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1.
Nat Commun ; 11(1): 4905, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32999288

RESUMO

Transcription factor (TF) IIIC is a conserved eukaryotic six-subunit protein complex with dual function. It serves as a general TF for most RNA polymerase (Pol) III genes by recruiting TFIIIB, but it is also involved in chromatin organization and regulation of Pol II genes through interaction with CTCF and condensin II. Here, we report the structure of the S. cerevisiae TFIIIC subcomplex τA, which contains the most conserved subunits of TFIIIC and is responsible for recruitment of TFIIIB and transcription start site (TSS) selection at Pol III genes. We show that τA binding to its promoter is auto-inhibited by a disordered acidic tail of subunit τ95. We further provide a negative-stain reconstruction of τA bound to the TFIIIB subunits Brf1 and TBP. This shows that a ruler element in τA achieves positioning of TFIIIB upstream of the TSS, and suggests remodeling of the complex during assembly of TFIIIB by TFIIIC.


Assuntos
Regulação Fúngica da Expressão Gênica , RNA Polimerase III/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae/genética , Fatores de Transcrição TFIII/ultraestrutura , Animais , Linhagem Celular , Microscopia Crioeletrônica , DNA Fúngico/genética , DNA Fúngico/metabolismo , Genes Fúngicos/genética , Insetos , Domínios Proteicos , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIIIB/genética , Fator de Transcrição TFIIIB/isolamento & purificação , Fator de Transcrição TFIIIB/metabolismo , Fatores de Transcrição TFIII/genética , Fatores de Transcrição TFIII/isolamento & purificação , Fatores de Transcrição TFIII/metabolismo , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética
2.
PLoS One ; 15(8): e0238452, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32866191

RESUMO

The filamentous fungus Acremonium chrysogenum is the main industrial producer of cephalosporin C (CPC), one of the major precursors for manufacturing of cephalosporin antibiotics. The plasma membrane H+-ATPase (PMA) plays a key role in numerous fungal physiological processes. Previously we observed a decrease of PMA activity in A. chrysogenum overproducing strain RNCM 408D (HY) as compared to the level the wild-type strain A. chrysogenum ATCC 11550. Here we report the relationship between PMA activity and CPC biosynthesis in A. chrysogenum strains. The elevation of PMA activity in HY strain through overexpression of PMA1 from Saccharomyces cerevisiae, under the control of the constitutive gpdA promoter from Aspergillus nidulans, results in a 1.2 to 10-fold decrease in CPC production, shift in beta-lactam intermediates content, and is accompanied by the decrease in cef genes expression in the fermentation process; the characteristic colony morphology on agar media is also changed. The level of PMA activity in A. chrysogenum HY OE::PMA1 strains has been increased by 50-100%, up to the level observed in WT strain, and was interrelated with ATP consumption; the more PMA activity is elevated, the more ATP level is depleted. The reduced PMA activity in A. chrysogenum HY strain may be one of the selected events during classical strain improvement, aimed at elevating the ATP content available for CPC production.


Assuntos
Acremonium/metabolismo , Membrana Celular/metabolismo , Cefalosporinas/biossíntese , Cefalosporinas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Adenosina Trifosfatases/metabolismo , Meios de Cultura/metabolismo , Fermentação/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , beta-Lactamas/metabolismo
3.
Mol Genet Genomics ; 295(6): 1489-1500, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32948893

