Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 46
Filtrar
1.
Proc Natl Acad Sci U S A ; 118(48)2021 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-34815343

RESUMO

Ribosomes translate RNA into proteins. The protein synthesis inhibitor cycloheximide (CHX) is widely used to inhibit eukaryotic ribosomes engaged in translation elongation. However, the lack of structural data for actively translating polyribosomes stalled by CHX leaves unanswered the question of which elongation step is inhibited. We elucidated CHX's mechanism of action based on the cryo-electron microscopy structure of actively translating Neurospora crassa ribosomes bound with CHX at 2.7-Å resolution. The ribosome structure from this filamentous fungus contains clearly resolved ribosomal protein eL28, like higher eukaryotes but unlike budding yeast, which lacks eL28. Despite some differences in overall structures, the ribosomes from Neurospora, yeast, and humans all contain a highly conserved CHX binding site. We also sequenced classic Neurospora CHX-resistant alleles. These mutations, including one at a residue not previously observed to affect CHX resistance in eukaryotes, were in the large subunit proteins uL15 and eL42 that are part of the CHX-binding pocket. In addition to A-site transfer RNA (tRNA), P-site tRNA, messenger RNA, and CHX that are associated with the translating N. crassa ribosome, spermidine is present near the CHX binding site close to the E site on the large subunit. The tRNAs in the peptidyl transferase center are in the A/A site and the P/P site. The nascent peptide is attached to the A-site tRNA and not to the P-site tRNA. The structural and functional data obtained show that CHX arrests the ribosome in the classical PRE translocation state and does not interfere with A-site reactivity.


Assuntos
Cicloeximida/farmacologia , Neurospora/fisiologia , Ribossomos/metabolismo , Alelos , Sítios de Ligação , Sequência Conservada , Microscopia Crioeletrônica , Fungos/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Modelos Moleculares , Conformação Molecular , Mutação , Neurospora crassa/metabolismo , Elongação Traducional da Cadeia Peptídica , Peptídeos/química , Peptidil Transferases/química , Polirribossomos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Inibidores da Síntese de Proteínas , RNA de Transferência/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/química
2.
Proc Natl Acad Sci U S A ; 117(20): 10935-10945, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32355000

RESUMO

The circadian clock in eukaryotes controls transcriptional and posttranscriptional events, including regulation of the levels and phosphorylation state of translation factors. However, the mechanisms underlying clock control of translation initiation, and the impact of this potential regulation on rhythmic protein synthesis, were not known. We show that inhibitory phosphorylation of eIF2α (P-eIF2α), a conserved translation initiation factor, is clock controlled in Neurospora crassa, peaking during the subjective day. Cycling P-eIF2α levels required rhythmic activation of the eIF2α kinase CPC-3 (the homolog of yeast and mammalian GCN2), and rhythmic activation of CPC-3 was abolished under conditions in which the levels of charged tRNAs were altered. Clock-controlled accumulation of P-eIF2α led to reduced translation during the day in vitro and was necessary for the rhythmic synthesis of select proteins in vivo. Finally, loss of rhythmic P-eIF2α levels led to reduced linear growth rates, supporting the idea that partitioning translation to specific times of day provides a growth advantage to the organism. Together, these results reveal a fundamental mechanism by which the clock regulates rhythmic protein production, and provide key insights into how rhythmic translation, cellular energy, stress, and nutrient metabolism are linked through the levels of charged versus uncharged tRNAs.


Assuntos
Relógios Circadianos/fisiologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Ritmo Circadiano , Fator de Iniciação 2 em Eucariotos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Neurospora crassa/crescimento & desenvolvimento , Neurospora crassa/metabolismo , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , eIF-2 Quinase/metabolismo
3.
Proc Natl Acad Sci U S A ; 116(21): 10435-10440, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31048503

