Transcription of a yeast ribosomal RNA minigene in Saccharomyces cerevisiae.
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ABSTRACT:
SUBMITTER: Quincey RV
PROVIDER: S-EPMC1144458 | biostudies-other | 1984 Dec
REPOSITORIES: biostudies-other
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Project description:Stress-induced strand breaks in rRNA have been observed in many organisms, but the mechanisms by which they originate are not well-understood. Here we show that a chemical rather than an enzymatic mechanism initiates rRNA cleavages during oxidative stress in yeast (Saccharomyces cerevisiae). We used cells lacking the mitochondrial glutaredoxin Grx5 to demonstrate that oxidant-induced cleavage formation in 25S rRNA correlates with intracellular iron levels. Sequestering free iron by chemical or genetic means decreased the extent of rRNA degradation and relieved the hypersensitivity of grx5Δ cells to the oxidants. Importantly, subjecting purified ribosomes to an in vitro iron/ascorbate reaction precisely recapitulated the 25S rRNA cleavage pattern observed in cells, indicating that redox activity of the ribosome-bound iron is responsible for the strand breaks in the rRNA. In summary, our findings provide evidence that oxidative stress-associated rRNA cleavages can occur through rRNA strand scission by redox-active, ribosome-bound iron that potentially promotes Fenton reaction-induced hydroxyl radical production, implicating intracellular iron as a key determinant of the effects of oxidative stress on ribosomes. We propose that iron binding to specific ribosome elements primes rRNA for cleavages that may play a role in redox-sensitive tuning of the ribosome function in stressed cells.
| S-EPMC6139556 | biostudies-literature
Project description:A considerable body of evidence links together mitochondrial dysfunctions, toxic action of metalloid oxyanions, and system and neurodegenerative disorders. In this study we have used the model yeast Saccharomyces cerevisiae to investigate the genetic determinants associated with tellurite resistance/sensitivity. Nitrosoguanidine-induced K2TeO3-resistant mutants were isolated, and one of these mutants, named Sc57-Te5R, was characterized. Both random spore analysis and tetrad analysis and growth of heterozygous (TeS/Te5R) diploid from Sc57-Te5R mutant revealed that nuclear and recessive mutation(s) was responsible for the resistance. To get insight into the mechanisms responsible for K2TeO3-resistance, RNA microarray analyses were performed with K2TeO3-treated and untreated Sc57-Te5R cells. A total of 372 differentially expressed loci were identified corresponding to 6.37% of the S. cerevisiae transcriptome. Of these, 288 transcripts were up-regulated upon K2TeO3 treatment. About half of up-regulated transcripts were associated with the following molecular functions: oxidoreductase activity, structural constituent of cell wall, transporter activity. Comparative whole-genome sequencing allowed us to identify nucleotide variants distinguishing Sc57-Te5R from parental strain Sc57. We detected 15 CDS-inactivating mutations, and found that 3 of them affected genes coding mitochondrial ribosomal proteins (MRPL44 and NAM9) and mitochondrial ribosomal biogenesis (GEP3) pointing out to alteration of mitochondrial ribosome as main determinant of tellurite resistance.
| S-EPMC6089990 | biostudies-literature
Project description:Effect of FLO8 or MSS11 deletion and -overexpression on yeast transcript profiles compared to wild type in laboratory yeast strains Σ1278b and S288c.
2011-09-21 | GSE17716 | GEO
Project description:A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation.
| S-EPMC4750496 | biostudies-literature
Project description:Yeast was transformed with eight recombinants that contained an rRNA minigene and upstream elements of rDNA in different orientations in the multi-copy yeast-Escherichia coli shuttle vector, pJDB207. The effect of these elements of upstream rDNA on the initiation of transcription of the minigene at the site for rRNA biosynthesis was determined by using an S1 nuclease mapping procedure to measure the abundance of the minigene transcript in RNA from the yeast transformants. Transcription of the minigene was enhanced 3-fold by DNA within a 2.2 kb element more than 1.5 kb upstream from the initiation site. Inversion of the 2.2 kb element decreased expression of the minigene by 40%. This 2.2 kb element contained approx. 500 bp from the 25S rRNA coding region at the 3' end of the preceding rRNA gene and 1 kb of adjacent nontranscribed spacer rDNA. The enhancing activity was independent of interference from readthrough that might have contributed to the 7-fold decrease in minigene expression caused by removing all rDNA upstream from -209 bp.
| S-EPMC1152859 | biostudies-other
Project description:Saccharomyces cerevisiae (strain A224A) has an abnormal distribution of cytoplasmic ribosomal subunits when grown at 36 degrees C, with sucrose-gradient analysis of extracts revealing an apparent excess of material sedimenting at 60 S. This abnormality is not observed at either 23 degrees C or 30 degrees C. At 36 degrees C the defect(s) is expressed as a slowed conversion of 20 S ribosomal precursor RNA to mature 18 S rRNA, although the corresponding maturation of 27 S ribosomal precursor RNA to mature 25 S rRNA is normal. Studies on this yeast strain and on mutants derived from it may help to elucidate the role(s) of individual ribosomal components in controlling ribosome biogenesis in eukaryotes.