RESUMO

Glucose, fructose and mannose are the preferred carbon/energy sources for the yeast Saccharomyces cerevisiae. Absence of preferred energy sources activates glucose derepression, which is regulated by the kinase Snf1. Snf1 phosphorylates the transcriptional repressor Mig1, which results in its exit from the nucleus and subsequent derepression of genes. In contrast, Snf1 is inactive when preferred carbon sources are available, which leads to dephosphorylation of Mig1 and its translocation to the nucleus where Mig1 acts as a transcription repressor. Here we revisit the role of the three hexose kinases, Hxk1, Hxk2 and Glk1, in glucose de/repression. We demonstrate that all three sugar kinases initially affect Mig1 nuclear localization upon addition of glucose, fructose and mannose. This initial import of Mig1 into the nucleus was temporary; for continuous nucleocytoplasmic shuttling of Mig1, Hxk2 is required in the presence of glucose and mannose and in the presence of fructose Hxk2 or Hxk1 is required. Our data suggest that Mig1 import following exposure to preferred energy sources is controlled via two different pathways, where (1) the initial import is regulated by signals derived from metabolism and (2) continuous shuttling is regulated by the Hxk2 and Hxk1 proteins. Mig1 nucleocytoplasmic shuttling appears to be important for the maintenance of the repressed state in which Hxk1/2 seems to play an essential role.


Assuntos
Núcleo Celular/metabolismo , Frutose/metabolismo , Glucose/metabolismo , Hexoquinase/metabolismo , Manose/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Regulação Fúngica da Expressão Gênica , Hexoquinase/genética , Fosforilação , Transporte Proteico , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
4.
Nat Commun ; 11(1): 4866, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978391

RESUMO

Mitochondria house evolutionarily conserved pathways of carbon and nitrogen metabolism that drive cellular energy production. Mitochondrial bioenergetics is regulated by calcium uptake through the mitochondrial calcium uniporter (MCU), a multi-protein complex whose assembly in the inner mitochondrial membrane is facilitated by the scaffold factor MCUR1. Intriguingly, many fungi that lack MCU contain MCUR1 homologs, suggesting alternate functions. Herein, we characterize Saccharomyces cerevisiae homologs Put6 and Put7 of MCUR1 as regulators of mitochondrial proline metabolism. Put6 and Put7 are tethered to the inner mitochondrial membrane in a large hetero-oligomeric complex, whose abundance is regulated by proline. Loss of this complex perturbs mitochondrial proline homeostasis and cellular redox balance. Yeast cells lacking either Put6 or Put7 exhibit a pronounced defect in proline utilization, which can be corrected by the heterologous expression of human MCUR1. Our work uncovers an unexpected role of MCUR1 homologs in mitochondrial proline metabolism.


Assuntos
Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Prolina/metabolismo , Saccharomyces cerevisiae/metabolismo , Canais de Cálcio , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Homeostase , Humanos , Proteínas de Membrana/genética , Redes e Vias Metabólicas/genética , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transcriptoma
5.
Mol Cell ; 79(6): 1051-1065.e10, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32877643

RESUMO

Mitochondria contain their own gene expression systems, including membrane-bound ribosomes dedicated to synthesizing a few hydrophobic subunits of the oxidative phosphorylation (OXPHOS) complexes. We used a proximity-dependent biotinylation technique, BioID, coupled with mass spectrometry to delineate in baker's yeast a comprehensive network of factors involved in biogenesis of mitochondrial encoded proteins. This mitochondrial gene expression network (MiGENet) encompasses proteins involved in transcription, RNA processing, translation, or protein biogenesis. Our analyses indicate the spatial organization of these processes, thereby revealing basic mechanistic principles and the proteins populating strategically important sites. For example, newly synthesized proteins are directly handed over to ribosomal tunnel exit-bound factors that mediate membrane insertion, co-factor acquisition, or their mounting into OXPHOS complexes in a special early assembly hub. Collectively, the data reveal the connectivity of mitochondrial gene expression, reflecting a unique tailoring of the mitochondrial gene expression system.


Assuntos
Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana/genética , Fosforilação Oxidativa , Biossíntese de Proteínas/genética , Saccharomyces cerevisiae/genética
6.
PLoS Genet ; 16(8): e1009005, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32841236

