RESUMO
Although lack of ADAR (adenosine deaminase acting on RNA) orthologs, genome-wide A-to-I editing occurs specifically during sexual reproduction in a number of filamentous ascomycetes, including Fusarium graminearum and Neurospora crassa. Unlike ADAR-mediated editing in animals, fungal A-to-I editing has a strong preference for hairpin loops and U at -1 position, which leads to frequent editing of UAG and UAA stop codons. Majority of RNA editing events in fungi are in the coding region and cause amino acid changes. Some of these editing events have been experimentally characterized for providing heterozygote and adaptive advantages in F. graminearum. Recent studies showed that FgTad2 and FgTad3, 2 ADAT (adenosine deaminase acting on tRNA) enzymes that normally catalyze the editing of A34 in the anticodon of tRNA during vegetative growth mediate A-to-I mRNA editing during sexual reproduction. Stage specificity of RNA editing is conferred by stage-specific expression of short transcript isoforms of FgTAD2 and FgTAD3 as well as cofactors such as AME1 and FIP5 that facilitate the editing of mRNA in perithecia. Taken together, fungal A-to-I RNA editing during sexual reproduction is catalyzed by ADATs and it has the same sequence and structural preferences with editing of A34 in tRNA.
Assuntos
Adenosina Desaminase , Edição de RNA , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ascomicetos/genética , RNA Fúngico/genética , RNA Fúngico/metabolismo , Adenosina/metabolismo , Adenosina/genética , Inosina/metabolismo , Inosina/genética , Fusarium/genética , Neurospora crassa/genéticaRESUMO
Factor-dependent termination uses molecular motors to remodel transcription machineries, but the associated mechanisms, especially in eukaryotes, are poorly understood. Here we use single-molecule fluorescence assays to characterize in real time the composition and the catalytic states of Saccharomyces cerevisiae transcription termination complexes remodeled by Sen1 helicase. We confirm that Sen1 takes the RNA transcript as its substrate and translocates along it by hydrolyzing multiple ATPs to form an intermediate with a stalled RNA polymerase II (Pol II) transcription elongation complex (TEC). We show that this intermediate dissociates upon hydrolysis of a single ATP leading to dissociation of Sen1 and RNA, after which Sen1 remains bound to the RNA. We find that Pol II ends up in a variety of states: dissociating from the DNA substrate, which is facilitated by transcription bubble rewinding, being retained to the DNA substrate, or diffusing along the DNA substrate. Our results provide a complete quantitative framework for understanding the mechanism of Sen1-dependent transcription termination in eukaryotes.
Assuntos
Trifosfato de Adenosina , DNA Helicases , RNA Polimerase II , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Imagem Individual de Molécula , Terminação da Transcrição Genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , RNA Polimerase II/metabolismo , Trifosfato de Adenosina/metabolismo , DNA Helicases/metabolismo , DNA Helicases/genética , Imagem Individual de Molécula/métodos , RNA Helicases/metabolismo , RNA Helicases/genética , Transcrição Gênica , RNA Fúngico/metabolismo , RNA Fúngico/genética , DNA Fúngico/metabolismo , DNA Fúngico/genética , HidróliseRESUMO
Nascent messenger RNA is endowed with a poly(A) tail that is subject to gradual deadenylation and subsequent degradation in the cytoplasm. Deadenylation and degradation rates are typically correlated, rendering it difficult to dissect the determinants governing each of these processes and the mechanistic basis of their coupling. Here we developed an approach that allows systematic, robust and multiplexed quantification of poly(A) tails in Saccharomyces cerevisiae. Our results suggest that mRNA deadenylation and degradation rates are decoupled during meiosis, and that transcript length is a major determinant of deadenylation rates and a key contributor to reshaping of poly(A) tail lengths. Meiosis-specific decoupling also leads to unique positive associations between poly(A) tail length and gene expression. The decoupling is associated with a focal localization pattern of the RNA degradation factor Xrn1, and can be phenocopied by Xrn1 deletion under nonmeiotic conditions. Importantly, the association of transcript length with deadenylation rates is conserved across eukaryotes. Our study uncovers a factor that shapes deadenylation rate and reveals a unique context in which degradation is decoupled from deadenylation.