RESUMO

Circadian clocks generate rhythms in cellular functions, including metabolism, to align biological processes with the 24-hour environment. Disruption of this alignment by shift work alters glucose homeostasis. Glucose homeostasis depends on signaling and allosteric control; however, the molecular mechanisms linking the clock to glucose homeostasis remain largely unknown. We investigated the molecular links between the clock and glycogen metabolism, a conserved glucose homeostatic process, in Neurospora crassa We find that glycogen synthase (gsn) mRNA, glycogen phosphorylase (gpn) mRNA, and glycogen levels, accumulate with a daily rhythm controlled by the circadian clock. Because the synthase and phosphorylase are critical to homeostasis, their roles in generating glycogen rhythms were investigated. We demonstrate that while gsn was necessary for glycogen production, constitutive gsn expression resulted in high and arrhythmic glycogen levels, and deletion of gpn abolished gsn mRNA rhythms and rhythmic glycogen accumulation. Furthermore, we show that gsn promoter activity is rhythmic and is directly controlled by core clock component white collar complex (WCC). We also discovered that WCC-regulated transcription factors, VOS-1 and CSP-1, modulate the phase and amplitude of rhythmic gsn mRNA, and these changes are similarly reflected in glycogen oscillations. Together, these data indicate the importance of clock-regulated gsn transcription over signaling or allosteric control of glycogen rhythms, a mechanism that is potentially conserved in mammals and critical to metabolic homeostasis.


Assuntos
Relógios Circadianos , Regulação da Expressão Gênica , Glicogênio Sintase/metabolismo , Glicogênio/metabolismo , Neurospora crassa/metabolismo , Proteínas Fúngicas/metabolismo , Glicogênio Sintase/genética , Neurospora crassa/genética
4.
Chemometr Intell Lab Syst ; 2122021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-35068632

RESUMO

BACKGROUND: The endogenous circadian clock, which controls daily rhythms in the expression of at least half of the mammalian genome, has a major influence on cell physiology. Consequently, disruption of the circadian system is associated with wide range of diseases including cancer. While several circadian clock genes have been associated with cancer progression, little is known about the survival when two or more platforms are considered together. Our goal was to determine if survival outcomes are associated with circadian clock function. To accomplish this goal, we developed a Bayesian hierarchical survival model coupled with the global local shrinkage prior and applied this model to available RNASeq and Copy Number Variation data to select significant circadian genes associates with cancer progression. RESULTS: Using a Bayesian shrinkage approach with the Bayesian accelerated failure time (AFT) model we showed the circadian clock associated gene DEC1 is positively correlated to survival outcome in breast cancer patients. The R package circgene implementing the methodology is available at https://github.com/MAITYA02/circgene. CONCLUSIONS: The proposed Bayesian hierarchical model is the first shrinkage prior based model in its kind which integrates two omics platforms to identify the significant circadian gene for cancer survival.

5.
BMC Cancer ; 19(1): 101, 2019 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-30674294

RESUMO

Following publication of the original article [1], we have been notified that the tagging of one of the author names was done incorrectly in the XML version of the paper. The online and pdf versions of this paper are not affected by the change. Original and corrected tagging can be seen below. The original article has been corrected.

6.
Methods ; 137: 11-19, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29294368

RESUMO

Cell-free protein synthesis (CFPS) can be used in many applications to produce polypeptides and to analyze mechanisms of mRNA translation. Here we describe how to make and use a CPFS system from the model filamentous fungus Neurospora crassa. The extensive genetic resources available in this system provide capacities to exploit robust CFPS for understanding translational control. Included are procedures for the growth and harvesting of cells, the preparation of cell-free extracts that serve as the source of the translational machinery in the CFPS and the preparation of synthetic mRNA to program the CFPS. Methods to accomplish cell-free translation and analyze protein synthesis, and to map positions of ribosomes on mRNAs by toeprinting, are described.


Assuntos
Sistema Livre de Células , Biologia Molecular/métodos , Neurospora crassa/genética , Biossíntese Peptídica/genética , Peptídeos/genética , RNA Mensageiro/genética , Ribossomos/genética
7.
Proc Natl Acad Sci U S A ; 113(34): 9605-10, 2016 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-27506798

