghYEtsU2CNC3MbYxt1Tw1EvIM7M3bJIh0GGPbbZ5jEoYNPMfKiNQyv7HtNm+DBGODRI4Z8bYUA7XYN8i/IUELM1BLYoMED/FamiXnHJNIbuNJK7YgSothG/PBD29qbGz72Q9vvEGclgYog40o5SAgT7TKUTAKRsEoGLkAAAK5JzC/DHRQAAAAAElFTkSuQmCC","orcid":"","institution":"VA Greater Los Angeles Healthcare System","correspondingAuthor":true,"prefix":"","firstName":"Jacob","middleName":"","lastName":"Fleischmann","suffix":""}],"badges":[],"createdAt":"2024-06-07 18:48:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4547749/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4547749/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59087037,"identity":"567e7961-87fa-42be-93fe-4d8a5b21230d","added_by":"auto","created_at":"2024-06-26 08:10:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":19484,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the growth curves from S. \u003cem\u003ecerevisiae\u003c/em\u003e wild type (S288C) and S. \u003cem\u003ecerevisiae\u003c/em\u003e TOR mutant (BY-28996). Cells were incubated in YPD medium at 30\u003csup\u003eo\u003c/sup\u003eC until stationary phase was reached.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4547749/v1/172102f15902885a59221a0f.png"},{"id":59085644,"identity":"d4dab5c3-8216-4830-9837-6540915d3a8f","added_by":"auto","created_at":"2024-06-26 07:54:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":38611,"visible":true,"origin":"","legend":"\u003cp\u003eTerminator percentage resistance in RNA from S. \u003cem\u003ecerevisiae\u003c/em\u003e wild type (S288C) and S. \u003cem\u003ecerevisiae\u003c/em\u003eTOR-mutant (BY-28996). Ratios of uncut to cut ribosomal 18S and 25S were obtained using a Bioanalyzer\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4547749/v1/c3a1603f36a269a5416707d0.png"},{"id":59085650,"identity":"a12f0726-1da9-4436-91db-a9f4983cc931","added_by":"auto","created_at":"2024-06-26 07:54:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":83023,"visible":true,"origin":"","legend":"\u003cp\u003e5’ end analysis of ribosomal RNA extracted from wild type cells under mid-log and stationary conditions. (\u003cstrong\u003eA\u003c/strong\u003e) Illustration of the possible 5’-end forms of 18S and 25S and the predicted outcome after enzyme treatment to both mid-log and stationary organisms. (\u003cstrong\u003eB\u003c/strong\u003e) Terminator resistance percentages from S. \u003cem\u003ecerevisiae\u003c/em\u003e wild type mid-log (WT ML) and stationary (WT STAT) were calculated after RNA was treated with alkaline phosphatase (AP) or without treatment.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4547749/v1/ee9a6bb844859780d1e84ee4.png"},{"id":59086401,"identity":"68dc077b-42bf-427b-9788-6dc356bf665e","added_by":"auto","created_at":"2024-06-26 08:02:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":80053,"visible":true,"origin":"","legend":"\u003cp\u003e5’ end analysis of ribosomal RNA extracted from wild type cells under mid-log and stationary conditions. (\u003cstrong\u003eA\u003c/strong\u003e) Illustration of the possible 5’-end forms of 18S and 25S and the predicted outcome after enzyme treatment to both mid-log and stationary organisms. (\u003cstrong\u003eB\u003c/strong\u003e) Terminator resistance percentages from S. \u003cem\u003ecerevisiae\u003c/em\u003ewild type mid-log (WT ML) and stationary (WT STAT) were calculated after RNA was treated with acid pyrophosphatase (Cap Clip) or without treatment.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4547749/v1/5b6ee437e6509a87c86b91b5.png"},{"id":59086403,"identity":"9322a6c1-47b3-44bf-a067-7bd7e08c53e2","added_by":"auto","created_at":"2024-06-26 08:02:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":100566,"visible":true,"origin":"","legend":"\u003cp\u003e5’ end analysis of ribosomal RNA extracted from wild type cells under mid-log and stationary conditions. \u0026nbsp;(\u003cstrong\u003eA\u003c/strong\u003e) Illustration of the possible 5’-end forms of 18S and 25S and the predicted outcome after enzyme treatment to both mid-log and stationary organisms. (\u003cstrong\u003eB\u003c/strong\u003e)Terminator resistance percentages from S. \u003cem\u003ecerevisiae\u003c/em\u003e wild type mid-log (WT ML) and stationary (WT STAT) were calculated after RNA was treated sequentially with AP and Cap Clip, or without treatment.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4547749/v1/88e5723d5dd4d4c27238d3fb.png"},{"id":59085648,"identity":"fe649a91-f547-4fd9-b766-c3a041d935ee","added_by":"auto","created_at":"2024-06-26 07:54:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":39382,"visible":true,"origin":"","legend":"\u003cp\u003e5’ end analysis of ribosomal RNA extracted from wild type cells exposed to rapamycin, and from TOR-mutant cells. Exonuclease resistance percentages from S. \u003cem\u003ecerevisiae\u003c/em\u003e wild type plus rapamycin (WT Rap) and TOR-mutant cells (BY-96) were calculated after RNA treatment with alkaline phosphatase (AP), acid pyrophosphatase (Cap Clip), both AP and Cap Clip.