RESUMO

Azole drugs are the most frequently used antifungal agents. The pathogenic yeast Candida glabrata acquires resistance to azole drugs via single amino acid substitution mutations eliciting a gain-of-function (GOF) hyperactive phenotype in the Pdr1 transcription factor. These GOF mutants constitutively drive high transcription of target genes such as the ATP-binding cassette transporter-encoding CDR1 locus. Previous characterization of Pdr1 has demonstrated that this factor is negatively controlled by the action of a central regulatory domain (CRD) of ~700 amino acids, in which GOF mutations are often found. Our earlier experiments demonstrated that a Pdr1 derivative in which the CRD was deleted gave rise to a transcriptional regulator that could not be maintained as the sole copy of PDR1 in the cell owing to its toxically high activity. Using a set of GOF PDR1 alleles from azole-resistant clinical isolates, we have analyzed the mechanisms acting to repress Pdr1 transcriptional activity. Our data support the view that Pdr1-dependent transactivation is mediated by a complex network of transcriptional coactivators interacting with the extreme C-terminal part of Pdr1. These coactivators include but are not limited to the Mediator component Med15A. Activity of this C-terminal domain is controlled by the CRD and requires multiple regions across the C-terminus for normal function. We also provide genetic evidence for an element within the transactivation domain that mediates the interaction of Pdr1 with coactivators on one hand while restricting Pdr1 activity on the other hand. These data indicate that GOF mutations in PDR1 block nonidentical negative inputs that would otherwise restrain Pdr1 transcriptional activation. The strong C-terminal transactivation domain of Pdr1 uses multiple different protein regions to recruit coactivators.


Assuntos
Candida glabrata/efeitos dos fármacos , Candidíase/tratamento farmacológico , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Antifúngicos/efeitos adversos , Antifúngicos/farmacologia , Azóis/efeitos adversos , Azóis/farmacologia , Candida glabrata/genética , Candida glabrata/patogenicidade , Candidíase/genética , Candidíase/microbiologia , Proteínas de Ligação a DNA , Farmacorresistência Fúngica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Humanos , Ativação Transcricional/efeitos dos fármacos
7.
PLoS One ; 15(7): e0234448, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735565

RESUMO

Soybean root rot is a typical soil-borne disease that severely affects the yield of soybean. Funneliformis mosseae is one of the arbuscular mycorrhizal fungi(AMF) dominant strains in soybean continuous cropping soil. The aim of this study was to providing an experimental basis for the study of the molecular mechanism underlying the alleviation of the obstacles associated with the continuous cropping of soybean by AMF. In this study, F. mosseae was inoculated in soil planted with soybean infected with Fusarium oxysporum. The results showed that the incidence of soybean root rot was significantly reduced after inoculation with F. mosseae. In F. mosseae-treated samples, the significantly upregulated genes encoded transmembrane protein in fungal cell membrane. The significantly downregulated genes encoded some proteins, which took part in composition of essential component of fungal cell wall; hydrolyse cellulose and hemicellulose. The DEGs in each treatment were enriched in antigen processing and presentation, carbon fixation in photosynthetic organisms, glycolysis/gluconeogenesis, the MAPK signalling pathway, protein processing in the endoplasmic reticulum and RNA degradation. Inoculation with F. mosseae could in a variety of ways to promote the growth, development of soybean and improve disease resistance. Such as help fungal build barriers to the disease resistance of host plant and enhance their pathogenicity; damaging the structure of the pathogen; protect plant tissues and so on. This study provides an experimental basis for further research on the molecular mechanism underlying the alleviation of challenges associated with the continuous cropping of soybean by AMF.


Assuntos
Fusarium/genética , Micorrizas/genética , Transcriptoma/genética , Fusarium/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Fúngica da Expressão Gênica/genética , Micorrizas/patogenicidade , Fotossíntese , Raízes de Plantas/metabolismo , Solo , Microbiologia do Solo , Soja/crescimento & desenvolvimento
8.
Nat Commun ; 11(1): 4206, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826896

RESUMO

Saccharomyces cerevisiae TBP associated factor 14 (Taf14) is a well-studied transcriptional regulator that controls diverse physiological processes and that physically interacts with at least seven nuclear complexes in yeast. Despite multiple previous Taf14 structural studies, the nature of its disparate transcriptional regulatory functions remains opaque. Here, we demonstrate that the extra-terminal (ET) domain of Taf14 (Taf14ET) recognizes a common motif in multiple transcriptional coactivator proteins from several nuclear complexes, including RSC, SWI/SNF, INO80, NuA3, TFIID, and TFIIF. Moreover, we show that such partner binding promotes liquid-liquid phase separation (LLPS) of Taf14ET, in a mechanism common to YEATS-associated ET domains (e.g., AF9ET) but not Bromo-associated ET domains from BET-family proteins. Thus, beyond identifying the molecular mechanism by which Taf14ET associates with many transcriptional regulators, our study suggests that Taf14 may function as a versatile nuclear hub that orchestrates transcriptional machineries to spatiotemporally regulate diverse cellular pathways.


Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIID/metabolismo , Proteínas de Transporte , Proteínas de Ciclo Celular/metabolismo , Análise por Conglomerados , Proteínas de Ligação a DNA , Epigenômica , Regulação Fúngica da Expressão Gênica , Modelos Moleculares , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fator de Transcrição TFIID/química , Fator de Transcrição TFIID/genética , Fatores de Transcrição/metabolismo
9.
Nat Commun ; 11(1): 4212, 2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32839469

RESUMO

Phosphatases, together with kinases and transcription factors, are key components in cellular signalling networks. Here, we present a systematic functional analysis of the phosphatases in Cryptococcus neoformans, a fungal pathogen that causes life-threatening fungal meningoencephalitis. We analyse 230 signature-tagged mutant strains for 114 putative phosphatases under 30 distinct in vitro growth conditions, revealing at least one function for 60 of these proteins. Large-scale virulence and infectivity assays using insect and mouse models indicate roles in pathogenicity for 31 phosphatases involved in various processes such as thermotolerance, melanin and capsule production, stress responses, O-mannosylation, or retromer function. Notably, phosphatases Xpp1, Ssu72, Siw14, and Sit4 promote blood-brain barrier adhesion and crossing by C. neoformans. Together with our previous systematic studies of transcription factors and kinases, our results provide comprehensive insight into the pathobiological signalling circuitry of C. neoformans.


Assuntos
Cryptococcus neoformans/genética , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica/métodos , Genoma Fúngico/genética , Estudo de Associação Genômica Ampla/métodos , Monoéster Fosfórico Hidrolases/genética , Animais , Análise por Conglomerados , Criptococose/microbiologia , Cryptococcus neoformans/patogenicidade , Feminino , Proteínas Fúngicas/classificação , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Camundongos Endogâmicos , Monoéster Fosfórico Hidrolases/classificação , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases/classificação , Fosfotransferases/genética , Fosfotransferases/metabolismo , Transdução de Sinais/genética , Termotolerância/genética , Fatores de Transcrição/classificação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Virulência/genética
10.
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
11.
PLoS One ; 15(8): e0237273, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32760134

RESUMO

Uromyces appendiculatus is a major pathogen on common bean. Like other rust fungi, it uses effectors to influence its host plant. Effectors are assumed to possess characteristic expression profiles, reflecting their activity during the infection process. In order to determine expression profiles using RT-qPCR, stably expressed reference genes are necessary for normalization. These reference genes need to be tested. Using samples representing seven different developmental stages of the urediospore-based infection process we employed RT-qPCR to measure the expression of 14 candidate reference genes and determined the most suitable ones based on the range of Cq values and comparative calculations using the geNorm and NormFinder algorithms. Among the tested genes RPS14 had the smallest Cq range, followed by Elf1a and Elf3; geNorm rated Tub and UbcE2 best with CytB as a third and NormFinder found UbcE2, Tub and Elf3 as best reference genes. Combining these findings using equal weight for the rankings UbcE2, Elf3 and Tub can be considered the best reference genes. A combination of either two reference genes, UbcE2 and Tub or three reference genes, UbcE2, Tub, and Elf3 is recommended for normalization. However, differences between most genes were relatively small, so all tested genes can be considered suitable for normalization with the exception of RPS9, SDH, Ubc and PDK.


