RESUMO
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.
Assuntos
Proteínas de Transporte , Ribossomos , Proteínas de Transporte/genética , Divisão Celular , Fases de Leitura Aberta , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismoRESUMO
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.
Assuntos
Aminoácidos , Saccharomyces cerevisiae , Ciclo Celular , Aminoácidos/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Leucina/biossíntese , Glucose/metabolismo , Fase G1RESUMO
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.
Assuntos
Acetil-CoA Carboxilase/biossíntese , Regulação Fúngica da Expressão Gênica , Mitose , Biossíntese de Proteínas , Leveduras/crescimento & desenvolvimento , Leveduras/genética , Acetil-CoA Carboxilase/genética , Metabolismo dos Lipídeos , Fases de Leitura Aberta , Ribossomos/metabolismo , Leveduras/metabolismoRESUMO
Amino acid starvation activates the protein kinase Gcn2p, leading to changes in gene expression and translation. Gcn2p is activated by deacylated tRNA, which accumulates when tRNA aminoacylation is limited by lack of substrates or inhibition of synthesis. Pairing of amino acids and deacylated tRNAs is catalyzed by aminoacyl-tRNA synthetases, which use quality control pathways to maintain substrate specificity. Phenylalanyl-tRNA synthetase (PheRS) maintains specificity via an editing pathway that targets non-cognate Tyr-tRNAPhe. While the primary role of aaRS editing is to prevent misaminoacylation, we demonstrate editing of misaminoacylated tRNA is also required for detection of amino acid starvation by Gcn2p. Ablation of PheRS editing caused accumulation of Tyr-tRNAPhe (5%), but not deacylated tRNAPhe during amino acid starvation, limiting Gcn2p kinase activity and suppressing Gcn4p-dependent gene expression. While the PheRS-editing ablated strain grew 50% slower and displayed a 27-fold increase in the rate of mistranslation of Phe codons as Tyr compared to wild type, the increase in mistranslation was insufficient to activate an unfolded protein stress response. These findings show that during amino acid starvation a primary role of aaRS quality control is to help the cell mount an effective stress response, independent of the role of editing in maintaining translational accuracy.
Assuntos
Fenilalanina-tRNA Ligase/metabolismo , Edição de RNA , RNA de Transferência de Fenilalanina/metabolismo , Saccharomyces cerevisiae/metabolismo , Aminoacilação de RNA de Transferência , Resposta a Proteínas não Dobradas , Aminoácidos/metabolismo , Fenilalanina/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Estresse Fisiológico , Tirosina/metabolismoRESUMO
Understanding the causes and consequences of dynamic changes in the abundance and activity of cellular components during cell division is what most cell cycle studies are about. Here we focus on control of gene expression in the cell cycle at the level of translation. The advent of deep sequencing methodologies led to technologies that quantify the levels of all mRNAs that are bound by ribosomes and engaged in translation in the cell (Ingolia et al. Science 324:218-223, 2009). This approach has been applied recently to synchronous cell populations to find transcripts under translational control at different cell cycle phases (Blank et al. EMBO J 36:487-502, 2017; Stumpf et al. Mol Cell 52:574-582, 2013; Tanenbaum et al. Elife 4:e07957, 2015). These studies revealed new biology, but they also have limitations, pointing to challenges that need to be addressed in the future.
Assuntos
Pontos de Checagem do Ciclo Celular/genética , Ciclinas/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Ribossomos/genética , Divisão Celular , Ciclinas/metabolismo , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismoRESUMO
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.
