Translational control of MPS1 links protein synthesis with the initiation of cell division and spindle pole body duplication in Saccharomyces cerevisiae.

Protein synthesis underpins cell growth and controls when cells commit to a new round of cell division at a point in late G1 of the cell cycle called Start. Passage through Start also coincides with the duplication of the microtubule-organizing centers, the yeast spindle pole bodies, which will form the two poles of the mitotic spindle that segregates the chromosomes in mitosis. The conserved Mps1p kinase governs the duplication of the spindle pole body in Saccharomyces cerevisiae. Here, we show that the MPS1 transcript has a short upstream open reading frame that represses the synthesis of Mps1p. Mutating the MPS1 uORF makes the cells smaller, accelerates the appearance of Mps1p in late G1, and promotes completion of Start. Monitoring the spindle pole body in the cell cycle using structured illumination microscopy revealed that mutating the MPS1 uORF enabled cells to duplicate their spindle pole body earlier at a smaller cell size. The accelerated Start of MPS1 uORF mutants depends on the G1 cyclin Cln3p and the transcriptional repressor Whi5p but not on the Cln1,2p G1 cyclins. These results identify growth inputs in mechanisms that control duplication of the microtubule-organizing center and implicate these processes in the coupling of cell growth with division.

Late-life dietary folate restriction reduces biosynthetic processes without compromising healthspan in mice.

Folate is a vitamin required for cell growth and is present in fortified foods in the form of folic acid to prevent congenital abnormalities. The impact of low folate status on life-long health is poorly understood. We found that limiting folate levels with the folate antagonist methotrexate increased the lifespan of yeast and worms. We then restricted folate intake in aged mice and measured various health metrics, metabolites, and gene expression signatures. Limiting folate intake decreased anabolic biosynthetic processes in mice and enhanced metabolic plasticity. Despite reduced serum folate levels in mice with limited folic acid intake, these animals maintained their weight and adiposity late in life, and we did not observe adverse health outcomes. These results argue that the effectiveness of folate dietary interventions may vary depending on an individual's age and sex. A higher folate intake is advantageous during the early stages of life to support cell divisions needed for proper development. However, a lower folate intake later in life may result in healthier aging.

Targeting APEX2 to the mRNA encoding fatty acid synthase ß in yeast identifies interacting proteins that control its abundance in the cell cycle.

Profiling the repertoire of proteins associated with a given mRNA during the cell cycle is unstudied. Furthermore, it is easier to ask and answer what mRNAs a specific protein might bind to than the other way around. Here, we implemented an RNA-centric proximity labeling technology at different points in the cell cycle in highly synchronous yeast cultures. To understand how the abundance of FAS1, encoding fatty acid synthase, peaks late in the cell cycle, we identified proteins that interact with the FAS1 transcript in a cell cycle-dependent manner. We used dCas13d-APEX2 fusions to target FAS1 and label nearby proteins, which were then identified by mass spectrometry. The glycolytic enzyme Tdh3p, a known RNA-binding protein, interacted with the FAS1 mRNA, and it was necessary for the periodic abundance of Fas1p in the cell cycle. These results point to unexpected connections between major metabolic pathways. They also underscore the role of mRNA-protein interactions for gene expression during cell division.

Branched-chain amino acid synthesis is coupled to TOR activation early in the cell cycle in yeast.

How cells coordinate their metabolism with division determines the rate of cell proliferation. Dynamic patterns of metabolite synthesis during the cell cycle are unexplored. We report the first isotope tracing analysis in synchronous, growing budding yeast cells. Synthesis of leucine, a branched-chain amino acid (BCAA), increases through the G1 phase of the cell cycle, peaking later during DNA replication. Cells lacking Bat1, a mitochondrial aminotransferase that synthesizes BCAAs, grow slower, are smaller, and are delayed in the G1 phase, phenocopying cells in which the growth-promoting kinase complex TORC1 is moderately inhibited. Loss of Bat1 lowers the levels of BCAAs and reduces TORC1 activity. Exogenous provision of valine and, to a lesser extent, leucine to cells lacking Bat1 promotes cell division. Valine addition also increases TORC1 activity. In wild-type cells, TORC1 activity is dynamic in the cell cycle, starting low in early G1 but increasing later in the cell cycle. These results suggest a link between BCAA synthesis from glucose to TORC1 activation in the G1 phase of the cell cycle.

Translational control of one-carbon metabolism underpins ribosomal protein phenotypes in cell division and longevity.