RESUMO

The circadian clock has a profound effect on gene regulation, controlling rhythmic transcript accumulation for up to half of expressed genes in eukaryotes. Evidence also exists for clock control of mRNA translation, but the extent and mechanisms for this regulation are not known. In Neurospora crassa, the circadian clock generates daily rhythms in the activation of conserved mitogen-activated protein kinase (MAPK) pathways when cells are grown in constant conditions, including rhythmic activation of the well-characterized p38 osmosensing (OS) MAPK pathway. Rhythmic phosphorylation of the MAPK OS-2 (P-OS-2) leads to temporal control of downstream targets of OS-2. We show that osmotic stress in N. crassa induced the phosphorylation of a eukaryotic elongation factor-2 (eEF-2) kinase, radiation sensitivity complementing kinase-2 (RCK-2), and that RCK-2 is necessary for high-level phosphorylation of eEF-2, a key regulator of translation elongation. The levels of phosphorylated RCK-2 and phosphorylated eEF-2 cycle in abundance in wild-type cells but not in cells deleted for OS-2 or the core clock component FREQUENCY (FRQ). Translation extracts from cells grown in constant conditions show decreased translational activity in the late subjective morning, coincident with the peak in eEF-2 phosphorylation, and rhythmic translation of glutathione S-transferase (GST-3) from constitutive mRNA levels in vivo is dependent on circadian regulation of eEF-2 activity. In contrast, rhythms in phosphorylated eEF-2 levels are not necessary for rhythms in accumulation of the clock protein FRQ, indicating that clock control of eEF-2 activity promotes rhythmic translation of specific mRNAs.


Assuntos
Relógios Circadianos/genética , Quinase do Fator 2 de Elongação/genética , Regulação Fúngica da Expressão Gênica , Neurospora crassa/genética , Elongação Traducional da Cadeia Peptídica , RNA Mensageiro/genética , Quinase do Fator 2 de Elongação/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurospora crassa/crescimento & desenvolvimento , Neurospora crassa/metabolismo , Pressão Osmótica , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
8.
BMC Cancer ; 18(1): 43, 2018 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-29316898

RESUMO

BACKGROUND: The circadian clock is the basis for biological time keeping in eukaryotic organisms. The clock mechanism relies on biochemical signaling pathways to detect environmental stimuli and to regulate the expression of clock-controlled genes throughout the body. MAPK signaling pathways function in both circadian input and output pathways in mammals depending on the tissue; however, little is known about the role of p38 MAPK, an established tumor suppressor, in the mammalian circadian system. Increased expression and activity of p38 MAPK is correlated with poor prognosis in cancer, including glioblastoma multiforme; however, the toxicity of p38 MAPK inhibitors limits their clinical use. Here, we test if timed application of the specific p38 MAPK inhibitor VX-745 reduces glioma cell invasive properties in vitro. METHODS: The levels and rhythmic accumulation of active phosphorylated p38 MAPK in different cell lines were determined by western blots. Rhythmic luciferase activity from clock gene luciferase reporter cells lines was used to test the effect of p38 MAPK inhibition on clock properties as determined using the damped sine fit and Levenberg-Marquardt algorithm. Nonlinear regression and Akaike's information criteria were used to establish rhythmicity. Boyden chamber assays were used to measure glioma cell invasiveness following time-of-day-specific treatment with VX-745. Significant differences were established using t-tests. RESULTS: We demonstrate the activity of p38 MAPK cycles under control of the clock in mouse fibroblast and SCN cell lines. The levels of phosphorylated p38 MAPK were significantly reduced in clock-deficient cells, indicating that the circadian clock plays an important role in activation of this pathway. Inhibition of p38 MAPK activity with VX-745 led to cell-type-specific period changes in the molecular clock. In addition, phosphorylated p38 MAPK levels were rhythmic in HA glial cells, and high and arrhythmic in invasive IM3 glioma cells. We show that inhibition of p38 MAPK activity in IM3 cells at the time of day when the levels are normally low in HA cells under control of the circadian clock, significantly reduced IM3 invasiveness. CONCLUSIONS: Glioma treatment with p38 MAPK inhibitors may be more effective and less toxic if administered at the appropriate time of the day.


Assuntos
Proteínas CLOCK/genética , Relógios Circadianos/genética , Glioblastoma/tratamento farmacológico , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Linhagem da Célula/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Luciferases , Camundongos , Invasividade Neoplásica/genética , Fosforilação , Piridazinas/administração & dosagem , Pirimidinas/administração & dosagem , Transdução de Sinais/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética
9.
Proc Natl Acad Sci U S A ; 111(48): 16995-7002, 2014 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-25362047

