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1.
Nat Commun ; 11(1): 5723, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-33184262

RESUMO

The identification of cell borders ('segmentation') in microscopy images constitutes a bottleneck for large-scale experiments. For the model organism Saccharomyces cerevisiae, current segmentation methods face challenges when cells bud, crowd, or exhibit irregular features. We present a convolutional neural network (CNN) named YeaZ, the underlying training set of high-quality segmented yeast images (>10 000 cells) including mutants, stressed cells, and time courses, as well as a graphical user interface and a web application ( www.quantsysbio.com/data-and-software ) to efficiently employ, test, and expand the system. A key feature is a cell-cell boundary test which avoids the need for fluorescent markers. Our CNN is highly accurate, including for buds, and outperforms existing methods on benchmark images, indicating it transfers well to other conditions. To demonstrate how efficient large-scale image processing uncovers new biology, we analyze the geometries of ≈2200 wild-type and cyclin mutant cells and find that morphogenesis control occurs unexpectedly early and gradually.


Assuntos
Microscopia/métodos , Redes Neurais de Computação , Saccharomyces cerevisiae/citologia , Ciclo Celular , Processamento de Imagem Assistida por Computador/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Software
2.
Nat Commun ; 11(1): 5052, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33028817

RESUMO

The mechanism and function of autophagy as a highly-conserved bulk degradation pathway are well studied, but the physiological role of autophagy remains poorly understood. We show that autophagy is involved in the adaptation of Saccharomyces cerevisiae to respiratory growth through its recycling of serine. On respiratory media, growth onset, mitochondrial initiator tRNA modification and mitochondrial protein expression are delayed in autophagy defective cells, suggesting that mitochondrial one-carbon metabolism is perturbed in these cells. The supplementation of serine, which is a key one-carbon metabolite, is able to restore mitochondrial protein expression and alleviate delayed respiratory growth. These results indicate that autophagy-derived serine feeds into mitochondrial one-carbon metabolism, supporting the initiation of mitochondrial protein synthesis and allowing rapid adaptation to respiratory growth.


Assuntos
Adaptação Fisiológica , Autofagia/fisiologia , Proteínas Mitocondriais/biossíntese , Saccharomyces cerevisiae/fisiologia , Carbono/metabolismo , Respiração Celular/fisiologia , Mitocôndrias/metabolismo , Biossíntese de Proteínas/fisiologia , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/metabolismo
3.
Proc Natl Acad Sci U S A ; 117(45): 28191-28200, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33106417

RESUMO

Genomic alterations including single-base mutations, deletions and duplications, translocations, mitotic recombination events, and chromosome aneuploidy generate genetic diversity. We examined the rates of all of these genetic changes in a diploid strain of Saccharomyces cerevisiae by whole-genome sequencing of many independent isolates (n = 93) subcloned about 100 times in unstressed growth conditions. The most common alterations were point mutations and small (<100 bp) insertion/deletions (n = 1,337) and mitotic recombination events (n = 1,215). The diploid cells of most eukaryotes are heterozygous for many single-nucleotide polymorphisms (SNPs). During mitotic cell divisions, recombination can produce derivatives of these cells that have become homozygous for the polymorphisms, termed loss-of-heterozygosity (LOH) events. LOH events can change the phenotype of the cells and contribute to tumor formation in humans. We observed two types of LOH events: interstitial events (conversions) resulting in a short LOH tract (usually less than 15 kb) and terminal events (mostly cross-overs) in which the LOH tract extends to the end of the chromosome. These two types of LOH events had different distributions, suggesting that they may have initiated by different mechanisms. Based on our results, we present a method of calculating the probability of an LOH event for individual SNPs located throughout the genome. We also identified several hotspots for chromosomal rearrangements (large deletions and duplications). Our results provide insights into the relative importance of different types of genetic alterations produced during vegetative growth.