Assuntos
Meiose/genética , Estabilidade de RNA/genética , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Adenosina/química , Exorribonucleases/metabolismo , Expressão Gênica/genética , Poli A/química , RNA Mensageiro/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Aconitase, a highly conserved protein across all domains of life, functions in converting citrate to isocitrate in the tricarboxylic acid cycle. Cytosolic aconitase is also known to act as an iron regulatory protein in mammals, binding to the RNA hairpin structures known as iron-responsive elements within the untranslated regions of specific RNAs. Aconitase-2 (Aco2) in fission yeast is a fusion protein consisting of an aconitase and a mitochondrial ribosomal protein, bL21, residing not only in mitochondria but also in cytosol and the nucleus. To investigate the role of Aco2 in the nucleus and cytoplasm of fission yeast, we analyzed the transcriptome of aco2ΔN mutant that is deleted of nuclear localization signal (NLS). RNA sequencing revealed that the aco2ΔN mutation caused increase in mRNAs encoding iron uptake transporters, such as Str1, Str3, and Shu1. The half-lives of mRNAs for these genes were found to be significantly longer in the aco2ΔN mutant than the wild-type strain, suggesting the role of Aco2 in mRNA turnover. The three conserved cysteines required for the catalytic activity of aconitase were not necessary for this role. The UV cross-linking RNA immunoprecipitation analysis revealed that Aco2 directly bound to the mRNAs of iron uptake transporters. Aco2-mediated degradation of iron-uptake mRNAs appears to utilize exoribonuclease pathway that involves Rrp6 as evidenced by genetic interactions. These results reveal a novel role of non-mitochondrial aconitase protein in the mRNA turnover in fission yeast to fine-tune iron homeostasis, independent of regulation by transcriptional repressor Fep1.
Assuntos
Aconitato Hidratase/metabolismo , Proteínas de Transporte de Cátions/genética , Regulação Fúngica da Expressão Gênica , Ferro/metabolismo , RNA Fúngico/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Proteínas de Transporte de Cátions/metabolismo , Núcleo Celular/enzimologia , Citoplasma/enzimologia , Exorribonucleases/genética , Exorribonucleases/metabolismo , Fatores de Transcrição GATA/genética , Fatores de Transcrição GATA/metabolismo , Genes Fúngicos , Proteínas Reguladoras de Ferro/genética , Proteínas Reguladoras de Ferro/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Regulon , Ribonucleases/genética , Ribonucleases/metabolismo , Schizosaccharomyces/enzimologia , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
RNAs are key players in the cell, and to fulfil their functions, they are enzymatically modified. These modifications have been found to be dynamic and dependent on internal and external factors, such as stress. In this study we used nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) to address the question of which mechanisms allow the dynamic adaptation of RNA modifications during stress in the model organism S. cerevisiae. We found that both tRNA and rRNA transcription is stalled in yeast exposed to stressors such as H2O2, NaAsO2 or methyl methanesulfonate (MMS). From the absence of new transcripts, we concluded that most RNA modification profile changes observed to date are linked to changes happening on the pre-existing RNAs. We confirmed these changes, and we followed the fate of the pre-existing tRNAs and rRNAs during stress recovery. For MMS, we found previously described damage products in tRNA, and in addition, we found evidence for direct base methylation damage of 2'O-ribose methylated nucleosides in rRNA. While we found no evidence for increased RNA degradation after MMS exposure, we observed rapid loss of all methylation damages in all studied RNAs. With NAIL-MS we further established the modification speed in new tRNA and 18S and 25S rRNA from unstressed S. cerevisiae. During stress exposure, the placement of modifications was delayed overall. Only the tRNA modifications 1-methyladenosine and pseudouridine were incorporated as fast in stressed cells as in control cells. Similarly, 2'-O-methyladenosine in both 18S and 25S rRNA was unaffected by the stressor, but all other rRNA modifications were incorporated after a delay. In summary, we present mechanistic insights into stress-dependent RNA modification profiling in S. cerevisiae tRNA and rRNA.
