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.

One-carbon metabolic enzymes are regulated during cell division and make distinct contributions to the metabolome and cell cycle progression in Saccharomyces cerevisiae.

Enzymes of one-carbon (1C) metabolism play pivotal roles in proliferating cells. They are involved in the metabolism of amino acids, nucleotides, and lipids and the supply of all cellular methylations. However, there is limited information about how these enzymes are regulated during cell division and how cell cycle kinetics are affected in several loss-of-function mutants of 1C metabolism. Here, we report that the levels of the S. cerevisiae enzymes Ade17p and Cho2p, involved in the de novo synthesis of purines and phosphatidylcholine (PC), respectively, are cell cycle-regulated. Cells lacking Ade17p, Cho2p, or Shm2p (an enzyme that supplies 1C units from serine) have distinct alterations in size homeostasis and cell cycle kinetics. Loss of Ade17p leads to a specific delay at START, when cells commit to a new round of cell division, while loss of Shm2p has broader effects, reducing growth rate. Furthermore, the inability to synthesize PC de novo in cho2Δ cells delays START and reduces the coherence of nuclear elongation late in the cell cycle. Loss of Cho2p also leads to profound metabolite changes. Besides the expected changes in the lipidome, cho2Δ cells have reduced levels of amino acids, resembling cells shifted to poorer media. These results reveal the different ways that 1C metabolism allocates resources to affect cell proliferation at multiple cell cycle transitions.

mRNA-binding proteins and cell cycle progression.

Proteins that bind to each mRNA may affect the latter's abundance and location in the cell and how well ribosomes will translate that mRNA into a protein. Hence, mRNA-binding proteins (mRBPs) represent obvious control points in gene expression. Surprisingly, little is known about mRBPs and cell-cycle progression.

Undergraduate GPA Predicts Biochemistry PhD Completion and Is Associated with Time to Degree.

There is interest in admission criteria that predict future success in biomedical graduate school programs, but identifying predictors of PhD attainment is inherently complex. In particular, high noncompletion rates of PhD programs have long been recognized as a major crisis. Here, we present a quantitative analysis of the PhD students enrolled in the Department of Biochemistry and Biophysics at Texas A&M University between 1980 and 2010. The input variables included sex, country of citizenship, undergraduate grade point average (GPA), and Graduate Record Examination (GRE) scores (Verbal and Quantitative Reasoning). Only GPA was a significant predictor of PhD completion based on logistic regression. We also examined associations involving nonbinary measures of success (PhD duration, first author, and total number of publications) among students who completed a PhD. GPA was again associated with the PhD duration. No enrollment variable was strongly associated with publication output. Despite potential limitations, this analysis is the first to suggest an undergraduate GPA association with PhD completion in life sciences. These results from a large state university in a predominantly rural area expand the range of programs from which such analyses have been reported.

Translational control of lipogenesis links protein synthesis and phosphoinositide signaling with nuclear division in Saccharomyces cerevisiae.

Continuously dividing cells coordinate their growth and division. How fast cells grow in mass determines how fast they will multiply. Yet, there are few, if any, examples of a metabolic pathway that actively drives a cell cycle event instead of just being required for it. Here, we show that translational upregulation of lipogenic enzymes in Saccharomyces cerevisiae increased the abundance of lipids and promoted nuclear elongation and division. Derepressing translation of acetyl-CoA carboxylase and fatty acid synthase also suppressed cell cycle-related phenotypes, including delayed nuclear division, associated with Sec14p phosphatidylinositol transfer protein deficiencies, and the irregular nuclear morphologies of mutants defective in phosphatidylinositol 4-OH kinase activities. Our results show that increased lipogenesis drives a critical cell cycle landmark and report a phosphoinositide signaling axis in control of nuclear division. The broad conservation of these lipid metabolic and signaling pathways raises the possibility these activities similarly govern nuclear division in other eukaryotes.

Life span extension by glucose restriction is abrogated by methionine supplementation: Cross-talk between glucose and methionine and implication of methionine as a key regulator of life span.

Caloric restriction (CR) is known to extend life span across species; however, the molecular mechanisms are not well understood. We investigate the mechanism by which glucose restriction (GR) extends yeast replicative life span, by combining ribosome profiling and RNA-seq with microfluidic-based single-cell analysis. We discovered a cross-talk between glucose sensing and the regulation of intracellular methionine: GR down-regulated the transcription and translation of methionine biosynthetic enzymes and transporters, leading to a decreased intracellular methionine concentration; external supplementation of methionine cancels the life span extension by GR. Furthermore, genetic perturbations that decrease methionine synthesis/uptake extend life span. These observations suggest that intracellular methionine mediates the life span effects of various nutrient and genetic perturbations, and that the glucose-methionine cross-talk is a general mechanism for coordinating the nutrient status and the translation/growth of a cell. Our work also implicates proteasome as a downstream effector of the life span extension by GR.