RESUMO

Neurospora crassa has been for decades a principal model for filamentous fungal genetics and physiology as well as for understanding the mechanism of circadian clocks. Eukaryotic fungal and animal clocks comprise transcription-translation-based feedback loops that control rhythmic transcription of a substantial fraction of these transcriptomes, yielding the changes in protein abundance that mediate circadian regulation of physiology and metabolism: Understanding circadian control of gene expression is key to understanding eukaryotic, including fungal, physiology. Indeed, the isolation of clock-controlled genes (ccgs) was pioneered in Neurospora where circadian output begins with binding of the core circadian transcription factor WCC to a subset of ccg promoters, including those of many transcription factors. High temporal resolution (2-h) sampling over 48 h using RNA sequencing (RNA-Seq) identified circadianly expressed genes in Neurospora, revealing that from ∼10% to as much 40% of the transcriptome can be expressed under circadian control. Functional classifications of these genes revealed strong enrichment in pathways involving metabolism, protein synthesis, and stress responses; in broad terms, daytime metabolic potential favors catabolism, energy production, and precursor assembly, whereas night activities favor biosynthesis of cellular components and growth. Discriminative regular expression motif elicitation (DREME) identified key promoter motifs highly correlated with the temporal regulation of ccgs. Correlations between ccg abundance from RNA-Seq, the degree of ccg-promoter activation as reported by ccg-promoter-luciferase fusions, and binding of WCC as measured by ChIP-Seq, are not strong. Therefore, although circadian activation is critical to ccg rhythmicity, posttranscriptional regulation plays a major role in determining rhythmicity at the mRNA level.


Assuntos
Relógios Circadianos , Regulação Fúngica da Expressão Gênica , Neurospora crassa/genética , Transcriptoma/genética , Metabolismo Energético/genética , Retroalimentação Fisiológica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Neurospora crassa/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Transdução de Sinais/genética
10.
Eukaryot Cell ; 12(1): 59-69, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23125351

RESUMO

The circadian clock regulates the expression of many genes involved in a wide range of biological functions through output pathways such as mitogen-activated protein kinase (MAPK) pathways. We demonstrate here that the clock regulates the phosphorylation, and thus activation, of the MAPKs MAK-1 and MAK-2 in the filamentous fungus Neurospora crassa. In this study, we identified genetic targets of the MAK-1 pathway, which is homologous to the cell wall integrity pathway in Saccharomyces cerevisiae and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in mammals. When MAK-1 was deleted from Neurospora cells, vegetative growth was reduced and the transcript levels for over 500 genes were affected, with significant enrichment for genes involved in protein synthesis, biogenesis of cellular components, metabolism, energy production, and transcription. Additionally, of the ~500 genes affected by the disruption of MAK-1, more than 25% were previously identified as putative clock-controlled genes. We show that MAK-1 is necessary for robust rhythms of two morning-specific genes, i.e., ccg-1 and the mitochondrial phosphate carrier protein gene NCU07465. Additionally, we show clock regulation of a predicted chitin synthase gene, NCU04352, whose rhythmic accumulation is also dependent upon MAK-1. Together, these data establish a role for the MAK-1 pathway as an output pathway of the circadian clock and suggest a link between rhythmic MAK-1 activity and circadian control of cellular growth.


Assuntos
Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurospora crassa/enzimologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quitina Sintase/genética , Quitina Sintase/metabolismo , Relógios Circadianos/genética , Ritmo Circadiano , Ativação Enzimática , Proteínas Fúngicas/genética , Regulação Enzimológica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Neurospora crassa/genética , Neurospora crassa/crescimento & desenvolvimento , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Processamento de Proteína Pós-Traducional , Transcriptoma
11.
Fungal Genet Biol ; 49(2): 180-8, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22240319

RESUMO

The OS-pathway mitogen-activated protein kinase (MAPK) cascade of Neurospora crassa is responsible for adaptation to osmotic stress. Activation of the MAPK, OS-2, leads to the transcriptional induction of many genes involved in the osmotic stress response. We previously demonstrated that there is a circadian rhythm in the phosphorylation of OS-2 under constant non-stress inducing conditions. Additionally, several osmotic stress-induced genes are known to be regulated by the circadian clock. Therefore, we investigated if rhythms in activation of OS-2 lead to circadian rhythms in other known stress responsive targets. Here we identify three more osmotic stress induced genes as rhythmic: cat-1, gcy-1, and gcy-3. These genes encode a catalase and two predicted glycerol dehydrogenases thought to be involved in the production of glycerol. Rhythms in these genes depend upon the oscillator component FRQ. To investigate how the circadian signal is propagated to these stress induced genes, we examined the role of the OS-responsive transcription factor, ASL-1, in mediating circadian gene expression. We find that while the asl-1 transcript is induced by several stresses including an osmotic shock, asl-1 mRNA accumulation is not rhythmic. However, we show that ASL-1 is required for generating normal circadian rhythms of some OS-pathway responsive transcripts (bli-3, ccg-1, cat-1, gcy-1 and gcy-3) in the absence of an osmotic stress. These data are consistent with the possibility that post-transcriptional regulation of ASL-1 by the rhythmically activated OS-2 MAPK could play a role in generating rhythms in downstream targets.