Assuntos
Envelhecimento/patologia , Proliferação de Células , Senescência Celular , Mitose , Fatores Etários , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Genótipo , Humanos , Fenótipo , Transdução de SinaisRESUMO
The common non-steroidal anti-inflammatory drug ibuprofen has been associated with a reduced risk of some age-related pathologies. However, a general pro-longevity role for ibuprofen and its mechanistic basis remains unclear. Here we show that ibuprofen increased the lifespan of Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster, indicative of conserved eukaryotic longevity effects. Studies in yeast indicate that ibuprofen destabilizes the Tat2p permease and inhibits tryptophan uptake. Loss of Tat2p increased replicative lifespan (RLS), but ibuprofen did not increase RLS when Tat2p was stabilized or in an already long-lived strain background impaired for aromatic amino acid uptake. Concomitant with lifespan extension, ibuprofen moderately reduced cell size at birth, leading to a delay in the G1 phase of the cell cycle. Similar changes in cell cycle progression were evident in a large dataset of replicatively long-lived yeast deletion strains. These results point to fundamental cell cycle signatures linked with longevity, implicate aromatic amino acid import in aging and identify a largely safe drug that extends lifespan across different kingdoms of life.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Ibuprofeno/farmacologia , Longevidade/efeitos dos fármacos , Sistemas de Transporte de Aminoácidos/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/fisiologia , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/fisiologia , Avaliação Pré-Clínica de Medicamentos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Estabilidade Proteica , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Triptofano/metabolismoRESUMO
Upstream events that trigger initiation of cell division, at a point called START in yeast, determine the overall rates of cell proliferation. The identity and complete sequence of those events remain unknown. Previous studies relied mainly on cell size changes to identify systematically genes required for the timely completion of START. Here, we evaluated panels of non-essential single gene deletion strains for altered DNA content by flow cytometry. This analysis revealed that most gene deletions that altered cell cycle progression did not change cell size. Our results highlight a strong requirement for ribosomal biogenesis and protein synthesis for initiation of cell division. We also identified numerous factors that have not been previously implicated in cell cycle control mechanisms. We found that CBS, which catalyzes the synthesis of cystathionine from serine and homocysteine, advances START in two ways: by promoting cell growth, which requires CBS's catalytic activity, and by a separate function, which does not require CBS's catalytic activity. CBS defects cause disease in humans, and in animals CBS has vital, non-catalytic, unknown roles. Hence, our results may be relevant for human biology. Taken together, these findings significantly expand the range of factors required for the timely initiation of cell division. The systematic identification of non-essential regulators of cell division we describe will be a valuable resource for analysis of cell cycle progression in yeast and other organisms.
Assuntos
Divisão Celular/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Ribossomos , Saccharomyces cerevisiae , Proliferação de Células , Tamanho Celular , DNA/análise , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Homozigoto , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Proteins are the principal macromolecular constituent of proliferating cells, and protein synthesis is viewed as a primary metric of cell growth. While there are celebrated examples of proteins whose levels are periodic in the cell cycle (e.g., cyclins), the concentration of most proteins was not thought to change in the cell cycle, but some recent results challenge this notion. The 'bulk' protein is the focus of this article, specifically the rate of its synthesis, in the budding yeast Saccharomyces cerevisiae.
RESUMO
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 2 poles of the mitotic spindle that segregates the chromosomes in mitosis. The conserved Mps1p kinase governs the duplication of the spindle pole body (SPB) in Saccharomyces cerevisiae. Here, we show that the MPS1 transcript has a short upstream open reading frame (uORF) 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 SPB in the cell cycle using structured illumination microscopy revealed that mutating the MPS1 uORF enabled cells to duplicate their SPB 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.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Corpos Polares do Fuso , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Corpos Polares do Fuso/metabolismo , Corpos Polares do Fuso/genética , Ciclinas/metabolismo , Ciclinas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fases de Leitura Aberta , Biossíntese de Proteínas , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Regulação Fúngica da Expressão GênicaRESUMO
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.
Assuntos
Ácido Fólico , Longevidade , Animais , Ácido Fólico/administração & dosagem , Ácido Fólico/metabolismo , Camundongos , Masculino , Feminino , Envelhecimento/metabolismo , Dieta/métodos , Camundongos Endogâmicos C57BL , Metotrexato/farmacologia , Deficiência de Ácido Fólico/metabolismo , Caenorhabditis elegans , Saccharomyces cerevisiae/metabolismoRESUMO
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.
Assuntos
Técnicas de Cultura de Células/instrumentação , Senescência Celular , Técnicas Analíticas Microfluídicas/instrumentação , Saccharomyces cerevisiae/citologia , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Automação , Biologia Celular/instrumentação , Miniaturização , Modelos BiológicosRESUMO
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.
Assuntos
Carbono , Saccharomyces cerevisiae , Carbono/metabolismo , Divisão Celular , Ciclo Celular/genética , Metaboloma , Aminoácidos/metabolismoRESUMO
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.
Assuntos
Ácido Graxo Sintases , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , RNA Mensageiro/genética , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Ciclo Celular , Divisão CelularRESUMO
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.
Assuntos
Saccharomyces cerevisiaeRESUMO
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.