A long-standing problem is how cells that lack one of the highly similar ribosomal proteins (RPs) often display distinct phenotypes. Yeast and other organisms live longer when they lack specific ribosomal proteins, especially of the large 60S subunit of the ribosome. However, longevity is neither associated with the generation time of RP deletion mutants nor with bulk inhibition of protein synthesis. Here, we queried actively dividing RP mutants through the cell cycle. Our data link transcriptional, translational, and metabolic changes to phenotypes associated with the loss of paralogous RPs. We uncovered translational control of transcripts encoding enzymes of methionine and serine metabolism, which are part of one-carbon (1C) pathways. Cells lacking Rpl22Ap, which are long-lived, have lower levels of metabolites associated with 1C metabolism. Loss of 1C enzymes increased the longevity of wild type cells. 1C pathways exist in all organisms and targeting the relevant enzymes could represent longevity interventions.

Abundances of transcripts, proteins, and metabolites in the cell cycle of budding yeast reveal coordinate control of lipid metabolism.

Establishing the pattern of abundance of molecules of interest during cell division has been a long-standing goal of cell cycle studies. Here, for the first time in any system, we present experiment-matched datasets of the levels of RNAs, proteins, metabolites, and lipids from unarrested, growing, and synchronously dividing yeast cells. Overall, transcript and protein levels were correlated, but specific processes that appeared to change at the RNA level (e.g., ribosome biogenesis) did not do so at the protein level, and vice versa. We also found no significant changes in codon usage or the ribosome content during the cell cycle. We describe an unexpected mitotic peak in the abundance of ergosterol and thiamine biosynthesis enzymes. Although the levels of several metabolites changed in the cell cycle, by far the most significant changes were in the lipid repertoire, with phospholipids and triglycerides peaking strongly late in the cell cycle. Our findings provide an integrated view of the abundance of biomolecules in the eukaryotic cell cycle and point to a coordinate mitotic control of lipid metabolism.

Perturbations of Transcription and Gene Expression-Associated Processes Alter Distribution of Cell Size Values in Saccharomyces cerevisiae.

The question of what determines whether cells are big or small has been the focus of many studies because it is thought that such determinants underpin the coupling of cell growth with cell division. In contrast, what determines the overall pattern of how cell size is distributed within a population of wild type or mutant cells has received little attention. Knowing how cell size varies around a characteristic pattern could shed light on the processes that generate such a pattern and provide a criterion to identify its genetic basis. Here, we show that cell size values of wild type Saccharomyces cerevisiae cells fit a gamma distribution, in haploid and diploid cells, and under different growth conditions. To identify genes that influence this pattern, we analyzed the cell size distributions of all single-gene deletion strains in Saccharomyces cerevisiae We found that yeast strains which deviate the most from the gamma distribution are enriched for those lacking gene products functioning in gene expression, especially those in transcription or transcription-linked processes. We also show that cell size is increased in mutants carrying altered activity substitutions in Rpo21p/Rpb1, the largest subunit of RNA polymerase II (Pol II). Lastly, the size distribution of cells carrying extreme altered activity Pol II substitutions deviated from the expected gamma distribution. Our results are consistent with the idea that genetic defects in widely acting transcription factors or Pol II itself compromise both cell size homeostasis and how the size of individual cells is distributed in a population.

Scaling of G1 Duration with Population Doubling Time by a Cyclin in Saccharomyces cerevisiae.

The longer cells stay in particular phases of the cell cycle, the longer it will take these cell populations to increase. However, the above qualitative description has very little predictive value, unless it can be codified mathematically. A quantitative relation that defines the population doubling time (Td) as a function of the time eukaryotic cells spend in specific cell cycle phases would be instrumental for estimating rates of cell proliferation and for evaluating introduced perturbations. Here, we show that in human cells, the length of the G1 phase (TG1) regressed on Td with a slope of ≈0.75, while in the yeast Saccharomyces cerevisiae, the slope was slightly smaller, at ≈0.60. On the other hand, cell size was not strongly associated with Td or TG1 in cell cultures that were proliferating at different rates. Furthermore, we show that levels of the yeast G1 cyclin Cln3p were positively associated with rates of cell proliferation over a broad range, at least in part through translational control mediated by a short upstream ORF (uORF) in the CLN3 transcript. Cln3p was also necessary for the proper scaling between TG1 and Td In contrast, yeast lacking the Whi5p transcriptional repressor maintained the scaling between TG1 and Td These data reveal fundamental scaling relationships between the duration of eukaryotic cell cycle phases and rates of cell proliferation, point to the necessary role of Cln3p in these relationships in yeast, and provide a mechanistic basis linking Cln3p levels to proliferation rates and the scaling of G1 with doubling time.