Assuntos
Ritmo Circadiano/genética , Proteínas Fúngicas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurospora crassa/genética , Catalase/genética , Catalase/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Sistema de Sinalização das MAP Quinases/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Neurospora crassa/metabolismo , Pressão Osmótica/fisiologia , Desidrogenase do Álcool de Açúcar/genética , Desidrogenase do Álcool de Açúcar/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
12.
F1000Res ; 11: 1556, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-37841830

RESUMO

Background: In Neurospora crassa, the circadian clock controls rhythmic mRNA translation initiation through regulation of the eIF2α kinase CPC-3 (the homolog of yeast and mammalian GCN2). Active CPC-3 phosphorylates and inactivates eIF2α, leading to higher phosphorylated eIF2α (P-eIF2α) levels and reduced translation initiation during the subjective day. This daytime activation of CPC-3 is driven by its binding to uncharged tRNA, and uncharged tRNA levels peak during the day under control of the circadian clock. The daily rhythm in uncharged tRNA levels could arise from rhythmic amino acid levels or aminoacyl-tRNA synthetase (aaRSs) levels. Methods: To determine if and how the clock potentially controls rhythms in aspartyl-tRNA synthetase (AspRS) and glutaminyl-tRNA synthetase (GlnRS), both observed to be rhythmic in circadian genomic datasets, transcriptional and translational fusions to luciferase were generated. These luciferase reporter fusions were examined in wild type (WT), clock mutant Δ frq, and clock-controlled transcription factor deletion strains. Results: Translational and transcriptional fusions of AspRS and GlnRS to luciferase confirmed that their protein levels are clock-controlled with peak levels at night. Moreover, clock-controlled transcription factors NCU00275 and ADV-1 drive robust rhythmic protein expression of AspRS and GlnRS, respectively. Conclusions: These data support a model whereby coordinate clock control of select aaRSs drives rhythms in uncharged tRNAs, leading to rhythmic CPC-3 activation, and rhythms in translation of specific mRNAs.


Assuntos
Relógios Circadianos , Neurospora crassa , Animais , Relógios Circadianos/genética , Neurospora crassa/genética , Neurospora crassa/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Fatores de Transcrição/metabolismo , Saccharomyces cerevisiae/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , Luciferases/genética , Luciferases/metabolismo , Ligases/genética , Ligases/metabolismo , Mamíferos/genética , Mamíferos/metabolismo
13.
Cell Rep ; 41(13): 111879, 2022 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-36577368

RESUMO

Phosphorylation of Neurospora crassa eukaryotic initiation factor 2 α (eIF2α), a conserved translation initiation factor, is clock controlled. To determine the impact of rhythmic eIF2α phosphorylation on translation, we performed temporal ribosome profiling and RNA sequencing (RNA-seq) in wild-type (WT), clock mutant Δfrq, eIF2α kinase mutant Δcpc-3, and constitutively active cpc-3c cells. About 14% of mRNAs are rhythmically translated in WT cells, and translation rhythms for ∼30% of these mRNAs, which we named circadian translation-initiation-controlled genes (cTICs), are dependent on the clock and CPC-3. Most cTICs are expressed from arrhythmic mRNAs and contain a P-body (PB) localization motif in their 5' leader sequence. Deletion of SNR-1, a component of cytoplasmic messenger ribonucleoprotein granules (cmRNPgs) that include PBs and stress granules (SGs), and the PB motif on one of the cTIC mRNAs, zip-1, significantly alters zip-1 rhythmic translation. These results reveal that the clock regulates rhythmic translation of specific mRNAs through rhythmic eIF2α activity and cmRNPg metabolism.