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4547749/v1/a3ead168f05c05f9f4fedb5d.png"},{"id":59085649,"identity":"00d2e049-9de8-4c70-b54f-799049695e01","added_by":"auto","created_at":"2024-06-26 07:54:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":301847,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of RNA transcription in \u003cem\u003eS. cerevisiae\u003c/em\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) 4-Thiouracil was added to the cultures after organisms reached mid-log or stationary phase for 1, 2 and 3 hours. Thiol-specific biotinylated RNA (1µg) was separated on agarose, transferred onto a charged nylon membrane and detected with streptavidin-HRP. \u0026nbsp;(\u003cstrong\u003eB\u003c/strong\u003e) Exonuclease resistance in newly synthesized RNA. Ribosomal RNA from mid-log (ML) and stationary (STAT) S. \u003cem\u003ecerevisiae\u003c/em\u003e grown in the presence of thiouracil was labelled with MTSA-biotin and separated on a 1.2% agar gel, stained with SYBR-gold dye (\u003cstrong\u003ei\u003c/strong\u003e), transferred onto nitrocellulose membrane followed by methylene blue staining (\u003cstrong\u003eii)\u003c/strong\u003e and finally, the biotinylated RNA was detected by HRP-Streptavidin (\u003cstrong\u003eiii)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4547749/v1/d95cf88530cf798d2006211a.png"},{"id":66057384,"identity":"de12e9d7-9350-4c22-b4b6-fdf468f29eda","added_by":"auto","created_at":"2024-10-07 09:32:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1041892,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4547749/v1/c08f943a-e37a-4da1-aa0c-abd4333ccf4a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"18S and 25S ribosomal RNA molecules resistant to a 5'-monophosphate dependent exonuclease are produced by a mechanism independent of TOR","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn robustly growing cells, ribosome production consumes the greatest portion of the energy derived from cellular metabolism [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This process is highly sensitive to the availability of nutritional resources and is primarily regulated at the transcriptional level. Three RNA polymerases are utilized in this process [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. RNA polymerase I (RNAP I) transcribes multiple repeating ribosomal RNA genes (rDNAs) as 35S precursors, which are processed into 18S, 25S and 5.8S components. RNA polymerase II (RNAP II) transcribes ribosomal protein genes, and RNA polymerase III (RNAP III) copies 5.0S from the opposite strand of the intergenic sequences of rDNA. Typically, the 18S and 25S molecules emerging from the endonucleolytic activity of the processome have a single phosphate at their 5\u0026rsquo;end, represented by the phosphate group connecting the 3\u0026rsquo; position of one ribose to the 5\u0026rsquo; position of the next ribose [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This makes them vulnerable both in vivo and in vitro to attack by 5\u0026prime; \u0026rarr; 3 exonucleases that require 5\u0026prime;-monophosphates [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Examples of such nucleases are Terminator (Lucigen) and XrnI (New England Biolabs), which are commercially available for the purpose of degrading rRNA from total RNA extracted from cells. In studies with the pathogenic yeast \u003cem\u003eCandida albicans\u003c/em\u003e, we unexpectedly found that the cells produced exonuclease-resistant 18S and 25S molecules [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These molecules appeared as the cells approached the stationary growth phase with decreasing nutrient availability. We further detected these genes in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, including in polymerase-switched cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], which lack RNAPI activity.\u003c/p\u003e \u003cp\u003eThese molecule have at least one extra phosphate at their 5\u0026rsquo; end, making them susceptible to exonuclease digestion after being treated with decapping enzymes like tobacco acid pyrophosphatase(TAP) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. TAP cuts between two phosphates exclusively, thus leaving a single phosphate at the 5\u0026rsquo; end of these molecules, making them vulnerable to exonucleases. Having more than one phosphate at the 5\u0026rsquo;-end of an RNA is observed in newly transcribed molecules, as RNA polymerases initiate transcription with triphosphate nucleotides [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. It is unlikely that these resistant 18S and 25S molecules are newly transcribed. It is more likely that a modification occurs as the cells experience nutritional deprivation, making them resistant to exonuclease degradation. This would be of some advantage to the cells in maintaining ribosome generation with dwindling nutritional sources.