Assuntos
Processamento Pós-Transcricional do RNA , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/fisiologia , Estresse Fisiológico/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Pseudouridina/metabolismo , RNA Ribossômico/metabolismo , RNA Ribossômico 18S/metabolismo , RNA de Transferência/metabolismoRESUMO
Gene deletion and gene expression alteration can lead to growth defects that are amplified or reduced when a second mutation is present in the same cells. We performed 154 genetic interaction mapping (GIM) screens with query mutants related with RNA metabolism and estimated the growth rates of about 700 000 double mutant Saccharomyces cerevisiae strains. The tested targets included the gene deletion collection and 900 strains in which essential genes were affected by mRNA destabilization (DAmP). To analyze the results, we developed RECAP, a strategy that validates genetic interaction profiles by comparison with gene co-citation frequency, and identified links between 1471 genes and 117 biological processes. In addition to these large-scale results, we validated both enhancement and suppression of slow growth measured for specific RNA-related pathways. Thus, negative genetic interactions identified a role for the OCA inositol polyphosphate hydrolase complex in mRNA translation initiation. By analysis of suppressors, we found that Puf4, a Pumilio family RNA binding protein, inhibits ribosomal protein Rpl9 function, by acting on a conserved UGUAcauUA motif located downstream the stop codon of the RPL9B mRNA. Altogether, the results and their analysis should represent a useful resource for discovery of gene function in yeast.
Assuntos
Genes Fúngicos , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/genética , Alelos , Deleção de Genes , Pleiotropia Genética , Fosfatos de Inositol/metabolismo , Iniciação Traducional da Cadeia Peptídica , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologiaRESUMO
N6-methyladenosine (m6A) is a modification that plays pivotal roles in RNA metabolism and function, although its functions in spliceosomal U6 snRNA remain unknown. To elucidate its role, we conduct a large-scale transcriptome analysis of a Schizosaccharomyces pombe strain lacking this modification and found a global change of pre-mRNA splicing. The most significantly impacted introns are enriched for adenosine at the fourth position pairing the m6A in U6 snRNA, and exon sequences weakly recognized by U5 snRNA. This suggests cooperative recognition of 5' splice site by U6 and U5 snRNPs, and also a role of m6A facilitating efficient recognition of the splice sites weakly interacting with U5 snRNA, indicating that U6 snRNA m6A relaxes the 5' exon constraint and allows protein sequence diversity along with explosively increasing number of introns over the course of eukaryotic evolution.
Assuntos
Regulação Fúngica da Expressão Gênica , Splicing de RNA , RNA Fúngico/metabolismo , RNA Nuclear Pequeno/metabolismo , Schizosaccharomyces/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Éxons/genética , Espectrometria de Massas , Metiltransferases/genética , Metiltransferases/isolamento & purificação , Metiltransferases/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Sítios de Splice de RNA/genética , RNA Fúngico/genética , RNA Nuclear Pequeno/genética , RNA-Seq , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6 , Ribonucleoproteína Nuclear Pequena U5 , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Nonavailability of glucose as a carbon source results in glyoxylate pathway activation, which metabolizes nonfermentable carbon for energy generation in Saccharomyces cerevisiae. Ucc1p of S. cerevisiae inhibits activation of the glyoxylate pathway by targeting Cit2p, a key glyoxylate enzyme for ubiquitin-mediated proteasomal degradation when glucose is available as a carbon source. Sro9p, a La-motif protein involved in RNA biogenesis, interacts physically with the messenger RNA of UCC1; however, its functional relevance is yet to be discovered. This study presents binary epistatic interaction between UCC1 and SRO9, with functional implication on the growth rate, response to genotoxic stress, resistance to apoptosis, and petite mutation. Cells with ucc1Δsro9Δ, as their genetic background, exhibit alteration in morphology, improvement in growth rate, resistance to apoptosis, and petite mutation. Moreover, the study indicates a cross-link between ubiquitin-proteasome system and RNA biogenesis and metabolism, with applications in industrial fermentation and screening for cancer therapeutics.