Assuntos
Basidiomycota/genética , Phaseolus/microbiologia , Doenças das Plantas/microbiologia , Transcriptoma , Basidiomycota/fisiologia , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Interações Hospedeiro-Patógeno
12.
Mol Cell ; 79(6): 963-977.e3, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32735772

RESUMO

Autophagic degradation of the endoplasmic reticulum (ER-phagy) is triggered by ER stress in diverse organisms. However, molecular mechanisms governing ER stress-induced ER-phagy remain insufficiently understood. Here we report that ER stress-induced ER-phagy in the fission yeast Schizosaccharomyces pombe requires Epr1, a soluble Atg8-interacting ER-phagy receptor. Epr1 localizes to the ER through interacting with integral ER membrane proteins VAPs. Bridging an Atg8-VAP association is the main ER-phagy role of Epr1, as it can be bypassed by an artificial Atg8-VAP tether. VAPs contribute to ER-phagy not only by tethering Atg8 to the ER membrane, but also by maintaining the ER-plasma membrane contact. Epr1 is upregulated during ER stress by the unfolded protein response (UPR) regulator Ire1. Loss of Epr1 reduces survival against ER stress. Conversely, increasing Epr1 expression suppresses the ER-phagy defect and ER stress sensitivity of cells lacking Ire1. Our findings expand and deepen the molecular understanding of ER-phagy.


Assuntos
Estresse do Retículo Endoplasmático/genética , Endorribonucleases/genética , Proteínas R-SNARE/genética , Autofagossomos/metabolismo , Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/genética , Retículo Endoplasmático/genética , Regulação Fúngica da Expressão Gênica/genética , Proteólise , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Resposta a Proteínas não Dobradas/genética
13.
PLoS One ; 15(8): e0237540, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32804965

RESUMO

The yeast MAP kinase Hog1 pathway activates transcription of several hundreds genes. Large-scale gene expression and DNA binding assays suggest that most Hog1-induced genes are regulated by the transcriptional activators Msn2/4, Hot1 and Sko1. These studies also revealed the target genes of each activator and the putative binding sites on their promoters. In a previous study we identified a group of genes, which we considered the bona fide targets of Hog1, because they were induced in response to expression of intrinsically active mutant of Hog1, in the absence of any stress. We previously analyzed the promoter of the most highly induced gene, STL1, and noticed that some promoter properties were different from those proposed by large-scale data. We therefore continue to study promoters individually and present here analyses of promoters of more Hog1's targets, RTC3, HSP12, DAK1 and ALD3. We report that RTC3 and HSP12 promoters are robust and are induced, to different degrees, even in cells lacking all four activators. DAK1 and ALD3 promoters are not robust and fully depend on a single activator, DAK1 on Sko1 and ALD3 on Msn2/4. Most of these observations could not be inferred from the large-scale data. Msn2/4 are involved in regulating all four promoters. It was assumed, therefore, that the promoters are spontaneously active in ras2Δ cells, in which Msn2/4 are known to be de-repressed. Intriguingly, the promoters were not active in BY4741ras2Δ cells, but were de-repressed, as expected, in ras2Δ cells of other genetic backgrounds. This study describes two phenomena. One, some Hog1's target promoters are most robust, backupped by many activators. Second, in contrast to most laboratory strains, the widely used BY4741 strain does not induce Msn2/4 activity when the Ras/cAMP cascade is downregulated.


Assuntos
Proteínas de Choque Térmico/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Fatores de Transcrição de Zíper de Leucina Básica/genética , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Choque Térmico/química , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética
14.
Mol Cell ; 79(6): 991-1007.e4, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32780990

RESUMO

Stress granules (SGs) are condensates of mRNPs that form in response to stress. SGs arise by multivalent protein-protein, protein-RNA, and RNA-RNA interactions. However, the role of RNA-RNA interactions in SG assembly remains understudied. Here, we describe a yeast SG reconstitution system that faithfully recapitulates SG assembly in response to trigger RNAs. SGs assembled by stem-loop RNA triggers are ATP-sensitive, regulated by helicase/chaperone activity, and exhibit the hallmarks of maturation observed for SG proteins that phase-separate in vitro. Additionally, the fraction of total RNA that phase-separates in vitro is sufficient to trigger SG formation. However, condensation of NFT1 mRNA, an enriched transcript in this population, can only assemble an incomplete SG. These results suggest that networks of distinct transcripts are required to form a canonical SG and provide a platform for dissecting the interplay between the transcriptome and ATP-dependent remodeling in SG formation.


