Measurements of mRNA abundance and decay for two strains 211 (wt) and 212 (mutant)
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ABSTRACT: For each strain two time courses for mRNA abundance: Oxidative and MMS and two time courses for decay: reference decay and following oxidative stress We used Affymetrix microarrays to quantify changes in mRNA abundance following oxidative stress and DNA damage stress and also decay following transcription inhibition with and without oxidative stress. Each of the experiments were done for both strains 211 (wt) and 212 (mutant)
Project description:This SuperSeries is composed of the following subset Series:; GSE12220: Changes in Saccharomyces cerevisiae mRNA abundance following oxidative stress and DNA damage stress; GSE12221: Decay profiles of Saccharomyces cerevisiae mRNAs following oxidative stress and DNA damage Experiment Overall Design: Refer to individual Series
Project description:Analysis of mRNA stability in S. cerevisiae in connection to hyperosmotic stress at different time points following mild hyperosmotic shock in Saccharomyces cerevisiae cells.
Project description:We subjected yeast to two stresses, oxidative stress, which under current settings induces a fast and transient response in mRNA abundance, and DNA damage, which triggers a slow enduring response. Using microarrays, we performed a transcriptional arrest experiment to measure genome-wide mRNA decay profiles under each condition. Genome-wide decay kinetics in each condition were compared to decay experiments that were performed in a reference condition (only transcription inhibition without an additional stress) to quantify changes in mRNA stability in each condition. We found condition-specific changes in mRNA decay rates and coordination between mRNA production and degradation. In the transient response, most induced genes were surprisingly destabilized, while repressed genes were somewhat stabilized, exhibiting counteraction between production and degradation. This strategy can reconcile high steady-state level with short response time among induced genes. In contrast, the stress that induces the slow response displays the more expected behavior, whereby most induced genes are stabilized, and repressed genes destabilized. Our results show genome-wide interplay between mRNA production and degradation, and that alternative modes of such interplay determine the kinetics of the transcriptome in response to stress. Experiment Overall Design: We used Affymetrix microarrays to measure the decay profiles of all genes following transcription inhibition in four separate experiments. In two reference experiments, only transcription inhibtion was applied. In two other experiments, an additional stress was applied prior to transcription inhibition: oxidative stress (0.3mM hydrogen peroxide) or DNA damage (0.1% methyl methanesulfonate).
Project description:For each strain two time courses for mRNA abundance: Oxidative and MMS and two time courses for decay: reference decay and following oxidative stress
Project description:We subjected yeast to two stresses, oxidative stress, which under current settings induces a fast and transient response in mRNA abundance, and DNA damage, which triggers a slow enduring response. Using microarrays we performed a conventional quantification of change in mRNA abundance. Experiment Overall Design: We used Affymetrix microarrays to quantify changes in mRNA abundance following oxidative stress (using hydrogen peroxide) and DNA damage stress (using methyl methanesulfonate) during a three-hour time course (0, 30 min, 60 min, 100 min, 140 min, 180 min).
Project description:Precise control of mRNA decay is fundamental for robust yet not exaggerated inflammatory responses to pathogens. Parameters determining the specificity and extent of mRNA degradation within the entire inflammation-associated transcriptome remain incompletely understood. Using transcriptome-wide high resolution occupancy assessment of the mRNA-destabilizing protein TTP, a major inflammation-limiting factor, we qualitatively and quantitatively characterize TTP binding positions and functionally relate them to TTP-dependent mRNA decay in immunostimulated macrophages. We identify pervasive TTP binding with incompletely penetrant linkage to mRNA destabilization. A necessary but not sufficient feature of TTP-mediated mRNA destabilization is binding to 3â?? untranslated regions (UTRs). Mapping of binding positions of the mRNA-stabilizing protein HuR in activated macrophages revealed that TTP and HuR binding sites in 3â?? UTRs occur mostly in different transcripts implicating only a limited co-regulation of inflammatory mRNAs by these proteins. Remarkably, we identify robust and widespread TTP binding to introns of stable transcripts. Nuclear TTP is associated with spliced-out introns and maintained in the nucleus throughout the inflammatory response. Our study establishes a functional annotation of binding positions dictating TTP-dependent mRNA decay in immunostimulated macrophages. The findings allow navigating the transcriptome-wide landscape of RNA elements controlling inflammation. Experiment comparing RNA decay rates in WT and TTP-/- macrophages at LPS 3 h and 6 h. Transcription was blocked with actinomycin D for 0, 45 or 90 min. Decay rates was calculated using linear model.
Project description:We measured mRNA levels of two yeast species (S.cerevisiae and S.paradoxus) and their hybrid, at four time-points (0, 20min, 40min, 60min) following transcription arrest using 1,10-Phenantroline (150ug/ml). This data was used to infer mRNA degradation rates of orthologous genes, study the divergence of mRNA degradation rates and the contribution of cis and trans mutations. For each of the two biological repeats and each of the four time point, poly(A) mRNAs of the two species was pooled and labeled with cy3 while hybrid poly(A) mRNA was labeled with cy5 and these were hybridized to our custom two-species microarray (Agilent) with four subarrays.
Project description:We estimated global mRNA half-lives by blocking transcription and measuring mRNA levels at different times after transcriptional shut-off.
Project description:Dinoflagellates have evolved a nuclear organization unlike that of any other eukaryotic group. Recent studies find a predominance of post-transcriptional control of dinoflagellate gene expression. This study investigated regulation of the environmental stress response in the red tide dinoflagellate, Karenia brevis using an Agilent custom oligonucleotide microarray. K. brevis cultures were exposed to 5°C or 10°C heat shock, or three different sources of oxidative stress: 60 µM H2O2, 10 mM NaNO2, or 12 µM PbCl2 over acute time courses. Ribosomal genes, genes involved in RNA processing, translation, and chaperones were among the classes of genes consistently downregulated across treatments, although within these functional classes the same genes did not always respond to different stressors. Genes involved in the photosystem and mitochondrial and chloroplast ATP generation dominated the down-regulated genes. Heat shock and oxidative stress response genes were not induced under any treatment, even under conditions that resulted in decreased viability. We subsequently identified the presence of a trans-spliced leader sequence on many stress response gene transcripts, which suggests that they may be transcribed constitutively and their expression regulated at the level of translation. Cultures of were grown to mid-log phase. For each treatment, five replicate untreated control cultures and five replicate treated cultures were harvested at several time points following treatment. The following time courses were used: 5°C heat shock - 5, 15, 30, 60, and 240 min; 10°C heat shock - 60 min; 60 µM H2O2 - 5, 15, 30, 60, and 240 min; 10 mM NaNO2 -1, 4, and 7 hours; 12 µM PbCl2 -1, 4, and 7 hours. For each treatment, RNA was pooled from the controls and treated cultures at each timepoint. Two color arrays were then run comparing each the transcriptome at timepoint with the pooled control for that treatment. A technical dye swap array was run at each timepoint.
Project description:Human articular chondrocytes were isolated from normal or osteoarthritic tissue. RNA decay was measured across the transcriptome in these cells by microarray analysis following an actinomycin D chase for 0, 1, 3 and 5 hours. Normalisation was conducted by quantile normalising each set of four decay curve points (i.e. 0, 1, 3 and 5 hour samples for a given donor's cells) independently of the other data. This meant that each decay curve is normalised independently of the others.