Assuntos
Glioxilatos/metabolismo , Proteínas dos Microfilamentos , RNA Fúngico , RNA Mensageiro , Proteínas de Ligação a RNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
RNA helicases remodel the spliceosome to enable pre-mRNA splicing, but their binding and mechanism of action remain poorly understood. To define helicase-RNA contacts in specific spliceosomal states, we develop purified spliceosome iCLIP (psiCLIP), which reveals dynamic helicase-RNA contacts during splicing catalysis. The helicase Prp16 binds along the entire available single-stranded RNA region between the branchpoint and 3'-splice site, while Prp22 binds diffusely downstream of the branchpoint before exon ligation, but then switches to more narrow binding in the downstream exon after exon ligation, arguing against a mechanism of processive translocation. Depletion of the exon-ligation factor Prp18 destabilizes Prp22 binding to the pre-mRNA, suggesting that proofreading by Prp22 may sense the stability of the spliceosome during exon ligation. Thus, psiCLIP complements structural studies by providing key insights into the binding and proofreading activity of spliceosomal RNA helicases.
Assuntos
Éxons , RNA Helicases/química , RNA Helicases/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Autoantígenos/química , Autoantígenos/metabolismo , Microscopia Crioeletrônica , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Modelos Moleculares , Precursores de RNA/química , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA Fúngico/metabolismo , Proteínas Recombinantes , Ribonucleoproteína Nuclear Pequena U5/química , Ribonucleoproteína Nuclear Pequena U5/genética , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/químicaRESUMO
Aflatoxin is a group of polyketide-derived carcinogenic and mutagenic secondary metabolites produced by Aspergillus flavus that negatively impact global food security and threaten the health of both humans and livestock. Aflatoxin biosynthesis is strongly affected by the fungal developmental stage, cultivation conditions, and environmental stress. In this study, a novel float culture method was used to examine the direct responses of the A. flavus transcriptome to temperature stress, oxidative stress, and their dual effects during the aflatoxin production stage. The transcriptomic response of A. flavus illustrated that the co-regulation of different secondary metabolic pathways likely contributes to maintaining cellular homeostasis and promoting cell survival under stress conditions. In particular, aflatoxin biosynthetic gene expression was downregulated, while genes encoding secondary metabolites with antioxidant properties, such as kojic acid and imizoquins, were upregulated under stress conditions. Multiple mitochondrial function-related genes, including those encoding NADH:ubiquinone oxidoreductase, ubiquinol-cytochrome C reductase, and alternative oxidase, were differentially expressed. These data can provide insights into the important mechanisms through which secondary metabolism in A. flavus is co-regulated and facilitate the deployment of various approaches for the effective control and prevention of aflatoxin contamination in food crops.
Assuntos
Aflatoxinas/biossíntese , Aspergillus flavus/genética , Produtos Agrícolas/microbiologia , Regulação Fúngica da Expressão Gênica , Aflatoxinas/análise , Aflatoxinas/toxicidade , Aspergillus flavus/metabolismo , Biologia Computacional , Microbiologia de Alimentos , Resposta ao Choque Térmico/genética , Temperatura Alta/efeitos adversos , Redes e Vias Metabólicas/genética , Estresse Oxidativo/genética , Pironas/metabolismo , RNA Fúngico/isolamento & purificação , RNA Fúngico/metabolismo , RNA-Seq , Metabolismo Secundário/genética , Esporos FúngicosRESUMO
The mRNA export flux through nuclear pore complexes (NPC) changes under DNA manipulation and hence affects protein translation. However, monitoring the flux of a specific mRNA in single live cell is beyond reach of traditional techniques. We developed a fluorescence-based detection method for measuring the export flux of mRNA through NPC in single live cell using a snapshot image, which had been tested on exogenous genes' expression in HeLa cells, with transfection or infection, and endogenous genes' expression in yeast cells, during incubation and carbon catabolite repression. With its speediness, explicitness and noninvasiveness, we believe that it would be valuable in direct monitoring of gene behavior, and the understanding of gene regulation at a single cell level.