Assuntos
Grânulos Citoplasmáticos/genética , Ribonucleoproteínas/genética , Estresse Fisiológico/genética , Transcriptoma/genética , Trifosfato de Adenosina/genética , Linhagem Celular , Regulação Fúngica da Expressão Gênica/genética , Humanos , RNA/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética
15.
PLoS Genet ; 16(7): e1008611, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32658892

RESUMO

Epimutations in fungal pathogens are emerging as novel phenomena that could explain the fast-developing resistance to antifungal drugs and other stresses. These epimutations are generated by RNA interference (RNAi) mechanisms that transiently silence specific genes to overcome stressful stimuli. The early-diverging fungus Mucor circinelloides exercises a fine control over two interacting RNAi pathways to produce epimutants: the canonical RNAi pathway and a new RNAi degradative pathway. The latter is considered a non-canonical RNAi pathway (NCRIP) because it relies on RNA-dependent RNA polymerases (RdRPs) and a novel ribonuclease III-like named R3B2 to degrade target transcripts. Here in this work, we uncovered the role of NCRIP in regulating virulence processes and transposon movements through key components of the pathway, RdRP1 and R3B2. Mutants in these genes are unable to launch a proper virulence response to macrophage phagocytosis, resulting in a decreased virulence potential. The transcriptomic profile of rdrp1Δ and r3b2Δ mutants revealed a pre-exposure adaptation to the stressful phagosomal environment even when the strains are not confronted by macrophages. These results suggest that NCRIP represses key targets during regular growth and releases its control when a stressful environment challenges the fungus. NCRIP interacts with the RNAi canonical core to protect genome stability by controlling the expression of centromeric retrotransposable elements. In the absence of NCRIP, these retrotransposons are robustly repressed by the canonical RNAi machinery; thus, supporting the antagonistic role of NCRIP in containing the epimutational pathway. Both interacting RNAi pathways might be essential to govern host-pathogen interactions through transient adaptations, contributing to the unique traits of the emerging infection mucormycosis.


Assuntos
Mucorales/genética , Mucormicose/genética , Interferência de RNA , Ribonuclease III/genética , Antifúngicos/farmacologia , Farmacorresistência Fúngica/genética , Epigênese Genética/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Instabilidade Genômica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Mucorales/patogenicidade , Mucormicose/microbiologia , Mutação/genética , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Virulência/genética
16.
PLoS Genet ; 16(6): e1008865, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32603360

RESUMO

Fpr1 (FK506-sensitive proline rotamase 1), a protein of the FKBP12 (FK506-binding protein 12 kDa) family in Saccharomyces cerevisiae, is a primary target for the immunosuppressive agents FK506 and rapamycin. Fpr1 inhibits calcineurin and TORC1 (target of rapamycin complex 1) when bound to FK506 and rapamycin, respectively. Although Fpr1 is recognised to play a crucial role in the efficacy of these drugs, its physiological functions remain unclear. In a hmo1Δ (high mobility group family 1-deleted) yeast strain, deletion of FPR1 induced severe growth defects, which could be alleviated by increasing the copy number of RPL25 (ribosome protein of the large subunit 25), suggesting that RPL25 expression was affected in hmo1Δfpr1Δ cells. In the current study, extensive chromatin immunoprecipitation (ChIP) and ChIP-sequencing analyses revealed that Fpr1 associates specifically with the upstream activating sequences of nearly all RPG (ribosomal protein gene) promoters, presumably in a manner dependent on Rap1 (repressor/activator site binding protein 1). Intriguingly, Fpr1 promotes the binding of Fhl1/Ifh1 (forkhead-like 1/interacts with forkhead 1), two key regulators of RPG transcription, to certain RPG promoters independently of and/or cooperatively with Hmo1. Furthermore, mutation analyses of Fpr1 indicated that for transcriptional function on RPG promoters, Fpr1 requires its N-terminal domain and the binding surface for rapamycin, but not peptidyl-prolyl isomerase activity. Notably, Fpr1 orthologues from other species also inhibit TORC1 when bound to rapamycin, but do not regulate transcription in yeast, which suggests that these two functions of Fpr1 are independent of each other.