Assuntos
Relógios Circadianos , Relógios Circadianos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Grânulos Citoplasmáticos/metabolismo , Biossíntese de Proteínas , Fator de Iniciação 2 em Eucariotos/metabolismo
14.
Nature ; 438(7071): 1105-15, 2005 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-16372000

RESUMO

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.


Assuntos
Aspergillus fumigatus/genética , Aspergillus nidulans/genética , Aspergillus oryzae/genética , Genoma Fúngico/genética , Genômica , Aspergillus fumigatus/fisiologia , Aspergillus nidulans/fisiologia , Aspergillus oryzae/fisiologia , Sequência de Bases , Sequência Consenso/genética , Sequência Conservada/genética , Evolução Molecular , Genes Fúngicos Tipo Acasalamento/genética , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Filogenia , Proteoma/genética , Sequências Reguladoras de Ácido Nucleico/genética , Análise de Sequência de DNA , Sintenia/genética
15.
Eukaryot Cell ; 9(10): 1549-56, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20675579

RESUMO

Light signaling pathways and circadian clocks are inextricably linked and have profound effects on behavior in most organisms. Here, we used chromatin immunoprecipitation (ChIP) sequencing to uncover direct targets of the Neurospora crassa circadian regulator White Collar Complex (WCC). The WCC is a blue-light receptor and the key transcription factor of the circadian oscillator. It controls a transcriptional network that regulates ∼20% of all genes, generating daily rhythms and responses to light. We found that in response to light, WCC binds to hundreds of genomic regions, including the promoters of previously identified clock- and light-regulated genes. We show that WCC directly controls the expression of 24 transcription factor genes, including the clock-controlled adv-1 gene, which controls a circadian output pathway required for daily rhythms in development. Our findings provide links between the key circadian activator and effectors in downstream regulatory pathways.


Assuntos
Relógios Circadianos , Regulação Fúngica da Expressão Gênica , Luz , Neurospora crassa/fisiologia , Transdução de Sinais , Fatores de Transcrição/metabolismo , Imunoprecipitação da Cromatina , Ritmo Circadiano , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Redes Reguladoras de Genes , Genoma Fúngico/genética , Sequenciamento de Nucleotídeos em Larga Escala , Neurospora crassa/genética , Neurospora crassa/metabolismo , Reação em Cadeia da Polimerase , Fatores de Transcrição/genética
16.
mBio ; 12(3)2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006661

RESUMO

The circadian clock controls the phosphorylation and activity of eukaryotic translation initiation factor 2α (eIF2α). In Neurospora crassa, the clock drives a daytime peak in the activity of the eIF2α kinase CPC-3, the homolog of yeast and mammalian GCN2 kinase. This leads to increased levels of phosphorylated eIF2α (P-eIF2α) and reduced mRNA translation initiation during the day. We hypothesized that rhythmic eIF2α activity also requires dephosphorylation of P-eIF2α at night by phosphatases. In support of this hypothesis, we show that mutation of N. crassa PPP-1, a homolog of the yeast eIF2α phosphatase GLC7, leads to high and arrhythmic P-eIF2α levels, while maintaining core circadian oscillator function. PPP-1 levels are clock-controlled, peaking in the early evening, and rhythmic PPP-1 levels are necessary for rhythmic P-eIF2α accumulation. Deletion of the N terminus of N. crassa eIF2γ, the region necessary for eIF2γ interaction with GLC7 in yeast, led to high and arrhythmic P-eIF2α levels. These data supported that N. crassa eIF2γ functions to recruit PPP-1 to dephosphorylate eIF2α at night. Thus, in addition to the activity of CPC-3 kinase, circadian clock regulation of eIF2α activity requires dephosphorylation by PPP-1 phosphatase at night. These data show how the circadian clock controls the activity a central regulator of translation, critical for cellular metabolism and growth control, through the temporal coordination of phosphorylation and dephosphorylation events.IMPORTANCE Circadian clock control of mRNA translation contributes to the daily cycling of a significant proportion of the cellular protein synthesis, but how this is accomplished is not understood. We discovered that the clock in the model fungus Neurospora crassa regulates rhythms in protein synthesis by controlling the phosphorylation and dephosphorylation of a conserved translation initiation factor eIF2α. During the day, N. crassa eIF2α is phosphorylated and inactivated by CPC-3 kinase. At night, a clock-controlled phosphatase, PPP-1, dephosphorylates and activates eIF2α, leading to increased nighttime protein synthesis. Translation requires significant cellular energy; thus, partitioning translation to the night by the clock provides a mechanism to coordinate energy metabolism with protein synthesis and cellular growth.