Assuntos
Transporte Ativo do Núcleo Celular , Poro Nuclear/metabolismo , RNA Mensageiro/metabolismo , Repressão Catabólica , Dependovirus/genética , Dependovirus/metabolismo , Expressão Gênica , Genes Fúngicos , Células HeLa , Humanos , Kluyveromyces/genética , Kluyveromyces/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Biológicos , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Viral/genética , RNA Viral/metabolismo , Análise de Célula Única , Transfecção , Proteína Vermelha FluorescenteRESUMO
RNA editing is an important posttranscriptional process that alters the genetic information of RNA encoded by genomic DNA. Adenosine-to-inosine (A-to-I) editing is the most prevalent type of RNA editing in animal kingdom, catalyzed by adenosine deaminases acting on RNA (ADARs). Recently, genome-wide A-to-I RNA editing is discovered in fungi, involving adenosine deamination mechanisms distinct from animals. Aiming to draw more attention to RNA editing in fungi, here we discuss the considerations for deep sequencing data preparation and the available various methods for detecting RNA editing, with a special emphasis on their usability for fungal RNA editing detection. We describe computational protocols for the identification of candidate RNA editing sites in fungi by using two software packages REDItools and RES-Scanner with RNA sequencing (RNA-Seq) and genomic DNA sequencing (DNA-Seq) data.
Assuntos
Biologia Computacional/métodos , Fungos/genética , Genoma Fúngico , Edição de RNA/genética , RNA Fúngico/genética , Software , Adenosina/química , Adenosina/genética , Fungos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Inosina/química , Inosina/genética , RNA Fúngico/metabolismoRESUMO
During pathogen interaction with the host, several mechanisms are used to favor or inhibit the infectious process; one is called nutritional immunity, characterized by restriction of micronutrients to pathogens. Several studies on fungi of the Paracoccidioides complex, have demonstrated that these pathogens remodel their metabolic pathways to overcome the hostile condition imposed by the host. However, molecular mechanisms that control the regulation of those metabolic changes are not fully understood. Therefore, this work characterizes the expression profile of miRNAs during iron deprivation and describes metabolic pathways putatively regulated by those molecules. Through analysis of RNAseq, 45 miRNAs were identified and eight presented alterations in the expression profile during iron deprivation. Among the differentially regulated miRNAs, five were more abundant in yeast cells during iron deprivation and interestingly, the analyses of genes potentially regulated by those five miRNAs, pointed to metabolic pathways as oxidative phosphorylation, altered in response to iron deprivation. In addition, miRNAs with more abundance in iron presence, have as target genes encoding transcriptional factors related to iron homeostasis and uptake. Therefore, we suggest that miRNAs produced by Paracoccidioides brasiliensis may contribute to the adaptive responses of this fungus in iron starvation environment.
Assuntos
Regulação Fúngica da Expressão Gênica , Ferro/metabolismo , MicroRNAs/metabolismo , Paracoccidioides/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Homeostase , Humanos , MicroRNAs/genética , Paracoccidioides/metabolismo , Paracoccidioidomicose/microbiologia , RNA Fúngico/genética , RNA Fúngico/metabolismoRESUMO
Protein biosynthesis is fundamental to cellular life and requires the efficient functioning of the translational machinery. At the center of this machinery is the ribosome, a ribonucleoprotein complex that depends heavily on Mg2+ for structure. Recent work has indicated that other metal cations can substitute for Mg2+, raising questions about the role different metals may play in the maintenance of the ribosome under oxidative stress conditions. Here, we assess ribosomal integrity following oxidative stress both in vitro and in cells to elucidate details of the interactions between Fe2+ and the ribosome and identify Mn2+ as a factor capable of attenuating oxidant-induced Fe2+-mediated degradation of rRNA. We report that Fe2+ promotes degradation of all rRNA species of the yeast ribosome and that it is bound directly to RNA molecules. Furthermore, we demonstrate that Mn2+ competes with Fe2+ for rRNA-binding sites and that protection of ribosomes from Fe2+-mediated rRNA hydrolysis correlates with the restoration of cell viability. Our data, therefore, suggest a relationship between these two transition metals in controlling ribosome stability under oxidative stress.