Assuntos
Cromossomos Fúngicos/genética , Mutação/genética , Saccharomyces cerevisiae/genética , Mapeamento Cromossômico , Diploide , Conversão Gênica/genética , Rearranjo Gênico/genética , Perda de Heterozigosidade/genética , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Saccharomyces cerevisiae/citologia
4.
BMC Bioinformatics ; 21(1): 472, 2020 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-33087041

RESUMO

BACKGROUND: Optimality principles have been used to explain the structure and behavior of living matter at different levels of organization, from basic phenomena at the molecular level, up to complex dynamics in whole populations. Most of these studies have assumed a single-criteria approach. Such optimality principles have been justified from an evolutionary perspective. In the context of the cell, previous studies have shown how dynamics of gene expression in small metabolic models can be explained assuming that cells have developed optimal adaptation strategies. Most of these works have considered rather simplified representations, such as small linear pathways, or reduced networks with a single branching point, and a single objective for the optimality criteria. RESULTS: Here we consider the extension of this approach to more realistic scenarios, i.e. biochemical pathways of arbitrary size and structure. We first show that exploiting optimality principles for these networks poses great challenges due to the complexity of the associated optimal control problems. Second, in order to surmount such challenges, we present a computational framework which has been designed with scalability and efficiency in mind, including mechanisms to avoid the most common pitfalls. Third, we illustrate its performance with several case studies considering the central carbon metabolism of S. cerevisiae and B. subtilis. In particular, we consider metabolic dynamics during nutrient shift experiments. CONCLUSIONS: We show how multi-objective optimal control can be used to predict temporal profiles of enzyme activation and metabolite concentrations in complex metabolic pathways. Further, we also show how to consider general cost/benefit trade-offs. In this study we have considered metabolic pathways, but this computational framework can also be applied to analyze the dynamics of other complex pathways, such as signal transduction or gene regulatory networks.


Assuntos
Biologia Computacional/métodos , Redes e Vias Metabólicas , Redes Reguladoras de Genes , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Transcriptoma
5.
PLoS One ; 15(9): e0233197, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32946445

RESUMO

Levels of protein translation by ribosomes are governed both by features of the translation machinery as well as sequence properties of the mRNAs themselves. We focus here on a striking three-nucleotide periodicity, characterized by overrepresentation of GCN codons and underrepresentation of G at the second position of codons, that is observed in Open Reading Frames (ORFs) of mRNAs. Our examination of mRNA sequences in Saccharomyces cerevisiae revealed that this periodicity is particularly pronounced in the initial codons-the ramp region-of ORFs of genes with high protein expression. It is also found in mRNA sequences immediately following non-standard AUG start sites, located upstream or downstream of the standard annotated start sites of genes. To explore the possible influences of the ramp GCN periodicity on translation efficiency, we tested edited ramps with accentuated or depressed periodicity in two test genes, SKN7 and HMT1. Greater conformance to (GCN)n was found to significantly depress translation, whereas disrupting conformance had neutral or positive effects on translation. Our recent Molecular Dynamics analysis of a subsystem of translocating ribosomes in yeast revealed an interaction surface that H-bonds to the +1 codon that is about to enter the ribosome decoding center A site. The surface, comprised of 16S/18S rRNA C1054 and A1196 (E. coli numbering) and R146 of ribosomal protein Rps3, preferentially interacts with GCN codons, and we hypothesize that modulation of this mRNA-ribosome interaction may underlie GCN-mediated regulation of protein translation. Integration of our expression studies with large-scale reporter studies of ramp sequence variants suggests a model in which the C1054-A1196-R146 (CAR) interaction surface can act as both an accelerator and braking system for ribosome translation.


Assuntos
Códon de Iniciação/genética , Biossíntese de Proteínas/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Composição de Bases/genética , Códon de Iniciação/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Simulação de Dinâmica Molecular , Fases de Leitura Aberta/genética , Proteína-Arginina N-Metiltransferases/biossíntese , Proteína-Arginina N-Metiltransferases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
6.
Mol Cell ; 80(2): 311-326.e4, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32970994