Assuntos
Relógios Circadianos/genética , Fator de Iniciação 2 em Eucariotos/genética , Proteínas Fúngicas/genética , Neurospora crassa/genética , Neurospora crassa/metabolismo , Biossíntese de Proteínas/genética , Proteína Fosfatase 1/metabolismo , Fator de Iniciação 2 em Eucariotos/classificação , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas Fúngicas/metabolismo , Neurospora crassa/enzimologia , Fosforilação , Biossíntese de Proteínas/fisiologia , Proteína Fosfatase 1/genética
17.
Proc Natl Acad Sci U S A ; 104(46): 18223-8, 2007 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-17984065

RESUMO

Circadian clocks are composed of central oscillators, input pathways that transduce external information to the oscillators, and output pathways that allow the oscillators to temporally regulate cellular processes. Little is known about the output pathways. In this study, we show that the Neurospora crassa osmosensing MAPK pathway, essential for osmotic stress responses, is a circadian output pathway that regulates daily rhythms in the expression of downstream genes. Rhythmic activation of the highly conserved stress-activated p38-type MAPK [Osmotically Sensitive-2 (OS-2)] by the N. crassa circadian clock allows anticipation and preparation for hyperosmotic stress and desiccation that begin at sunrise. These results suggest a conserved role for MAPK pathways in circadian rhythmicity.


Assuntos
Ritmo Circadiano , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Ativação Enzimática , Neurospora crassa/enzimologia , Pressão Osmótica , Fenótipo
18.
Fungal Biol ; 124(5): 235-252, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32389286

RESUMO

Stress is a normal part of life for fungi, which can survive in environments considered inhospitable or hostile for other organisms. Due to the ability of fungi to respond to, survive in, and transform the environment, even under severe stresses, many researchers are exploring the mechanisms that enable fungi to adapt to stress. The International Symposium on Fungal Stress (ISFUS) brings together leading scientists from around the world who research fungal stress. This article discusses presentations given at the third ISFUS, held in São José dos Campos, São Paulo, Brazil in 2019, thereby summarizing the state-of-the-art knowledge on fungal stress, a field that includes microbiology, agriculture, ecology, biotechnology, medicine, and astrobiology.


Assuntos
Fungos , Estresse Fisiológico , Brasil , Fungos/fisiologia
19.
Microbiol Mol Biol Rev ; 68(1): 1-108, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15007097

RESUMO

We present an analysis of over 1,100 of the approximately 10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa. Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease.


Assuntos
Proteínas Fúngicas/genética , Genoma Fúngico , Neurospora crassa , Animais , Biologia Computacional , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Humanos , Micoses/microbiologia , Neurospora crassa/química , Neurospora crassa/genética , Neurospora crassa/metabolismo , Neurospora crassa/patogenicidade , Doenças das Plantas/microbiologia
20.
J Biol Rhythms ; 21(6): 432-44, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17107934

RESUMO

Research in Neurospora crassa pioneered the isolation of clock-controlled genes (ccgs), and more than 180 ccgs have been identified that function in various aspects of the fungal life cycle. Many clock-controlled genes are associated with damage repair, stress responses, intermediary metabolism, protein synthesis, and development. The expression of most of these genes peaks just before dawn and appears to prepare the cells for the desiccation, mutagenesis, and stress caused by sunlight. Progress on characterization of the output signaling pathways from the circadian oscillator mechanism to the ccgs is discussed. The authors also review evidence suggesting that, similar to other clock model organisms, a connection exists between the redox state of the cell and the Neurospora clock. The authors speculate that the clock system may sense not only light but also the redox potential of the cell through one of the PAS domains of the core clock components WC-1 or WC-2.


Assuntos
Ritmo Circadiano/fisiologia , Neurospora crassa/fisiologia , Relógios Biológicos/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Regulação Fúngica da Expressão Gênica , Análise em Microsséries , Neurospora crassa/crescimento & desenvolvimento , Fatores de Transcrição/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA