Long non-coding RNA-mediated transcriptional interference of a permease gene confers drug tolerance in fission yeast.
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ABSTRACT:
SUBMITTER: Ard R
PROVIDER: S-EPMC4255232 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Microorganisms and plants produce siderophores, which function to transport environmental iron into cells as well as participate in cellular iron use and deposition. Their biological functions are diverse although their role in primary metabolism is poorly understood. Ferrichrome is a fungal-type siderophore synthesized by nonribosomal peptide synthetase (NRPS). Herein we show that ferrichrome induces adaptive growth of fission yeast on high ammonium media. Ammonium is a preferred nitrogen source as it suppresses uptake and catabolism of less preferred nitrogen sources such as leucine through a mechanism called nitrogen catabolite repression (NCR). Therefore, the growth of fission yeast mutant cells with leucine auxotrophy is suppressed in the presence of high concentrations of ammonium. This growth suppression was canceled by ferrichrome in a manner dependent on the amino acid transporter Cat1. Additionally, growth retardation of wild-type cells by excess ammonium was exacerbated by deleting the NRPS gene sib1, which is responsible for the biosynthesis of ferrichrome, suggesting that intrinsically produced ferrichrome functions in suppressing the metabolic action of ammonium. Furthermore, ferrichrome facilitated the growth of both wild-type and sib1-deficient cells under low glucose conditions. These results suggest that intracellular iron regulates primary metabolism, including NCR, which is mediated by siderophores.
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Project description:Eukaryotic genomes express numerous long intergenic non-coding RNAs (lincRNAs) that do not overlap any coding genes. Some lincRNAs function in various aspects of gene regulation, but it is not clear in general to what extent lincRNAs contribute to the information flow from genotype to phenotype. To explore this question, we systematically analysed cellular roles of lincRNAs in Schizosaccharomyces pombe. Using seamless CRISPR/Cas9-based genome editing, we deleted 141 lincRNA genes to broadly phenotype these mutants, together with 238 diverse coding-gene mutants for functional context. We applied high-throughput colony-based assays to determine mutant growth and viability in benign conditions and in response to 145 different nutrient, drug, and stress conditions. These analyses uncovered phenotypes for 47.5% of the lincRNAs and 96% of the protein-coding genes. For 110 lincRNA mutants, we also performed high-throughput microscopy and flow cytometry assays, linking 37% of these lincRNAs with cell-size and/or cell-cycle control. With all assays combined, we detected phenotypes for 84 (59.6%) of all lincRNA deletion mutants tested. For complementary functional inference, we analysed colony growth of strains ectopically overexpressing 113 lincRNA genes under 47 different conditions. Of these overexpression strains, 102 (90.3%) showed altered growth under certain conditions. Clustering analyses provided further functional clues and relationships for some of the lincRNAs. These rich phenomics datasets associate lincRNA mutants with hundreds of phenotypes, indicating that most of the lincRNAs analysed exert cellular functions in specific environmental or physiological contexts. This study provides groundwork to further dissect the roles of these lincRNAs in the relevant conditions.
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Project description:Long non-coding RNA repertoire and regulation by nuclear exosome, cytoplasmic exonuclease and RNAi in fission yeast
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Project description:BackgroundChanges in gene expression are hallmarks of cellular differentiation. Sexual differentiation in fission yeast (Schizosaccharomyces pombe) provides a model system for gene expression programs accompanying and driving cellular specialization. The expression of hundreds of genes is modulated in successive waves during meiosis and sporulation in S. pombe, and several known transcription factors are critical for these processes.ResultsWe used DNA microarrays to investigate meiotic gene regulation by examining transcriptomes after genetic perturbations (gene deletion and/or overexpression) of rep1, mei4, atf21 and atf31, which encode known transcription factors controlling sexual differentiation. This analysis reveals target genes at a genome-wide scale and uncovers combinatorial control by Atf21p and Atf31p. We also studied two transcription factors not previously implicated in sexual differentiation whose meiotic induction depended on Mei4p: Rsv2p induces stress-related genes during spore formation, while Rsv1p represses glucose-metabolism genes. Our data further reveal negative feedback interactions: both Rep1p and Mei4p not only activate specific gene expression waves (early and middle genes, respectively) but are also required for repression of genes induced in the previous waves (Ste11p-dependent and early genes, respectively).ConclusionThese data give insight into regulatory principles controlling the extensive gene expression program driving sexual differentiation and highlight sophisticated interactions and combinatorial control among transcription factors. Besides triggering simultaneous expression of gene waves, transcription factors also repress genes in the previous wave and induce other factors that in turn regulate a subsequent wave. These dependencies ensure an ordered and timely succession of transcriptional waves during cellular differentiation.
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Project description:The CRISPR-Cas12a is a class II, type V clustered regularly interspaced short palindromic repeat (CRISPR) system with both RNase and DNase activity. Compared to the CRISPR-Cas9 system, it recognizes T-rich PAM sequences and has the advantage of multiplex genomic editing. Here, in fission yeast Schizosaccharomyces pombe, we successfully implemented the CRISPR-Cas12a system for versatile genomic editing and manipulation. In addition to the rrk1 promoter, we used new pol II promoters from endogenous coding genes to express crRNA for Cas12a and obtained a much higher editing efficiency. This new design expands the promoter choices for potential applications in fission yeast and other organisms. In addition, we expressed a gRNA array using a strong constitutive pol II promoter. The array transcript is processed by Cas12a itself to release multiple mature crRNAs. With this construct, multiplex genomic editing of up to three loci was achieved from a single yeast transformation. We also built a CRISPR interference system using a DNase-dead Cas12a to significantly repress endogenous gene expression. Our study provides the first CRISPR-Cas12a toolkit for efficient and rapid genomic gene editing and regulation in fission yeast.
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Project description:BACKGROUND: Hydrogen sulfide (H(2)S) is a newly identified member of the small family of gasotransmitters that are endogenous gaseous signaling molecules that have a fundamental role in human biology and disease. Although it is a relatively recent discovery and the mechanism of H(2)S activity is not completely understood, it is known to be involved in a number of cellular processes; H(2)S can affect ion channels, transcription factors and protein kinases in mammals. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we have used fission yeast as a model organism to study the global gene expression profile in response to H(2)S by microarray. We initially measured the genome-wide transcriptional response of fission yeast to H(2)S. Through the functional classification of genes whose expression profile changed in response to H(2)S, we found that H(2)S mainly influences genes that encode putative or known stress proteins, membrane transporters, cell cycle/meiotic proteins, transcription factors and respiration protein in the mitochondrion. Our analysis showed that there was a significant overlap between the genes affected by H(2)S and the stress response. We identified that the target genes of the MAPK pathway respond to H(2)S; we also identified that a number of transporters respond to H(2)S, these include sugar/carbohydrate transporters, ion transporters, and amino acid transporters. We found many mitochondrial genes to be down regulated upon H(2)S treatment and that H(2)S can reduce mitochondrial oxygen consumption. CONCLUSION/SIGNIFICANCE: This study identifies potential molecular targets of the signaling molecule H(2)S in fission yeast and provides clues about the identity of homologues human proteins and will further the understanding of the cellular role of H(2)S in human diseases.
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