RESUMO

To determine whether double-strand break (DSB) mobility enhances the physical search for an ectopic template during homology-directed repair (HDR), we tested the effects of factors that control chromatin dynamics, including cohesin loading and kinetochore anchoring. The former but not the latter is altered in response to DSBs. Loss of the nonhistone high-mobility group protein Nhp6 reduces histone occupancy and increases chromatin movement, decompaction, and ectopic HDR. The loss of nucleosome remodeler INO80-C did the opposite. To see whether enhanced HDR depends on DSB mobility or the global chromatin response, we tested the ubiquitin ligase mutant uls1Δ, which selectively impairs local but not global movement in response to a DSB. Strand invasion occurs in uls1Δ cells with wild-type kinetics, arguing that global histone depletion rather than DSB movement is rate limiting for HDR. Impaired break movement in uls1Δ correlates with elevated MRX and cohesin loading, despite normal resection and checkpoint activation.


Assuntos
Quebras de DNA de Cadeia Dupla , Nucleossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Bleomicina/farmacologia , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , DNA Fúngico/metabolismo , Histonas/metabolismo , Modelos Biológicos , Fosforilação , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Corpos Polares do Fuso/metabolismo
7.
Proc Natl Acad Sci U S A ; 117(38): 23539-23547, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32907940

RESUMO

RNA movements and localization pervade biology, from embryonic development to disease. To identify RNAs at specific locations, we developed a strategy in which a uridine-adding enzyme is anchored to subcellular sites, where it directly marks RNAs with 3' terminal uridines. This localized RNA recording approach yields a record of RNA locations, and is validated through identification of RNAs localized selectively to the endoplasmic reticulum (ER) or mitochondria. We identify a broad dual localization pattern conserved from yeast to human cells, in which the same battery of mRNAs encounter both ER and mitochondria in both species, and include an mRNA encoding a key stress sensor. Subunits of many multiprotein complexes localize to both the ER and mitochondria, suggesting coordinated assembly. Noncoding RNAs in the course of RNA surveillance and processing encounter both organelles. By providing a record of RNA locations over time, the approach complements those that capture snapshots of instantaneous positions.


Assuntos
RNA Fúngico , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Humanos , Mitocôndrias/química , Mitocôndrias/metabolismo , RNA Fúngico/química , RNA Fúngico/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Uridina
8.
PLoS One ; 15(8): e0237982, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32817700

RESUMO

Some organisms, like Trichomonas vaginalis, contain mitochondria-related hydrogen-producing organelles, called hydrogenosomes. The protein targeting into these organelles is proposed to be similar to the well-studied mitochondria import. Indeed, S. cerevisiae mitochondria and T. vaginalis hydrogenosomes share some components of protein import complexes. However, it is still unknown whether targeting signals directing substrate proteins to hydrogenosomes can support in other eukaryotes specific mitochondrial localization. To address this issue, we investigated the intracellular localization of three hydrogenosomal tail-anchored proteins expressed in yeast cells. We observed that these proteins were targeted to both mitochondria and ER with a variable dependency on the mitochondrial MIM complex. Our results suggest that the targeting signal of TA proteins are only partially conserved between hydrogenosomes and yeast mitochondria.


Assuntos
Retículo Endoplasmático/metabolismo , Regulação Fúngica da Expressão Gênica , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Trichomonas vaginalis/citologia , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trichomonas vaginalis/genética , Trichomonas vaginalis/metabolismo
9.
Biosci Rep ; 40(9)2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32794578

RESUMO

Apoptosis is a form of programmed cell death which is essential for the growth of dividing human cells whereas, in contrast, it is deleterious for post-mitotic cells such as neurons. Bax and α-synuclein are two human proteins which play a role in the induction of neuronal apoptosis in neurodegenerative diseases like Alzheimer's and Parkinson's. Human Bax and α-synuclein also induce cell death when expressed in baker's yeast, Saccharomyces cerevisiae. Quite unexpectedly, the human α-synuclein gene had been identified as an inhibitor of pro-apoptotic Bax using a yeast-based screen of a human hippocampal cDNA library. Plasmids were constructed with different promoters, which allow expression of wildtype and Parkinson's disease (PD)-related mutant α-synuclein genes, from (i) multi-copy 2µ (episomal) plasmids and (ii) integrative plasmids that compel expression of genes from chromosomal sites in varying copy numbers (1-3). All α-synuclein-containing plasmids were introduced, through transformation, into a yeast strain which already contained a chromosomally integrated copy of Bax. It is for the first time that it was observed that, depending on gene dosage, only wildtype α-synuclein is anti-apoptotic while mutant α-synuclein is not. The results also indicate that wildtype α-synuclein has a remarkable ability to manifest two contrasting effects depending on its level of expression: (i) normally, it would negate apoptosis but (ii) when overexpressed, it tends to induce apoptosis which is probably what happens in PD.


