Project description:The structural complexity of nucleosomes underlies their functional versatility. Here we report a new type of complexity – nucleosome fragility, manifested as high sensitivity to micrococcal nuclease, in contrast to the common presumption that nucleosomes are similar in resistance to MNase digestion. Using differential MNase digestion of chromatin and high-throughput sequencing, we have identified a special group of nucleosomes termed fragile nucleosomes throughout the yeast genome, nearly one thousand of which are at previously determined “nucleosome free” loci. Nucleosome fragility is broadly implicated in multiple chromatin processes, including transcription, translocation and replication, in correspondence to specific physiological states of cells. In the environmental-stress-response genes, the presence of fragile nucleosomes prior to the occurrence of environmental changes suggests that nucleosome fragility poises genes for swift up-regulation in response to the environmental changes. We propose that nucleosome fragility underscores distinct functional statuses of the chromatin and provides a new dimension for portraying the landscape of genome organization.
Project description:Using RNA-Seq analysis of nonsense-mediated mRNA decay (NMD) mutant strains, we show that many Saccharomyces cerevisiae intron-containing genes exhibit usage of alternative splice sites, but most transcripts generated by splicing from these sites are non-functional because they introduce premature termination codons leading to transcript degradation by NMD. Analysis of splicing mutants combined with NMD inactivation revealed the role of specific splicing factors in governing the use of these alternative splice sites and identified novel functions for Prp17p in enhancing the use of branchpoint-proximal upstream 3’ splice sites and for Prp18p in suppressing the usage of a non-canonical AUG 3’-splice site. The use of non-productive alternative splice sites can limit the expression of some transcripts and can be increased in stress conditions in a promoter-dependent manner, contributing to the down-regulation of genes during stress. These results reveal that alternative splicing is frequent in S.cerevisiae but masked by RNA degradation and that the use of alternative splice sites is mostly aimed at controlling transcript levels rather than increasing proteome diversity.
Project description:The structural complexity of nucleosomes underlies their functional versatility. Here we report a new type of complexity – nucleosome fragility, manifested as high sensitivity to micrococcal nuclease, in contrast to the common presumption that nucleosomes are similar in resistance to MNase digestion. Using differential MNase digestion of chromatin and high-throughput sequencing, we have identified a special group of nucleosomes termed fragile nucleosomes throughout the yeast genome, nearly one thousand of which are at previously determined “nucleosome free” loci. Nucleosome fragility is broadly implicated in multiple chromatin processes, including transcription, translocation and replication, in correspondence to specific physiological states of cells. In the environmental-stress-response genes, the presence of fragile nucleosomes prior to the occurrence of environmental changes suggests that nucleosome fragility poises genes for swift up-regulation in response to the environmental changes. We propose that nucleosome fragility underscores distinct functional statuses of the chromatin and provides a new dimension for portraying the landscape of genome organization. Comparing nucleosome occupancy under different MNase digestion levels and growth conditions.
Project description:Using RNA-Seq analysis of nonsense-mediated mRNA decay (NMD) mutant strains, we show that many Saccharomyces cerevisiae intron-containing genes exhibit usage of alternative splice sites, but most transcripts generated by splicing from these sites are non-functional because they introduce premature termination codons leading to transcript degradation by NMD. Analysis of splicing mutants combined with NMD inactivation revealed the role of specific splicing factors in governing the use of these alternative splice sites and identified novel functions for Prp17p in enhancing the use of branchpoint-proximal upstream 3M-bM-^@M-^Y splice sites and for Prp18p in suppressing the usage of a non-canonical AUG 3M-bM-^@M-^Y-splice site. The use of non-productive alternative splice sites can limit the expression of some transcripts and can be increased in stress conditions in a promoter-dependent manner, contributing to the down-regulation of genes during stress. These results reveal that alternative splicing is frequent in S.cerevisiae but masked by RNA degradation and that the use of alternative splice sites is mostly aimed at controlling transcript levels rather than increasing proteome diversity. mRNA-Seq profiling of 3 mutants in the nonsense-mediated mRNA decay pathway and wildtype yeast
Project description:The accumulation of unfolded proteins in the lumen of the endoplasmic reticulum (ER) causes stress and induces the unfolded protein response (UPR) which is characterised in part by the transcriptional induction of genes involved in assisting protein folding. Translational responses to ER stress have been less well described and here we report on a genome-wide analysis of translational regulation in the response to the ER stress-inducing agent dithiothreitol (DTT) in Saccharomyces cerevisiae. Although the observed polysome profiles were similar under control and ER stress conditions microarray analysis identified transcipt-specific translational regulation. Genes with functions in ribosomal biogenesis and assembly were translationally repressed under ER stress. In contrast mRNAs for known UPR genes, including the UPR transcription factor HAC1, the ER-oxidoreductase ERO1 and the ER-associated protein degradation (ERAD) gene DER1 were enriched in polysomal fractions under ER stress conditions. In addition, we show that splicing of HAC1 mRNA is required for efficient ribosomal loading and that Gcn2p is required for normal HAC1 splicing, so shedding light on the role of this protein kinase in the UPR pathway. Keywords: stress response, translational analysis
Project description:While the core splicing machinery is highly conserved between budding yeast and mammals, the absence of alternative splicing in Saccharomyces cerevisiae raises the fundamental question of why introns have been retained in ~5% of the 6,000 genes. Because Ribosomal Protein-encoding Genes (RPGs) are highly over-represented in the set of intron-containing genes, we tested the hypothesis that splicing of these transcripts would be regulated under conditions where translation is impaired. Using a microarraybased strategy, we find that within minutes after the induction of amino acid starvation the splicing of the majority of RPGs is specifically inhibited. In response to an unrelated stress, exposure to toxic levels of ethanol, splicing of a different group of transcripts is inhibited, while the splicing of a third set is actually improved. We propose that regulation of splicing, like transcription, can afford rapid and specific changes in gene expression in response to the environment. Keywords: stress, environmental stress, time course, splicing, splicing-specific microarray
Project description:Reactive oxygen species, generated in vivo or exogenously encountered, constantly challenge living organisms. Oxidation of polyunsaturated fatty acids (PUFA), which are susceptible to oxidant attack, can lead to initiation of lipid peroxidation and in turn rapid production of toxic lipid hydroperoxides. Eukaryotic microorganisms such as Saccharomyces cerevisiae can survive harsh industrial conditions that contain high levels of the PUFA linoleic acid and its oxidised derivative, linoleic acid hydroperoxide (LoaOOH). The precise signalling and response mechanisms induced by yeast to overcome lipid hydroperoxide stress are ill understood. We used genome-wide microarrays to investigate the changes in gene expression of S. cerevisiae to LoaOOH-induced oxidative stress.