| S-EPMC1153648 | biostudies-other
Project description:Effect of either FLO8 or MSS11 deletion and -overexpression on yeast transcript profiles compared to wild type in laboratory yeast strains Σ1278b and S288c - also the effect of FLO11 (MUC1) overexpression in the Σ1278b genetic background The aim of this study was to (1) perform a repeat analysis (to improve statistical analysis of these data sets) similar to data submitted previously (GSE17716) and also (2) study the effect of FLO11 over-expression on the transcriptome. Background: The outer cell wall of the yeast Saccharomyces cerevisiae serves as the interface with the surrounding environment and defines cell-cell and cell-surface interactions. Many of these interactions are facilitated by specific adhesins that belong to the Flo protein family. This family of mannoproteins has been implicated in phenotypes such as flocculation and substrate adhesion as well as pseudohyphal growth. Genetic data strongly suggest that individual Flo proteins are responsible for many specific cellular adhesion phenotypes. However, it remains unclear whether such phenotypes are determined solely by the nature of the expressed FLO genes or rather the result of a combination of FLO gene expression and other cell wall properties and cell wall proteins. Mss11p has been shown to be a central element of FLO1 and FLO11 gene regulation and acts together with the cAMP-PKA-dependent transcription factor Flo8p. We use genome wide transcript analysis to identify genes that are direct ly or indirectly regulated by Mss11p in the genetic backgrounds: Sigma1278b and S288c. Sigma 1278b is the strain historically used for the study of pseudohyphae (FLO11 expression) but we also included S288c as this strain is widely used in the research community and was used to determine the first full genome sequence (Thus correspond with SGD information). We also compare this data with transcriptome data from Sigma 1278b yeast over-expressing FLO8 to compare similarities/differences between these two signalling factors. Finally the effect of FLO11 over-expression in Sigma1278b on global transcription is studied so that we can differentiate between "direct" gene targets of Flo8p or Mss11p, and those regulated as a result by the "indirect" effect caused by modified cell wall Flo11p levels.
2011-09-21 | GSE29371 | GEO
Project description:In the yeast Saccharomyces cerevisiae, L30 is one of many ribosomal proteins that is encoded by two functional genes. We have cloned and sequenced RPL30B, which shows strong homology to RPL30A. Use of mRNA as a template for a polymerase chain reaction demonstrated that RPL30B contains an intron in its 5' untranslated region. This intron has an unusual 5' splice site, C/GUAUGU. The genomic copies of RPL30A and RPL30B were disrupted by homologous recombination. Growth rates, primer extension, and two-dimensional ribosomal protein analyses of these disruption mutants suggested that RPL30A is responsible for the majority of L30 production. Surprisingly, meiosis of a diploid strain carrying one disrupted RPL30A and one disrupted RPL30B yielded four viable spores. Ribosomes from haploid cells carrying both disrupted genes had no detectable L30, yet such cells grew with a doubling time only 30% longer than that of wild-type cells. Furthermore, depletion of L30 did not alter the ratio of 60S to 40S ribosomal subunits, suggesting that there is no serious effect on the assembly of 60S subunits. Polysome profiles, however, suggest that the absence of L30 leads to the formation of stalled translation initiation complexes.
| S-EPMC361207 | biostudies-other
Project description:BackgroundReverse-engineering regulatory networks is one of the central challenges for computational biology. Many techniques have been developed to accomplish this by utilizing transcription factor binding data in conjunction with expression data. Of these approaches, several have focused on the reconstruction of the cell cycle regulatory network of Saccharomyces cerevisiae. The emphasis of these studies has been to model the relationships between transcription factors and their target genes. In contrast, here we focus on reverse-engineering the network of relationships among transcription factors that regulate the cell cycle in S. cerevisiae.ResultsWe have developed a technique to reverse-engineer networks of the time-dependent activities of transcription factors that regulate the cell cycle in S. cerevisiae. The model utilizes linear regression to first estimate the activities of transcription factors from expression time series and genome-wide transcription factor binding data. We then use least squares to construct a model of the time evolution of the activities. We validate our approach in two ways: by demonstrating that it accurately models expression data and by demonstrating that our reconstructed model is similar to previously-published models of transcriptional regulation of the cell cycle.ConclusionOur regression-based approach allows us to build a general model of transcriptional regulation of the yeast cell cycle that includes additional factors and couplings not reported in previously-published models. Our model could serve as a starting point for targeted experiments that test the predicted interactions. In the future, we plan to apply our technique to reverse-engineer other systems where both genome-wide time series expression data and transcription factor binding data are available.
| S-EPMC1570153 | biostudies-literature
Project description:Somatic mutations contribute to the development of age-associated disease. In earlier work, we found that, at high frequency, aging Saccharomyces cerevisiae diploid cells produce daughters without mitochondrial DNA, leading to loss of respiration competence and increased loss of heterozygosity (LOH) in the nuclear genome. Here we used the recently developed Mother Enrichment Program to ask whether aging cells that maintain the ability to produce respiration-competent daughters also experience increased genomic instability. We discovered that this population exhibits a distinct genomic instability phenotype that primarily affects the repeated ribosomal RNA gene array (rDNA array). As diploid cells passed their median replicative life span, recombination rates between rDNA arrays on homologous chromosomes progressively increased, resulting in mutational events that generated LOH at >300 contiguous open reading frames on the right arm of chromosome XII. We show that, while these recombination events were dependent on the replication fork block protein Fob1, the aging process that underlies this phenotype is Fob1-independent. Furthermore, we provide evidence that this aging process is not driven by mechanisms that modulate rDNA recombination in young cells, including loss of cohesion within the rDNA array or loss of Sir2 function. Instead, we suggest that the age-associated increase in rDNA recombination is a response to increasing DNA replication stress generated in aging cells.
| S-EPMC3060066 | biostudies-literature