Assuntos
Apoptose/fisiologia , Saccharomyces cerevisiae/citologia , alfa-Sinucleína/genética , Proteína X Associada a bcl-2/metabolismo , Variações do Número de Cópias de DNA , Galactoquinase/genética , Dosagem de Genes , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Humanos , Potencial da Membrana Mitocondrial , Microrganismos Geneticamente Modificados , Mutação , Doença de Parkinson/genética , Plasmídeos , Regiões Promotoras Genéticas , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , alfa-Sinucleína/metabolismo , Proteína X Associada a bcl-2/genética
10.
Nature ; 584(7821): 470-474, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32669712

RESUMO

The rate of cell growth is crucial for bacterial fitness and drives the allocation of bacterial resources, affecting, for example, the expression levels of proteins dedicated to metabolism and biosynthesis1,2. It is unclear, however, what ultimately determines growth rates in different environmental conditions. Moreover, increasing evidence suggests that other objectives are also important3-7, such as the rate of physiological adaptation to changing environments8,9. A common challenge for cells is that these objectives cannot be independently optimized, and maximizing one often reduces another. Many such trade-offs have indeed been hypothesized on the basis of qualitative correlative studies8-11. Here we report a trade-off between steady-state growth rate and physiological adaptability in Escherichia coli, observed when a growing culture is abruptly shifted from a preferred carbon source such as glucose to fermentation products such as acetate. These metabolic transitions, common for enteric bacteria, are often accompanied by multi-hour lags before growth resumes. Metabolomic analysis reveals that long lags result from the depletion of key metabolites that follows the sudden reversal in the central carbon flux owing to the imposed nutrient shifts. A model of sequential flux limitation not only explains the observed trade-off between growth and adaptability, but also allows quantitative predictions regarding the universal occurrence of such tradeoffs, based on the opposing enzyme requirements of glycolysis versus gluconeogenesis. We validate these predictions experimentally for many different nutrient shifts in E. coli, as well as for other respiro-fermentative microorganisms, including Bacillus subtilis and Saccharomyces cerevisiae.


Assuntos
Adaptação Fisiológica , Meio Ambiente , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Acetatos/metabolismo , Bacillus subtilis/citologia , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Divisão Celular , Escherichia coli/enzimologia , Escherichia coli/genética , Fermentação , Gluconeogênese , Glucose/metabolismo , Glicólise , Metabolômica , Modelos Biológicos , Mutação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo
11.
J Biosci Bioeng ; 130(4): 367-373, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32646632

RESUMO

Cross hybridization breeding of sake yeasts is hampered by difficulty in acquisition of haploid cells through sporulation. We previously demonstrated that typical sake yeast strains were defective in meiotic chromosome recombination, which caused poor sporulation and loss of spore viability. In this study, we screened a single copy plasmid genomic DNA library of the laboratory Saccharomyces cerevisiae GRF88 for genes that might complement the meiotic recombination defect of UTCAH-3, a strain derived from the sake yeast Kyokai no. 7 (K7). We identified the SPO11 gene of the laboratory strain (ScSPO11), encoding a meiosis-specific endonuclease that catalyzes DNA double-strand breaks required for meiotic recombination, as a gene that restored meiotic recombination and spore viability of UTCAH-3. K7SPO11 could not restore sporulation efficiency and spore viability of UTCAH-3 and a laboratory strain BY4743 spo11Δ/spo11Δ, indicating that K7SPO11 is not functional. Sequence analysis of the SPO11 genes of various Kyokai sake yeasts (K1, and K3-K10) revealed that the K7 group of sake yeasts (K6, K7, K9, and K10) had a mutual missense mutation (C73T) in addition to other three common mutations present in all Kyokai yeasts tested. ScSPO11C73T created through in vitro mutagenesis could not restore spore viability of BY4743 spo11Δ/spo11Δ. On the other hand, K8SPO11, which have the three common mutations except for C73T could restore spore viability of BY4743 spo11Δ/spo11Δ. These results suggest that C73T might be a causative mutation of recombination defect in K7SPO11. Moreover, we found that the introduction of ScRIM15 restored sporulation efficiency but not spore viability.