Translational control of lipogenic enzymes in the cell cycle of synchronous, growing yeast cells.

Translational control during cell division determines when cells start a new cell cycle, how fast they complete it, the number of successive divisions, and how cells coordinate proliferation with available nutrients. The translational efficiencies of mRNAs in cells progressing synchronously through the mitotic cell cycle, while preserving the coupling of cell division with cell growth, remain uninvestigated. We now report comprehensive ribosome profiling of a yeast cell size series from the time of cell birth, to identify mRNAs under periodic translational control. The data reveal coordinate translational activation of mRNAs encoding lipogenic enzymes late in the cell cycle including Acc1p, the rate-limiting enzyme acetyl-CoA carboxylase. An upstream open reading frame (uORF) confers the translational control of ACC1 and adjusts Acc1p protein levels in different nutrients. The ACC1 uORF is relevant for cell division because its ablation delays cell cycle progression, reduces cell size, and suppresses the replicative longevity of cells lacking the Sch9p protein kinase regulator of ribosome biogenesis. These findings establish an unexpected relationship between lipogenesis and protein synthesis in mitotic cell divisions.

Mitotic entry in the presence of DNA damage is a widespread property of aneuploidy in yeast.

Genetic instability is a hallmark of aneuploidy in budding and fission yeast. All aneuploid yeast strains analyzed to date harbor elevated levels of Rad52-GFP foci, a sign of DNA damage. Here we investigate how continuously elevated levels of DNA damage affect aneuploid cells. We show that Rad52-GFP foci form during S phase, consistent with the observation that DNA replication initiation and elongation are impaired in some aneuploid yeast strains. We furthermore find that although DNA damage is low in aneuploid cells, it nevertheless has dramatic consequences. Many aneuploid yeast strains adapt to DNA damage and undergo mitosis despite the presence of unrepaired DNA leading to cell death. Wild-type cells exposed to low levels of DNA damage exhibit a similar phenotype, indicating that adaptation to low levels of unrepaired DNA is a general property of the cell's response to DNA damage. Our results indicate that by causing low levels of DNA damage, whole-chromosome aneuploidies lead to DNA breaks that persist into mitosis. Such breaks provide the substrate for translocations and deletions that are a hallmark of cancer.

Aneuploidy drives genomic instability in yeast.

Aneuploidy decreases cellular fitness, yet it is also associated with cancer, a disease of enhanced proliferative capacity. To investigate one mechanism by which aneuploidy could contribute to tumorigenesis, we examined the effects of aneuploidy on genomic stability. We analyzed 13 budding yeast strains that carry extra copies of single chromosomes and found that all aneuploid strains exhibited one or more forms of genomic instability. Most strains displayed increased chromosome loss and mitotic recombination, as well as defective DNA damage repair. Aneuploid fission yeast strains also exhibited defects in mitotic recombination. Aneuploidy-induced genomic instability could facilitate the development of genetic alterations that drive malignant growth in cancer.

The unfolded protein response is not necessary for the G1/S transition, but it is required for chromosome maintenance in Saccharomyces cerevisiae.

BACKGROUND: The unfolded protein response (UPR) is a eukaryotic signaling pathway, from the endoplasmic reticulum (ER) to the nucleus. Protein misfolding in the ER triggers the UPR. Accumulating evidence links the UPR in diverse aspects of cellular homeostasis. The UPR responds to the overall protein synthesis capacity and metabolic fluxes of the cell. Because the coupling of metabolism with cell division governs when cells start dividing, here we examined the role of UPR signaling in the timing of initiation of cell division and cell cycle progression, in the yeast Saccharomyces cerevisiae. METHODOLOGY/PRINCIPAL FINDINGS: We report that cells lacking the ER-resident stress sensor Ire1p, which cannot trigger the UPR, nonetheless completed the G1/S transition on time. Furthermore, loss of UPR signaling neither affected the nutrient and growth rate dependence of the G1/S transition, nor the metabolic oscillations that yeast cells display in defined steady-state conditions. Remarkably, however, loss of UPR signaling led to hypersensitivity to genotoxic stress and a ten-fold increase in chromosome loss. CONCLUSIONS/SIGNIFICANCE: Taken together, our results strongly suggest that UPR signaling is not necessary for the normal coupling of metabolism with cell division, but it has a role in genome maintenance. These results add to previous work that linked the UPR with cytokinesis in yeast. UPR signaling is conserved in all eukaryotes, and it malfunctions in a variety of diseases, including cancer. Therefore, our findings may be relevant to other systems, including humans.

Sulfur metabolism actively promotes initiation of cell division in yeast.