Assuntos
Ferro/metabolismo , Manganês/metabolismo , Estresse Oxidativo , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Ribossomos/genética , Saccharomyces cerevisiae/genéticaRESUMO
Cadmium (Cd) is a toxic metal occurring in the environment naturally. Almond mushroom (Agaricus brasiliensis) is a well-known cultivated edible and medicinal mushroom. In the past few decades, Cd accumulation in A.brasiliensis has received increasing attention. However, the molecular mechanisms of Cd-accumulation in A. brasiliensis are still unclear. In this paper, a comparative transcriptome of two A.brasiliensis strains with contrasting Cd accumulation and tolerance was performed to identify Cd-responsive genes possibly responsible for low Cd-accumulation and high Cd-tolerance. Using low Cd-accumulating and Cd-tolerant (J77) and high Cd-accumulating and Cd-sensitive (J1) A.brasiliensis strains, we investigated 0, 2 and 5 mg L-1 Cd-effects on mycelium growth, Cd-accumulation and transcriptome revealed by RNA-Seq. A total of 57,884 unigenes were obtained. Far less Cd-responsive genes were identified in J77 mycelia than those in J1 mycelia (e.g., ABC transporters, ZIP Zn transporter, Glutathione S-transferase and Cation efflux (CE) family). The higher Cd-accumulation in J1 mycelia might be due to Cd-induced upregulation of ZIP Zn transporter. Cd impaired cell wall, cell cycle, DNA replication and repair, thus decreasing J1 mycelium growth. Cd-stimulated production of sulfur-containing compounds, polysaccharides, organic acids, trehalose, ATP and NADPH, and sequestration of Cd might be adaptive responses of J1 mycelia to the increased Cd-accumulation. DNA replication and repair had better stability under 2 mg L-1 Cd, but greater positive modifications under 5 mg L-1 Cd. Better stability of DNA replication and repair, better cell wall and cell cycle stability might account for the higher Cd-tolerance of J77 mycelia. Our findings provide a comprehensive set of DEGs influenced by Cd stress; and shed light on molecular mechanism of A.brasiliensis Cd accumulation and Cd tolerance.
Assuntos
Agaricus/metabolismo , Cádmio/metabolismo , Transcriptoma , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Agaricus/efeitos dos fármacos , Agaricus/genética , Cádmio/toxicidade , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Tolerância a Medicamentos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Micélio/química , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimento , Polissacarídeos/metabolismo , RNA Fúngico/química , RNA Fúngico/metabolismo , RNA-SeqRESUMO
Controlling efficiency and fidelity in the early stage of mitochondrial DNA transcription is crucial for regulating cellular energy metabolism. Conformational transitions of the transcription initiation complex must be central for such control, but how the conformational dynamics progress throughout transcription initiation remains unknown. Here, we use single-molecule fluorescence resonance energy transfer techniques to examine the conformational dynamics of the transcriptional system of yeast mitochondria with single-base resolution. We show that the yeast mitochondrial transcriptional complex dynamically transitions among closed, open, and scrunched states throughout the initiation stage. Then abruptly at position +8, the dynamic states of initiation make a sharp irreversible transition to an unbent conformation with associated promoter release. Remarkably, stalled initiation complexes remain in dynamic scrunching and unscrunching states without dissociating the RNA transcript, implying the existence of backtracking transitions with possible regulatory roles. The dynamic landscape of transcription initiation suggests a kinetically driven regulation of mitochondrial transcription.
Assuntos
Mitocôndrias/genética , Saccharomyces cerevisiae/genética , Iniciação da Transcrição Genética , Trifosfato de Adenosina , DNA Fúngico/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula/métodos , Elongação da Transcrição Genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Stress granules are condensates of non-translating mRNAs and proteins involved in the stress response and neurodegenerative diseases. Stress granules form in part through intermolecular RNA-RNA interactions, and to better understand how RNA-based condensation occurs, we demonstrate that RNA is effectively recruited to the surfaces of RNA or RNP condensates in vitro. We demonstrate that, through ATP-dependent RNA binding, the DEAD-box protein eIF4A reduces RNA condensation in vitro and limits stress granule formation in cells. This defines a function for eIF4A to limit intermolecular RNA-RNA interactions in cells. These results establish an important role for eIF4A, and potentially other DEAD-box proteins, as ATP-dependent RNA chaperones that limit the condensation of RNA, analogous to the function of proteins like HSP70 in combatting protein aggregates.
Assuntos
RNA Helicases DEAD-box/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/metabolismo , RNA Helicases/metabolismo , RNA Fúngico/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Células HeLa , Humanos , Microscopia Confocal , Ligação Proteica , RNA Fúngico/isolamento & purificação , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Imagem com Lapso de TempoRESUMO
The 5' untranslated regions (5' UTR) of mRNAs play an important role in the eukaryotic translation initiation process. Additional levels of translational regulation may be mediated through interactions between structured mRNAs that can adopt interchangeable secondary or tertiary structures and the regulatory protein/RNA factors or components of the translational apparatus. Here we report a regulatory function of the 5' UTR mRNA of the spe2 gene (SAM decarboxylase) in polyamine metabolism of the fission yeast Schizosaccharomyces pombe Reporter assays, biochemical experiments, and mutational analysis demonstrate that this 5' UTR mRNA of spe2 can bind to spermidine to regulate translation. A tertiary structure transition in the 5' UTR RNA upon spermidine binding is essential for translation regulation. This study provides biochemical evidence for spermidine binding to regulate translation of the spe2 gene through interactions with the 5' UTR mRNA. The identification of such a regulatory RNA that is directly associated with an essential eukaryotic metabolic process suggests that other ligand-binding RNAs may also contribute to eukaryotic gene regulation.
Assuntos
Regiões 5' não Traduzidas , Adenosilmetionina Descarboxilase/genética , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/metabolismo , Schizosaccharomyces/genética , Espermidina/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Ligantes , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , Schizosaccharomyces/metabolismoRESUMO
The 5' untranslated region (5' UTR) of eukaryotic mRNA plays an important role in translation. Here we report the function of the 5' UTR mRNA of S-adenosylmethionine synthetase (sam1) in translational modulation in the presence of SAM in fission yeast Schizosaccharomyces pombe Reporter assays, binding and chemical probing experiments, and mutational analysis show that the 5' UTR mRNA of sam1 binds to SAM to effect translation. Translational modulation is dependent on a tertiary structure transition in the RNA upon SAM binding. The characterization of such an RNA that is directly associated with an essential metabolic process in eukaryotes provides additional evidence that ligand binding by RNAs plays an important role in eukaryotic gene regulation.
Assuntos
Regiões 5' não Traduzidas , Metionina Adenosiltransferase/genética , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/metabolismo , S-Adenosilmetionina/metabolismo , Schizosaccharomyces/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Ligantes , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , Schizosaccharomyces/metabolismoRESUMO
Pre-messenger RNA splicing involves multi-step assembly of the large spliceosome complexes that catalyse the two consecutive trans-esterification reactions, resulting in intron removal. There is evidence that proof-reading mechanisms monitor the fidelity of this complex process. Transcripts that fail these fidelity tests are thought to be directed to degradation pathways, permitting the splicing factors to be recycled. While studying the roles of splicing factors in vivo, in budding yeast, we performed targeted depletion of individual proteins, and analysed the effect on co-transcriptional spliceosome assembly and splicing efficiency. Unexpectedly, depleting factors such as Prp16 or Prp22, that are known to function at the second catalytic step or later in the splicing pathway, resulted in a defect in the first step of splicing, and accumulation of arrested spliceosomes. Through a kinetic analysis of newly synthesized RNA, we observed that a second step splicing defect (the primary defect) was rapidly followed by the first step of splicing defect. Our results show that knocking down a splicing factor can quickly lead to a recycling defect with splicing factors sequestered in stalled complexes, thereby limiting new rounds of splicing. We demonstrate that this 'feed-back' effect can be minimized by depleting the target protein more gradually or only partially, allowing a better separation between primary and secondary effects. Our findings indicate that splicing surveillance mechanisms may not always cope with spliceosome assembly defects, and suggest that work involving knock-down of splicing factors or components of other large complexes should be carefully monitored to avoid potentially misleading conclusions.