Assuntos
Proteínas de Grupo de Alta Mobilidade/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Calcineurina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Fatores de Transcrição Forkhead/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Proteínas de Grupo de Alta Mobilidade/genética , Peptidilprolil Isomerase/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Sirolimo/farmacologia , Tacrolimo/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Transcrição Genética
17.
PLoS Pathog ; 16(7): e1008645, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32667960

RESUMO

Aspergillus fumigatus is an opportunistic fungal pathogen that secretes an array of immune-modulatory molecules, including secondary metabolites (SMs), which contribute to enhancing fungal fitness and growth within the mammalian host. Gliotoxin (GT) is a SM that interferes with the function and recruitment of innate immune cells, which are essential for eliminating A. fumigatus during invasive infections. We identified a C6 Zn cluster-type transcription factor (TF), subsequently named RglT, important for A. fumigatus oxidative stress resistance, GT biosynthesis and self-protection. RglT regulates the expression of several gli genes of the GT biosynthetic gene cluster, including the oxidoreductase-encoding gene gliT, by directly binding to their respective promoter regions. Subsequently, RglT was shown to be important for virulence in a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA). Homologues of RglT and GliT are present in eurotiomycete and sordariomycete fungi, including the non-GT-producing fungus A. nidulans, where a conservation of function was described. Phylogenetically informed model testing led to an evolutionary scenario in which the GliT-based resistance mechanism is ancestral and RglT-mediated regulation of GliT occurred subsequently. In conclusion, this work describes the function of a previously uncharacterised TF in oxidative stress resistance, GT biosynthesis and self-protection in both GT-producing and non-producing Aspergillus species.


Assuntos
Aspergilose , Aspergillus fumigatus/patogenicidade , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Gliotoxina/biossíntese , Fatores de Transcrição/metabolismo , Animais , Aspergilose/metabolismo , Aspergilose/microbiologia , Aspergillus fumigatus/metabolismo , Camundongos , Estresse Oxidativo/fisiologia , Virulência/fisiologia
18.
PLoS Genet ; 16(7): e1008484, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32673313

RESUMO

Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-deoxyglucose were identified by whole genome sequencing. Missense alleles of the HXK2, REG1, GLC7 and SNF1 genes were shown to confer significant resistance to 2-deoxyglucose and all had the potential to alter the activity and or target selection of the Snf1 kinase signaling pathway. All three missense alleles in HXK2 resulted in significantly reduced catalytic activity. Addition of 2DG promotes endocytosis of the glucose transporter Hxt3. All but one of the 2DG-resistant strains reduced the 2DG-mediated hexose transporter endocytosis by increasing plasma membrane occupancy of the Hxt3 protein. Increased expression of the DOG (deoxyglucose) phosphatases has been associated with resistance to 2-deoxyglucose. Expression of both the DOG1 and DOG2 mRNA was elevated after treatment with 2-deoxyglucose but induction of these genes is not associated with 2DG-resistance. RNAseq analysis of the transcriptional response to 2DG showed large scale, genome-wide changes in mRNA abundance that were greatly reduced in the 2DG resistant strains. These findings suggest the common adaptive response to 2DG is to limit the magnitude of the response. Genetic studies of 2DG resistance using the dominant SNF1-G53R allele in cells that are genetically compromised in both the endocytosis and DOG pathways suggest that at least one more mechanism for conferring resistance to this glucose analog remains to be discovered.