\u003c/p\u003e \u003cp\u003eThe main regulators of cell metabolism in yeast are the Ser/Thr kinases residing in the protein complexes TORC1 and TORC2 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Complex 1 (TORC1) regulates ribosome biogenesis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and translation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], including the transcriptional activity of RNAPI [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The current understanding is that TORC1 downregulates the transcriptional activity of RNAPI in response to dwindling nutrient sources [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Thus, it was of interest to explore any possible role that TORC1 may play in relation to these exonuclease-resistant 18S and 25S genes. We show here that the production of such exonuclease-resistant 18S and 25S molecules, is not TOR dependent, as they are produced in rapamycin-suppressed cells and in TORC1-deleted mutants. We also show that thiouracil-labeled nascent RNAs that are transcribed and processed from 18S and 25S can be modified to become exonuclease resistant via a mechanism that has yet to be described.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eOrganisms\u003c/h2\u003e \u003cp\u003e \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e S288C (ATCC) and BY28996 (\u003cem\u003eMAT\u003c/em\u003e\u003cb\u003ea\u003c/b\u003e \u003cem\u003eura3-1 leu2-3, 112trp1-1 his3-11, 15 ade2-1 can1-100 tor1Δ::KanMX tor2Δ::HIS3 pRS316[TOR2]\u003c/em\u003e (YGRC/NBRP Japan)) were maintained in 50% glycerol in YPD broth (2% w/V tryptone, 1% w/v yeast extract, 2% w/v dextrose) at -80\u0026deg;C. The cells were activated in YPD broth at 30\u0026deg;C, maintained on Sabouraud dextrose agar at 4\u0026deg;C, and passaged every 4\u0026ndash;6 weeks up to 4\u0026ndash;5 times. Yeasts were lifted from the agar surface and grown in YPD broth for various lengths of time at 30\u0026deg;C. Yeast cell concentrations were established using a hemocytometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eGrowth Curve Analysis\u003c/h2\u003e \u003cp\u003eYeast cells (\u003cem\u003eS. cerevisiae\u003c/em\u003e S288C and BY28996) were collected at multiple time points, starting at 4 hours and ending at 48 hours. The cell concentration was calculated using a hemocytometer. The data were plotted using GraphPad Prism 9 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation\u003c/h2\u003e \u003cp\u003eThe cells were collected by centrifugation, washed with sterile phosphate-buffered saline (PBS) and placed on ice pending total RNA extraction. The cells were disrupted with RNase-free zirconia beads, and RNA was isolated using a RiboPure\u0026trade; RNA Purification Kit for Yeast (Invitrogen) according to the manufacturer\u0026rsquo;s instructions. RNA quantification and quality were assessed by using a Qubit 4 fluorometer and an Agilent 2100 Bioanalyzer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eRNA analysis\u003c/h2\u003e \u003cp\u003eExonuclease treated and nontreated RNA samples were loaded into an RNA 6000 Nano chip and analyzed with an Agilent 2100 bioanalyzer system (Agilent Technologies, INC). Electropherograms were used to calculate exonuclease resistance percentages by measuring the areas under the peaks of untreated (uncut) RNA and dividing it by the area of treated RNA (Supplementary Fig.)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAlkaline Phosphatase and Decapping Assays\u003c/h2\u003e \u003cp\u003eCap-Clip\u0026trade; acid pyrophosphatase (CellScript) and alkaline phosphatase calf intestinal (CIP) (New England Biolabs) were used according to the manufacturer\u0026rsquo;s instructions. RNA samples were treated with CIP only, CIP followed by CapClip or CapClip alone. All samples were purified by ethanol precipitation before proceeding to the exonuclease (Terminator) digestion assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e5\u0026rsquo;-Monophosphate-dependent Exonuclease Experiments and 5\u0026rsquo;-End Analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was treated with either Terminator (Biosearch Technologies) or XrnI (New England Biolabs) following the manufacturer\u0026rsquo;s protocol using the supplied Buffer A. The ratio of enzyme to substrate employed was 1 U per 1 \u0026micro;g of RNA to ensure adequate cleavage. RNase inhibitors were used in all the assays.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eRapamycin Assays\u003c/h2\u003e \u003cp\u003eYeast cells were incubated with 1 \u0026micro;g of rapamycin (Sigma‒Aldrich) per 1x10\u003csup\u003e6\u003c/sup\u003e cells. The incubation time was 6 hours at 30\u0026deg;C with constant shaking. After incubation, the cells were washed with PBS and used for the appropriate assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eThiouracil Experiments\u003c/h2\u003e \u003cp\u003eYeast cells were cultured in YPD medium until they reached the desired growth phase, either mid-log (5 hours) or stationary (16 hours). 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 prior to resuspension.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDetection of Thiouracil by blotting\u003c/h2\u003e \u003cp\u003eThiouracil (Sigma) incorporation was measured by biotin detection using MTSA-biotin-XX (Biotium). Biotinylation reactions included RNA (1 \u0026micro;M), 10 mM HEPES (pH 7.5), 1 mM EDTA, and 25 \u0026micro;M MTS-biotin (dissolved in DMSO at 250 \u0026micro;M).\u003c/p\u003e \u003cp\u003eRNA was separated on formaldehyde agarose gels (Lonza) and stained with SYBR Gold nucleic acid gel stain (Life Technologies) for 30 minutes. 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 \u003csup\u003eTM\u003c/sup\u003e (Prometheus) chemiluminescence substrate was used to detect the HRP signal. The film was developed with an SRX-101A Konica film processor.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eGrowth curve and production of resistant 18S and 25S in wild-type Saccharomyces and in TOR-deleted mutants.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDuring 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the growth curve of the TOR-mutant S. \u003cem\u003ecerevisiae\u003c/em\u003e (BY-28996) is similar to the wild-type \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e (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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The growth phase of TORC1-deleted BY-28996 was much longer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), 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.\u003c/p\u003e \u003cp\u003e \u003cb\u003e5\u0026rsquo;-End analysis of the exonuclease resistance of the 18S and 25S wild-type strains at different growth phases\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs mentioned, normally processed 18S and 25S molecules have a single phosphate at their 5\u0026rsquo; 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\u0026rsquo;-phosphate-dependent RNA exonucleases (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). One is the removal of the single phosphate at its 5\u0026rsquo; end, resulting in an OH at the 5\u0026rsquo; position of the first ribose. Another is that more phosphates are added at the 5\u0026rsquo; end. A third possibility is the addition of a structure other than a phosphate such as a cap, for example, to the 5\u0026rsquo; end phosphate. To determine which of these possibilities are most likely, we used digestion with alkaline phosphatase (AP) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), the decapping enzyme Cap-Clip, which is a pyrophosphatase that cuts only between phosphates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), and the combination of both enzymes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, 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\u0026rsquo; 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\u0026rsquo; end, thus making the exonuclease unable to digest the RNA. As the cells transition to the stationary phase, 30\u0026ndash;40% of them become resistant to exonucleases, indicating some change at the 5\u0026rsquo; end of these molecules. When they are digested with AP, they also become 100% resistant.\u003c/p\u003e \u003cp\u003eExonuclease resistance of RNA in stationary yeast can arrive from several ways as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. One possibility is that there might be more than one phosphate on the 5\u0026rsquo; 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.\u003c/p\u003e \u003cp\u003eWhen RNA from mid-log and stationary cells were treated with Cap-Clip (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eThe results of sequential digestion with AP and Cap-Clip are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Molecules from mid-log yeast, which are fully susceptible to exonucleases due to their 5\u0026rsquo;-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\u0026rsquo; 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\u0026rsquo;-phosphate. We know that resistant molecules have more than one phosphate at their 5\u0026rsquo; 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\u0026rsquo; end. Cap-Clip will make them susceptible to exonucleases, even with the additional structure. The remaining 60% of molecules with 5\u0026rsquo;-monophosphates will become exonuclease resistant by the initial AP removal of the phosphates.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEffects of rapamycin inhibition and TORC1 deletion on resistant 18S and 25S\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, 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 \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e ) 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.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eModification of the 18S and 25S molecules for exonuclease resistance\u003c/h2\u003e \u003cp\u003eAs our data regarding exonuclease resistance revealed a modification at the 5\u0026rsquo; 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA) 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB 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\u0026rsquo;-end modification system is active, both previously synthesized and newly formed ribosomal RNA molecules are targeted for these changes.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe have established several reports on the existence of exonuclease-resistant ribosomal 18S and 25S RNA molecules in yeast. Consistently, these molecules are produced as the nutritional support for cell growth diminishes, a phase in which TOR downregulates RNAPI activity. Our data showing the production of these molecules with TOR inhibition by rapamycin indicate that this process is independent of TOR or possibly suppressed by TOR, under conditions of adequate nutritional status. The consistent production of resistant 18S and 25S by TOR-deleted yeast cells adds additional support for TOR independence. Thiouracil labeling of nascent ribosomal RNAs showed that this conversion to exonuclease resistance involves both previously produced and processed molecules, as well as newly transcribed molecules. Thus, once this process is activated, RNA molecules with a single 5\u0026rsquo;-phosphate end become targets for modification.\u003c/p\u003e \u003cp\u003eThe mechanism by which these molecules become exonuclease resistant is unknown. The fact that these processed molecules can be made susceptible to a 5\u0026rsquo;-dependent exonuclease by a decapping reaction, indicates that any modification occurring at the 5\u0026rsquo;-end that initially makes them resistant to exonuclease digestion, at minimum, includes an added phosphate by a kinase reaction. Certainly, it is well established for recycling of mRNAs in the cytoplasm that molecules whose triphosphate 5\u0026rsquo;-end is lost to a monophosphate state become targets for 5\u0026rsquo;-kinases and guanylyltransferases for recovery [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. It is also well established that the polycistronic ribosomal RNA products of RNAPI transcription are processed to small and large ribosomal RNA components with a single phosphate at their 5\u0026rsquo; end, making them targets for such kinases [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, this alone does not explain why this resistance is detected exclusively as the cell approaches the stationary growth phase, and is not detected in the robust mid-log phase. We have also reported that this exonuclease resistance develops in the nucleus; thus, whatever this mechanism is, it is present there. This would be different from the capping of mRNA in the nucleus, which results from the removal of the terminal phosphate of the 5\u0026rsquo;-triphosphated RNA by RNA triphosphatase, resulting in biphosphate 5\u0026rsquo; ends, to which a guanylyl triphosphate is added by a transferase with the release of two phosphate molecules.\u003c/p\u003e \u003cp\u003eThere are genes in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e whose transcription is specifically activated in the stationary phase, and these genes need to be translated [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Thus, maintaining an adequate number of ribosomes is advantageous for the cell, and protecting 18S and 25S molecules from exonuclease digestion helps with this process. Indeed, such exonuclease-resistant ribosomal RNAs have been found in ribosomes isolated from \u003cem\u003eCandida albicans\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. It has also been proposed that yeast that approaches the stationary growth phase and the phase itself could be a possible model for the study of aging [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The conversion of otherwise susceptible to exonuclease-resistant ribosomal RNA components that we are describing is a phenomenon specific for the later stage of the yeast cycle, and defining the details of this process could be beneficial in this area of investigation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthical Approval and consent to participate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eConsent to Publication\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eConflict of interest\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eElias, Genevieve, Georgianna/ Atol Charitable Trust\u003c/p\u003e\n\u003cp\u003eThe Monica Lester Charitable Trust\u003c/p\u003e\n\u003cp\u003eAcknowledgment\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eAuthor Contribution\u003c/p\u003e\u003cp\u003eMR and JF contributed to the conception, design of the experiments, interpretation of the data and writing the manuscriptMR and BG contributed with the acquisition and analysis of the data\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWarner JR. The economics of ribosome biosynthesis in yeast. Trends Biochem Sci. 1999;24(11):437\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGutierrez-Santiago F, Navarro F. Transcription by the Three RNA Polymerases under the Control of the TOR Signaling Pathway in Saccharomyces cerevisiae. Biomolecules 2023, 13(4).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHenras AK, Plisson-Chastang C, O'Donohue MF, Chakraborty A, Gleizes PE. An overview of pre-ribosomal RNA processing in eukaryotes. Wiley Interdiscip Rev RNA. 2015;6(2):225\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJager D, Sharma CM, Thomsen J, Ehlers C, Vogel J, Schmitz RA. Deep sequencing analysis of the Methanosarcina mazei Go1 transcriptome in response to nitrogen availability. Proc Natl Acad Sci U S A. 2009;106(51):21878\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFleischmann J, Rocha MA. Nutrient depletion and TOR inhibition induce 18S and 25S ribosomal RNAs resistant to a 5'-phosphate-dependent exonuclease in Candida albicans and other yeasts. BMC Mol Biol. 2018;19(1):1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRocha MA, Gowda BS, Fleischmann J. RNAP II produces capped 18S and 25S ribosomal RNAs resistant to 5'-monophosphate dependent processive 5' to 3' exonuclease in polymerase switched Saccharomyces cerevisiae. BMC Mol Cell Biol. 2022;23(1):17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLockard RE, Rieser L, Vournakis JN. Labeling of eukaryotic messenger RNA 5' terminus with phosphorus \u0026ndash;\u0026thinsp;32: use of tobacco acid pyrophosphatase for removal of cap structures. Gene Amplif Anal. 1981;2:229\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAprikian P, Moorefield B, Reeder RH. New model for the yeast RNA polymerase I transcription cycle. Mol Cell Biol. 2001;21(15):4847\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorozumi Y, Shiozaki K. Conserved and Divergent Mechanisms That Control TORC1 in Yeasts and Mammals. Genes (Basel) 2021, 12(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuerra P, Vuillemenot LPE, van Oppen YB, Been M, Milias-Argeitis A. TORC1 and PKA activity towards ribosome biogenesis oscillates in synchrony with the budding yeast cell cycle. J Cell Sci 2022, 135(18).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Zheng X, Li G, Wang X. TORC1 Signaling in Fungi: From Yeasts to Filamentous Fungi. Microorganisms 2023, 11(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSengupta S, Peterson TR, Sabatini DM. Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress. Mol Cell. 2010;40(2):310\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrotman JB, Schoenberg DR. A recap of RNA recapping. Wiley Interdiscip Rev RNA. 2019;10(1):e1504.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWoolford JL Jr., Baserga SJ. Ribosome biogenesis in the yeast Saccharomyces cerevisiae. Genetics. 2013;195(3):643\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRiou C, Nicaud JM, Barre P, Gaillardin C. Stationary-phase gene expression in Saccharomyces cerevisiae during wine fermentation. Yeast. 1997;13(10):903\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFleischmann J, Rocha MA, Hauser PV, Gowda BS, Pilapil MGD. Exonuclease resistant 18S and 25S ribosomal RNA components in yeast are possibly newly transcribed by RNA polymerase II. BMC Mol Cell Biol. 2020;21(1):59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWerner-Washburne M, Roy S, Davidson GS. Aging and the survival of quiescent and non-quiescent cells in yeast stationary-phase cultures. Subcell Biochem. 2012;57:123\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4547749/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4547749/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIt has been previously shown that \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e yeast cells produce 18S and 25S ribosomal RNA components that are resistant to exonucleases and require a single phosphate at the 5\u0026rsquo;- end of the RNA. These molecules are produced during the stationary growth phase when TOR activity decreases. We wanted to further define the relationship between TOR and these resistant RNA molecules.\u003c/p\u003e \u003cp\u003eActive suppression of TOR activity by rapamycin results in the production of these molecules. Similarly, a TORC1-deleted mutant \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e produces resistant 18S and 25S in a steady fashion. Thiouracil labeling of these molecules showed that molecules previously produced during the logarithmic growth phase can be converted to this resistant state. Thiouracil uptake assays also revealed that fewer 18S and 25S genes are produced during the stationary phase. The decapping of these molecules converts them back to an exonuclease-sensitive state.\u003c/p\u003e \u003cp\u003eThese data indicate that the production of exonuclease resistance of 18S and 25S is independent of TOR activity and is perhaps suppressed when TOR is active. Decapping converts them back to an exonuclease-sensitive state, indicating that at the minimum, there is an additional phosphate at their 5\u0026rsquo;-end. These molecules likely allow the presence of some ribosomes in the nutritional decline phase to maintain protein production.\u003c/p\u003e","manuscriptTitle":"18S and 25S ribosomal RNA molecules resistant to a 5'-monophosphate dependent exonuclease are produced by a mechanism independent of TOR","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-26 07:54:02","doi":"10.21203/rs.3.rs-4547749/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a71672d4-e4bc-4b9c-8514-942dd6719196","owner":[],"postedDate":"June 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-07T09:24:03+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-26 07:54:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4547749","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4547749","identity":"rs-4547749","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}