Ribosomal proteins: mutant phenotypes by the numbers and associated gene expression changes.

Ribosomal proteins are highly conserved, many universally so among organisms. All ribosomal proteins are structural parts of the same molecular machine, the ribosome. However, when ribosomal proteins are mutated individually, they often lead to distinct and intriguing phenotypes, including specific human pathologies. This review is an attempt to collect and analyse all the reported phenotypes of each ribosomal protein mutant in several eukaryotes (Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Mus musculus, Homo sapiens). These phenotypes were processed with unbiased computational approaches to reveal associations between different phenotypes and the contributions of individual ribosomal protein genes. An overview of gene expression changes in ribosomal protein mutants, with emphasis on ribosome profiling studies, is also presented. The available data point to patterns that may account for most of the observed phenotypes. The information presented here may also inform future studies about the molecular basis of the phenotypes that arise from mutations in ribosomal proteins.

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.

Phenotypic Associations Among Cell Cycle Genes in Saccharomyces cerevisiae.

A long-standing effort in biology is to precisely define and group phenotypes that characterize a biological process, and the genes that underpin them. In Saccharomyces cerevisiae and other organisms, functional screens have generated rich lists of phenotypes associated with individual genes. However, it is often challenging to identify sets of phenotypes and genes that are most closely associated with a given biological process. Here, we focused on the 166 phenotypes arising from loss-of-function and the 86 phenotypes from gain-of-function mutations in 571 genes currently assigned to cell cycle-related ontologies in S. cerevisiae To reduce this complexity, we applied unbiased, computational approaches of correspondence analysis to identify a minimum set of phenotypic variables that accounts for as much of the variability in the data as possible. Loss-of-function phenotypes can be reduced to 20 dimensions, while gain-of-function ones to 14 dimensions. We also pinpoint the contributions of phenotypes and genes in each set. The approach we describe not only simplifies the categorization of phenotypes associated with cell cycle progression but might also potentially serve as a discovery tool for gene function.

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.

A Lipid Transfer Protein Signaling Axis Exerts Dual Control of Cell-Cycle and Membrane Trafficking Systems.

Kes1/Osh4 is a member of the conserved, but functionally enigmatic, oxysterol binding protein-related protein (ORP) superfamily that inhibits phosphatidylinositol transfer protein (Sec14)-dependent membrane trafficking through the trans-Golgi (TGN)/endosomal network. We now report that Kes1, and select other ORPs, execute cell-cycle control activities as functionally non-redundant inhibitors of the G1/S transition when cells confront nutrient-poor environments and promote replicative aging. Kes1-dependent cell-cycle regulation requires the Greatwall/MASTL kinase ortholog Rim15, and is opposed by Sec14 activity in a mechanism independent of Kes1/Sec14 bulk membrane-trafficking functions. Moreover, the data identify Kes1 as a non-histone target for NuA4 through which this lysine acetyltransferase co-modulates membrane-trafficking and cell-cycle activities. We propose the Sec14/Kes1 lipid-exchange protein pair constitutes part of the mechanism for integrating TGN/endosomal lipid signaling with cell-cycle progression and hypothesize that ORPs define a family of stage-specific cell-cycle control factors that execute tumor-suppressor-like functions.

Proteins associated with the doubling time of the NCI-60 cancer cell lines.

The varied nature of human cancers is recapitulated, at least to some extent, in the diverse NCI-60 panel of human cancer cell lines. Here, I used a basic, continuous variable of proliferating cells, their doubling time, to stratify the proteome across the NCI-60 cell lines. Among >7000 proteins quantified in the NCI-60 panel previously, the levels of 84 proteins increase in cells that proliferate slowly. This set overlapped with the hallmark molecular signature "epithelial-mesenchymal transition (EMT)" (p value = 1.1E-07). Conversely, the levels of 105 proteins increased in cells that proliferate faster and overlapped with the molecular signatures for "MYC targets V1" (p value = 3.8E-38) and "E2F targets" (p value = 2.4E-34). These data for the first time identify proteins whose levels are dynamically associated with doubling time, but not necessarily with cancer type origins, and argue for the incorporation of doubling time measurements in cell line-based profiling studies.

Unbalanced Growth, Senescence and Aging.

Usually, cells balance their growth with their division. Coordinating growth inputs with cell division ensures the proper timing of division when sufficient cell material is available and affects the overall rate of cell proliferation. At a very fundamental level, cellular replicative lifespan-defined as the number of times a cell can divide, is a manifestation of cell cycle control. Hence, control of mitotic cell divisions, especially when the commitment is made to a new round of cell division, is intimately linked to replicative aging of cells. In this chapter, we review our current understanding, and its shortcomings, of how unbalanced growth and division, can dramatically influence the proliferative potential of cells, often leading to cellular and organismal aging phenotypes. The interplay between growth and division also underpins cellular senescence (i.e., inability to divide) and quiescence, when cells exit the cell cycle but still retain their ability to divide.