Project description:Reprogramming a non-methylotrophic industrial host, such as Saccharomyces cerevisiae, to a synthetic methylotroph reprents a huge challenge due to the complex regulation in yeast. Through TMC strategy together with ALE strategy, we completed a strict synthetic methylotrophic yeast that could use methanol as the sole carbon source. However, how cells respond to methanol and remodel cellular metabolic network on methanol were not clear. Therefore, genome-scale transcriptional analysis was performed to unravel the cellular reprograming mechanisms underlying the improved growth phenotype.
Project description:Cells adjust their transcriptional state to accommodate environmental and genetic perturbations. An open question is to what extent transcriptional response to perturbations has been specifically selected along evolution. To test the possibility that transcriptional reprogramming does not need to be 'pre-designed' in order to lead to an adaptive metabolic state on physiological time scales, we confronted yeast cells with a novel challenge they had not previously encountered. We rewired the genome by recruiting an essential gene, HIS3 from the histidine biosynthesis pathway to a foreign regulatory system, the GAL network responsible for galactose utilization. Switching medium to glucose in a chemostat caused repression of the essential gene and presented the cells with a severe challenge. Using genome-wide expression arrays we show that a global transcriptional reprogramming (>1200 genes) is induced immediately following the switch into the challenging environment, allowing adaptation of the cell population. A larger environmental pressure applied directly on the recruited HIS3 gene by a competitive inhibitor, led to significantly larger expression correlations among hundreds of genes residing in different functional modules, showing that the global transcriptional response underlies the adaptation process. The correlated transcriptional pattern relaxed to the adaptive state over a long period (~10 generations). Our results show that transcriptional plasticity, involving an enhanced response of a sizeable fraction of the genome, is a natural property of the regulatory network allowing it to overcome unforeseen challenges. Keywords: time course, chemostat
Project description:The vast landscape of RNA-protein interactions at the heart of post-transcriptional regulation remains largely unexplored. Indeed it is likely that, even in yeast, a substantial fraction of the regulatory RNA-binding proteins (RBPs) remain to be discovered. Systematic experimental methods can play a key role in discovering these RBPs - most of the known yeast RBPs lack RNA-binding domains that might enable this activity to be predicted. We describe here a new proteome-wide approach to identify RNA-protein interactions based on in vitro binding of RNA samples to yeast protein microarrays that represent over 80% of the yeast proteome. We used this procedure to screen for novel RBPs and RNA-protein interactions. A complementary mass spectrometry technique also identified proteins that associate with yeast mRNAs. Both the protein microarray and mass spectrometry methods successfully identify previously annotated RBPs, suggesting that other proteins identified in these assays might be novel RBPs. Of 35 putative novel RBPs identified by either or both of these methods, 12, including 75% of the eight most highly-ranked candidates, reproducibly associated with specific cellular RNAs. Surprisingly, most of the 12 newly discovered RBPs were enzymes. Functional characteristics of the RNA targets of some of the novel RBPs suggest coordinated post-transcriptional regulation of subunits of protein complexes and a possible link between mRNA trafficking and vesicle transport. Our results suggest that many more RBPs still remain to be identified and provide a set of candidates for further investigation.
Project description:The vast landscape of RNA-protein interactions at the heart of post-transcriptional regulation remains largely unexplored. Indeed it is likely that, even in yeast, a substantial fraction of the regulatory RNA-binding proteins (RBPs) remain to be discovered. Systematic experimental methods can play a key role in discovering these RBPs - most of the known yeast RBPs lack RNA-binding domains that might enable this activity to be predicted. We describe here a new proteome-wide approach to identify RNA-protein interactions based on in vitro binding of RNA samples to yeast protein microarrays that represent over 80% of the yeast proteome. We used this procedure to screen for novel RBPs and RNA-protein interactions. A complementary mass spectrometry technique also identified proteins that associate with yeast mRNAs. Both the protein microarray and mass spectrometry methods successfully identify previously annotated RBPs, suggesting that other proteins identified in these assays might be novel RBPs. Of 35 putative novel RBPs identified by either or both of these methods, 12, including 75% of the eight most highly-ranked candidates, reproducibly associated with specific cellular RNAs. Surprisingly, most of the 12 newly discovered RBPs were enzymes. Functional characteristics of the RNA targets of some of the novel RBPs suggest coordinated post-transcriptional regulation of subunits of protein complexes and a possible link between mRNA trafficking and vesicle transport. Our results suggest that many more RBPs still remain to be identified and provide a set of candidates for further investigation. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc.
Project description:Genetic and environmental stresses are known factors that influence an organisms phenotype. Time is rarely taken into consideration when studying phenotypic changes in cells. Time-resolved microarray data revealed genome-wide transcriptional changes in yeast strain CEN.PK122 oscillating with~2 periods. We mapped the global patterns of transcriptional oscillationsinto a 3-dimensional map to represent different cellular phenotypes ofoscillation period. This map shows the dynamic nature of transcripts through time and concentration space, and that they are ordered and coupled to each other.
Project description:We analyzed genome-wide transcriptional changes in the S. cerevisiae strain BY4742 upon exposure to NP to identify differentially expressed genes, biological processes, metabolic pathways, and cellular compartments affected by this compound. For these analyses, we focused on two NP exposure scenarios: (1) exposure to a low inhibitory concentration (resulting in <15% reduction in cell number), and (2) exposure to a high inhibitory concentration (resulting in >65% reduction in cell number).
Project description:Oxidative stress is experienced by all aerobic organisms and results in cellular damage. The damage caused during oxidative stress is particular to the oxidant challenge faced, and so too is the induced stress response. The eukaryote Saccharomyces cerevisiae is sensitive to low concentrations of the lipid hydroperoxide - linoleic acid hydroperoxide (LoaOOH) - and its response is unique relative to other peroxide treatments. Part of the yeast response to LoaOOH includes a change in the cellular requirement for nutrients, such as sulfur, nitrogen and various metal ions. The metabolism of sulfur is involved in antioxidant defence, although the role nitrogen during oxidative stress is not well understood. Investigating the response induced by yeast to overcome LoaOOH exposure, with a particular focus on nitrogen metabolism, will lead to greater understanding of how eukaryotes survive lipid hydroperoxide-induced stress, and associated lipid peroxidation, which occurs in the presence of polyunsaturated fatty acids. We used genome-wide microarrays to investigate the changes in gene expression of S. cerevisiae (Dal80Δ) to LoaOOH-induced oxidative stress.