BACKGROUND: Sulfur metabolism is required for initiation of cell division, but whether or not it can actively promote cell division remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that yeast cells with more mtDNA have an expanded reductive phase of their metabolic cycle and an increased sulfur metabolic flux. We also show that in wild type cells manipulations of sulfur metabolic flux phenocopy the enhanced growth rate of cells with more mtDNA. Furthermore, introduction of a hyperactive cystathionine-beta-synthase (CBS) allele in wild type cells accelerates initiation of DNA replication. CONCLUSIONS/SIGNIFICANCE: Our results reveal a novel connection between a key sulfur metabolic enzyme, CBS, and the cell cycle. Since the analogous hyperactive CBS allele in human CBS suppresses other disease-causing CBS mutations, our findings may be relevant for human pathology. Taken together, our results demonstrate the importance of sulfur metabolism in actively promoting initiation of cell division.

An increase in mitochondrial DNA promotes nuclear DNA replication in yeast.

Coordination between cellular metabolism and DNA replication determines when cells initiate division. It has been assumed that metabolism only plays a permissive role in cell division. While blocking metabolism arrests cell division, it is not known whether an up-regulation of metabolic reactions accelerates cell cycle transitions. Here, we show that increasing the amount of mitochondrial DNA accelerates overall cell proliferation and promotes nuclear DNA replication, in a nutrient-dependent manner. The Sir2p NAD+-dependent de-acetylase antagonizes this mitochondrial role. We found that cells with increased mitochondrial DNA have reduced Sir2p levels bound at origins of DNA replication in the nucleus, accompanied with increased levels of K9, K14-acetylated histone H3 at those origins. Our results demonstrate an active role of mitochondrial processes in the control of cell division. They also suggest that cellular metabolism may impact on chromatin modifications to regulate the activity of origins of DNA replication.

The Dcr2p phosphatase destabilizes Sic1p in Saccharomyces cerevisiae.

Initiation of cell division is controlled by an irreversible switch. In Saccharomyces cerevisiae degradation of the Sic1p protein, an inhibitor of mitotic cyclin/cyclin-dependent kinase complexes, takes place before initiation of DNA replication, at a point called START. Sic1p is phosphorylated by multiple kinases, which can differentially affect the stability of Sic1p. How phosphorylations that stabilize Sic1p are reversed is unknown. Here we show that the Dcr2p phosphatase functionally and physically interacts with Sic1p. Over-expression of Dcr2p destabilizes Sic1p and leads to phenotypes associated with destabilized Sic1p, such as genome instability. Our results identify a novel factor that affects the stability of Sic1p, possibly contributing to mechanisms that trigger initiation of cell division.

CDK control of membrane-bound organelle homeostasis.

In eukaryotes, the copy number and size of any given organelle compartment remain constant in dividing cells, underlying a tight coordination between cell division and organelle homeostasis. However, in most cases the mechanisms for this coordination remain mysterious. Here we outline a few cases where the cell cycle machinery directly impacts on organelle homeostasis, with emphasis on the control of vacuolar (lysosomal) copy number and size in budding yeast. We also discuss aspects of organelle biology that can profoundly affect certain cell cycle parameters, such as cell size.

Bem1p, a scaffold signaling protein, mediates cyclin-dependent control of vacuolar homeostasis in Saccharomyces cerevisiae.

How proliferating cells maintain the copy number and overall size of their organelles is not clear. We had previously reported that in the budding yeast Saccharomyces cerevisiae the G1 cyclin Cln3p is required for vacuolar (lysosomal) homotypic fusion and loss of Cln3p leads to vacuolar fragmentation. The Cdc42p GTPase is also required for vacuole fusion. Here we show that the scaffold protein Bem1p, a critical regulator of Cdc42p activity, is a downstream effector of Cln3p and the cyclin-dependent kinase (Cdk) Cdc28p. Our results suggest that Bem1p is phosphorylated in a Cdk-dependent manner to promote vacuole fusion. Replacing Ser72 with Asp, to mimic phosphorylation at an optimal Cdk-consensus site located in the first SH3 domain of Bem1p, suppressed vacuolar fragmentation in cells lacking Cln3p. Using in vivo and in vitro assays, we found that Cln3p was unable to promote vacuole fusion in the absence of Bem1p or in the presence of a nonphosphorylatable Bem1p-Ser72Ala mutant. Furthermore, activation of Cdc42p also suppressed vacuolar fragmentation in the absence of Cln3p. Our results provide a mechanism that links cyclin-dependent kinase activity with vacuole fusion through Bem1p and the Cdc42p GTPase cycle.