Assuntos
Metabolismo Energético/genética , Glucose/metabolismo , Hexoquinase/genética , Monoéster Fosfórico Hidrolases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Desoxiglucose/efeitos adversos , Desoxiglucose/farmacologia , Endocitose/efeitos dos fármacos , Endocitose/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas Facilitadoras de Transporte de Glucose/genética , Humanos , Mutação/genética , Proteína Fosfatase 1/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Transdução de Sinais/efeitos dos fármacos , Sequenciamento Completo do Genoma
19.
PLoS One ; 15(7): e0236429, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730288

RESUMO

The soilborne fungus Gaeumannomyces tritici (G. tritici) causes the take-all disease on wheat roots. Ambient pH has been shown to be critical in different steps of G. tritici life cycle such as survival in bulk soil, saprophytic growth, and pathogenicity on plants. There are however intra-specific variations and we previously found two types of G. tritici strains that grow preferentially either at acidic pH or at neutral/alkaline pH; gene expression involved in pH-signal transduction pathway and pathogenesis was differentially regulated in two strains representative of these types. To go deeper in the description of the genetic pathways and the understanding of this adaptative mechanism, transcriptome sequencing was achieved on two strains (PG6 and PG38) which displayed opposite growth profiles in two pH conditions (acidic and neutral). PG6, growing better at acidic pH, overexpressed in this condition genes related to cell proliferation. In contrast, PG38, which grew better at neutral pH, overexpressed in this condition genes involved in fatty acids and amino acid metabolisms, and genes potentially related to pathogenesis. This strain also expressed stress resistance mechanisms at both pH, to assert a convenient growth under various ambient pH conditions. These differences in metabolic pathway expression between strains at different pH might buffer the effect of field or soil variation in wheat fields, and explain the success of the pathogen.


Assuntos
Ascomicetos/genética , Transcriptoma/genética , Ascomicetos/crescimento & desenvolvimento , Ascomicetos/isolamento & purificação , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Ontologia Genética , Genes Fúngicos , Concentração de Íons de Hidrogênio , Micélio/crescimento & desenvolvimento , Especificidade da Espécie , Triticum
20.
Proc Natl Acad Sci U S A ; 117(29): 17122-17129, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32632014

RESUMO

Symbioses of bacteria with fungi have only recently been described and are poorly understood. In the symbiosis of Mycetohabitans (formerly Burkholderia) rhizoxinica with the fungus Rhizopus microsporus, bacterial type III (T3) secretion is known to be essential. Proteins resembling T3-secreted transcription activator-like (TAL) effectors of plant pathogenic bacteria are encoded in the three sequenced Mycetohabitans spp. genomes. TAL effectors nuclear-localize in plants, where they bind and activate genes important in disease. The Burkholderia TAL-like (Btl) proteins bind DNA but lack the N- and C-terminal regions, in which TAL effectors harbor their T3 and nuclear localization signals, and activation domain. We characterized a Btl protein, Btl19-13, and found that, despite the structural differences, it can be T3-secreted and can nuclear-localize. A btl19 -13 gene knockout did not prevent the bacterium from infecting the fungus, but the fungus became less tolerant to cell membrane stress. Btl19-13 did not alter transcription in a plant-based reporter assay, but 15 R. microsporus genes were differentially expressed in comparisons both of the fungus infected with the wild-type bacterium vs. the mutant and with the mutant vs. a complemented strain. Southern blotting revealed btl genes in 14 diverse Mycetohabitans isolates. However, banding patterns and available sequences suggest variation, and the btl19-13 phenotype could not be rescued by a btl gene from a different strain. Our findings support the conclusion that Btl proteins are effectors that act on host DNA and play important but varied or possibly host genotype-specific roles in the M. rhizoxinica-R. microsporus symbiosis.


Assuntos
Burkholderia , Rhizopus , Simbiose/genética , Efetores Semelhantes a Ativadores de Transcrição , Burkholderia/genética , Burkholderia/metabolismo , Burkholderia/fisiologia , Regulação Fúngica da Expressão Gênica/genética , Rhizopus/genética , Rhizopus/metabolismo , Estresse Fisiológico/genética , Efetores Semelhantes a Ativadores de Transcrição/genética , Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Transcriptoma/genética , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo
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