Assuntos
Bebidas Alcoólicas/microbiologia , Endodesoxirribonucleases/genética , Meiose/genética , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Clonagem Molecular , Quebras de DNA de Cadeia Dupla , Mutação , Saccharomyces cerevisiae/citologia
12.
PLoS One ; 15(7): e0233755, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628677

RESUMO

Systems biology aims at holistically understanding the complexity of biological systems. In particular, nowadays with the broad availability of gene expression measurements, systems biology challenges the deciphering of the genetic cell machinery from them. In order to help researchers, reverse engineer the genetic cell machinery from these noisy datasets, interactive exploratory clustering methods, pipelines and gene clustering tools have to be specifically developed. Prior methods/tools for time series data, however, do not have the following four major ingredients in analytic and methodological view point: (i) principled time-series feature extraction methods, (ii) variety of manifold learning methods for capturing high-level view of the dataset, (iii) high-end automatic structure extraction, and (iv) friendliness to the biological user community. With a view to meet the requirements, we present AGCT (A Geometric Clustering Tool), a software package used to unravel the complex architecture of large-scale, non-necessarily synchronized time-series gene expression data. AGCT capture signals on exhaustive wavelet expansions of the data, which are then embedded on a low-dimensional non-linear map using manifold learning algorithms, where geometric proximity captures potential interactions. Post-processing techniques, including hard and soft information geometric clustering algorithms, facilitate the summarizing of the complete map as a smaller number of principal factors which can then be formally identified using embedded statistical inference techniques. Three-dimension interactive visualization and scenario recording over the processing helps to reproduce data analysis results without additional time. Analysis of the whole-cell Yeast Metabolic Cycle (YMC) moreover, Yeast Cell Cycle (YCC) datasets demonstrate AGCT's ability to accurately dissect all stages of metabolism and the cell cycle progression, independently of the time course and the number of patterns related to the signal. Analysis of Pentachlorophenol iduced dataset demonstrat how AGCT dissects data to identify two networks: Interferon signaling and NRF2-signaling networks.


Assuntos
Expressão Gênica , Software , Biologia de Sistemas/métodos , Análise de Ondaletas , Algoritmos , Animais , Ciclo Celular/genética , Biologia Computacional/métodos , Conjuntos de Dados como Assunto , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Cadeias de Markov , Camundongos , Pentaclorofenol/farmacologia , Pentaclorofenol/envenenamento , Distribuição Aleatória , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Biologia de Sistemas/estatística & dados numéricos
13.
Proc Natl Acad Sci U S A ; 117(31): 18530-18539, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690699

RESUMO

Endoplasmic reticulum (ER) macroautophagy (hereafter called ER-phagy) uses autophagy receptors to selectively degrade ER domains in response to starvation or the accumulation of aggregation-prone proteins. Autophagy receptors package the ER into autophagosomes by binding to the ubiquitin-like yeast protein Atg8 (LC3 in mammals), which is needed for autophagosome formation. In budding yeast, cortical and cytoplasmic ER-phagy requires the autophagy receptor Atg40. While different ER autophagy receptors have been identified, little is known about other components of the ER-phagy machinery. In an effort to identify these components, we screened the genome-wide library of viable yeast deletion mutants for defects in the degradation of cortical ER following treatment with rapamycin, a drug that mimics starvation. Among the mutants we identified was vps13Δ. While yeast has one gene that encodes the phospholipid transporter VPS13, humans have four vacuolar protein-sorting (VPS) protein 13 isoforms. Mutations in all four human isoforms have been linked to different neurological disorders, including Parkinson's disease. Our findings have shown that Vps13 acts after Atg40 engages the autophagy machinery. Vps13 resides at contact sites between the ER and several organelles, including late endosomes. In the absence of Vps13, the cortical ER marker Rtn1 accumulated at late endosomes, and a dramatic decrease in ER packaging into autophagosomes was observed. Together, these studies suggest a role for Vps13 in the sequestration of the ER into autophagosomes at late endosomes. These observations may have important implications for understanding Parkinson's and other neurological diseases.


Assuntos
Autofagossomos/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Autofagia , Linhagem Celular , Retículo Endoplasmático/genética , Endossomos/genética , Endossomos/metabolismo , Humanos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
14.
Proc Natl Acad Sci U S A ; 117(31): 18869-18879, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32675233

RESUMO

Metabolic modeling and machine learning are key components in the emerging next generation of systems and synthetic biology tools, targeting the genotype-phenotype-environment relationship. Rather than being used in isolation, it is becoming clear that their value is maximized when they are combined. However, the potential of integrating these two frameworks for omic data augmentation and integration is largely unexplored. We propose, rigorously assess, and compare machine-learning-based data integration techniques, combining gene expression profiles with computationally generated metabolic flux data to predict yeast cell growth. To this end, we create strain-specific metabolic models for 1,143 Saccharomyces cerevisiae mutants and we test 27 machine-learning methods, incorporating state-of-the-art feature selection and multiview learning approaches. We propose a multiview neural network using fluxomic and transcriptomic data, showing that the former increases the predictive accuracy of the latter and reveals functional patterns that are not directly deducible from gene expression alone. We test the proposed neural network on a further 86 strains generated in a different experiment, therefore verifying its robustness to an additional independent dataset. Finally, we show that introducing mechanistic flux features improves the predictions also for knockout strains whose genes were not modeled in the metabolic reconstruction. Our results thus demonstrate that fusing experimental cues with in silico models, based on known biochemistry, can contribute with disjoint information toward biologically informed and interpretable machine learning. Overall, this study provides tools for understanding and manipulating complex phenotypes, increasing both the prediction accuracy and the extent of discernible mechanistic biological insights.


Assuntos
Aprendizado de Máquina , Análise do Fluxo Metabólico/métodos , Saccharomyces cerevisiae , Biologia de Sistemas/métodos , Modelos Biológicos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcriptoma
15.
Nat Commun ; 11(1): 3122, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561742

RESUMO

During nuclear surveillance in yeast, the RNA exosome functions together with the TRAMP complexes. These include the DEAH-box RNA helicase Mtr4 together with an RNA-binding protein (Air1 or Air2) and a poly(A) polymerase (Trf4 or Trf5). To better determine how RNA substrates are targeted, we analyzed protein and RNA interactions for TRAMP components. Mass spectrometry identified three distinct TRAMP complexes formed in vivo. These complexes preferentially assemble on different classes of transcripts. Unexpectedly, on many substrates, including pre-rRNAs and pre-mRNAs, binding specificity is apparently conferred by Trf4 and Trf5. Clustering of mRNAs by TRAMP association shows co-enrichment for mRNAs with functionally related products, supporting the significance of surveillance in regulating gene expression. We compared binding sites of TRAMP components with multiple nuclear RNA binding proteins, revealing preferential colocalization of subsets of factors. TRF5 deletion reduces Mtr4 recruitment and increases RNA abundance for mRNAs specifically showing high Trf5 binding.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Espectrometria de Massas , Mutação , Mapeamento de Interação de Proteínas , Precursores de RNA/metabolismo , Estabilidade de RNA , RNA-Seq , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato/genética
16.
Nat Commun ; 11(1): 2790, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32493904

RESUMO

Age-dependent changes in metabolism can manifest as cellular lipid accumulation, but how this accumulation is regulated or impacts longevity is poorly understood. We find that Saccharomyces cerevisiae accumulate lipid droplets (LDs) during aging. We also find that over-expressing BNA2, the first Biosynthesis of NAD+ (kynurenine) pathway gene, reduces LD accumulation during aging and extends lifespan. Mechanistically, this LD accumulation during aging is not linked to NAD+ levels, but is anti-correlated with metabolites of the shikimate and aromatic amino acid biosynthesis (SA) pathways (upstream of BNA2), which produce tryptophan (the Bna2p substrate). We provide evidence that over-expressed BNA2 skews glycolytic flux from LDs towards the SA-BNA pathways, effectively reducing LDs. Importantly, we find that accumulation of LDs does not shorten lifespan, but does protect aged cells against stress. Our findings reveal how lipid accumulation impacts longevity, and how aging cell metabolism can be rewired to modulate lipid accumulation independently from longevity.


Assuntos
Metabolismo dos Lipídeos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Vias Biossintéticas , Temperatura Baixa , Gotículas Lipídicas/metabolismo , Metaboloma , NAD/metabolismo , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Ácido Chiquímico/metabolismo , Estresse Fisiológico
17.
Nature ; 582(7810): 119-123, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32494069

RESUMO

The three-dimensional architecture of the genome governs its maintenance, expression and transmission. The cohesin protein complex organizes the genome by topologically linking distant loci, and is highly enriched in specialized chromosomal domains surrounding centromeres, called pericentromeres1-6. Here we report the three-dimensional structure of pericentromeres in budding yeast (Saccharomyces cerevisiae) and establish the relationship between genome organization and function. We find that convergent genes mark pericentromere borders and, together with core centromeres, define their structure and function by positioning cohesin. Centromeres load cohesin, and convergent genes at pericentromere borders trap it. Each side of the pericentromere is organized into a looped conformation, with border convergent genes at the base. Microtubule attachment extends a single pericentromere loop, size-limited by convergent genes at its borders. Reorienting genes at borders into a tandem configuration repositions cohesin, enlarges the pericentromere and impairs chromosome biorientation during mitosis. Thus, the linear arrangement of transcriptional units together with targeted cohesin loading shapes pericentromeres into a structure that is competent for chromosome segregation. Our results reveal the architecture of the chromosomal region within which kinetochores are embedded, as well as the restructuring caused by microtubule attachment. Furthermore, we establish a direct, causal relationship between the three-dimensional genome organization of a specific chromosomal domain and cellular function.


Assuntos
Centrômero/genética , Centrômero/metabolismo , Genes Fúngicos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/metabolismo , Centrômero/química , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Genoma Fúngico/genética , Viabilidade Microbiana/genética , Mitose/genética , Conformação Molecular
18.
Nature ; 582(7810): 124-128, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32494071

RESUMO

In most species, homologous chromosomes must recombine in order to segregate accurately during meiosis1. Because small chromosomes would be at risk of missegregation if recombination were randomly distributed, the double-strand breaks (DSBs) that initiate recombination are not located arbitrarily2. How the nonrandomness of DSB distributions is controlled is not understood, although several pathways are known to regulate the timing, location and number of DSBs. Meiotic DSBs are generated by Spo11 and accessory DSB proteins, including Rec114 and Mer2, which assemble on chromosomes3-7 and are nearly universal in eukaryotes8-11. Here we demonstrate how Saccharomyces cerevisiae integrates multiple temporally distinct pathways to regulate the binding of Rec114 and Mer2 to chromosomes, thereby controlling the duration of a DSB-competent state. The engagement of homologous chromosomes with each other regulates the dissociation of Rec114 and Mer2 later in prophase I, whereas the timing of replication and the proximity to centromeres or telomeres influence the accumulation of Rec114 and Mer2 early in prophase I. Another early mechanism enhances the binding of Rec114 and Mer2 specifically on the shortest chromosomes, and is subject to selection pressure to maintain the hyperrecombinogenic properties of these chromosomes. Thus, the karyotype of an organism and its risk of meiotic missegregation influence the shape and evolution of its recombination landscape. Our results provide a cohesive view of a multifaceted and evolutionarily constrained system that allocates DSBs to all pairs of homologous chromosomes.


Assuntos
Cromossomos Fúngicos/genética , Recombinação Homóloga , Meiose , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Centrômero/genética , Segregação de Cromossomos , Cromossomos Fúngicos/metabolismo , Quebras de DNA de Cadeia Dupla , Período de Replicação do DNA , Meiose/genética , Prófase Meiótica I/genética , Recombinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Telômero/genética , Fatores de Tempo
19.
N Biotechnol ; 58: 55-60, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32562862

RESUMO

Co-culture conditions are beneficial for study due to the advances which arise from symbiotic interactions and which cannot be replicated under pure culture conditions. Here, the focus is on the connection between two fungi - a yeast, Saccharomyces cerevisiae, and a filamentous fungus, Penicillium chrysogenum - in a yeast immobilization system termed' yeast biocapsules', where the yeast and filamentous fungus are strongly attached to one another, forming spherical structures. This co-culture condition hinders filamentous fungal biomass growth, while immobilization of yeast cells continues to increase. The effect of the co-culture condition on endometabolites or intracellular metabolites were tracked during the beginning and end of the yeast biocapsule formation period, and metabolites analyzed by Gas Chromatography-Mass Spectrometry Detector (GC-MSD). Distinct metabolite profiles were found between single culture conditions, involving each organism separately, and with the co-culture condition, where there were differences in 54 endometabolites. Specifically, co-culture condition compounds such as fructose, glycolic acid and glyceric acid were present in higher concentrations at the end of biocapsule formation. These results shed light on the mechanisms and biochemical impact of the interaction between the yeast and filamentous fungus and serve as a basis to apply and further develop yeast biocapsules as a new biotechnological tool with benefits for industry.


Assuntos
Cápsulas Fúngicas/metabolismo , Penicillium chrysogenum/metabolismo , Saccharomyces cerevisiae/metabolismo , Biomassa , Biotecnologia , Técnicas de Cocultura , Frutose/química , Frutose/metabolismo , Cápsulas Fúngicas/química , Cromatografia Gasosa-Espectrometria de Massas , Ácidos Glicéricos/química , Ácidos Glicéricos/metabolismo , Glicolatos/química , Glicolatos/metabolismo , Penicillium chrysogenum/química , Penicillium chrysogenum/citologia , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia
20.
Biochim Biophys Acta Gen Subj ; 1864(10): 129658, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32512168

RESUMO

BACKGROUND: Syntaxin-1A and Sso1 are syntaxin family SNARE proteins engaged in synaptic vesicle fusion and yeast exocytosis. The syntaxin-1A SNARE motif can form a fusogenic SNARE complex with Sso1 partners. However, a chimera in which the SNARE motif in syntaxin-1A is introduced into Sso1 was not functional in yeast because the chimera is retained in the ER. Through the analysis of the transport defect of Sso1/syntaxin-1A chimeric SNAREs, we found that their SNARE motifs have distinctive properties. METHODS: Sso1, syntaxin-1A, and Sso1/syntaxin-1A chimeric SNAREs were expressed in yeast cells and their localization and interaction with other SNAREs are analyzed. RESULTS: SNARE proteins containing the syntaxin-1A SNARE motif exhibit a transport defect because they form a cis-SNARE complex in the ER. Ectopic SNARE complex formation can be prevented in syntaxin-1A by binding to a Sec1/Munc-18-like (SM) protein. In contrast, the SNARE motif of Sso1 does not form an ectopic SNARE complex. Additionally, we found that the SNARE motif in syntaxin-1A, but not that in Sso1, self-interacts, even when it is in the inactive form and bound to the SM protein. CONCLUSIONS: The SNARE motif in syntaxin-1A, but not in Sso1, likely forms ectopic SNARE complex. Because of this property, the SM protein is necessary for syntaxin-1A to prevent its promiscuous assembly and to promote its export from the ER. GENERAL SIGNIFICANCE: Properties of SNARE motifs affect characteristics of SNARE proteins. The regulatory mechanisms of SNARE proteins are, in part, designed to handle such properties.


Assuntos
Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Exocitose , Fusão de Membrana , Ligação Proteica , Mapas de Interação de Proteínas , Proteínas SNARE/análise , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/análise
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