Glucose-depletion time-course experiment in Saccharomyces cerevisiae wild-type cells.
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE33098 | GEO | 2012/06/18
SECONDARY ACCESSION(S): PRJNA156579
REPOSITORIES: GEO
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Project description:To investigate the glucose regulatory system in Saccharomyces cerevisiae, we conducted a time-course in which glucose-depleted wildtype (WT) cells were inoculated into fresh media (SC, 2% glucose). Their subsequent transcriptional output was monitored over a period of five hours by DNA microarrays: samples for gene expression profiling were taken immediately after, as well as 3, 7.5, 15, 30, 60, 110, 150, and 300 minutes after inoculation into fresh medium. Transcripts upregulated are involved in translational processes such as the GO biological processes “ribosome biogenesis” and “ribosome localization”. Transcripts downregulated are enriched for the GO biological processes “cellular respiration” and various metabolism related processes. The time-course was used to verify the physiological relevance of gene expression profiles determined for individual deletions of glucose regulatory system components. Importantly, transcripts up- or downregulated in WT cells upon the addition of glucose are similarly up- or downregulated in deletion mutants that each lack a component of the glucose regulatory system. RNA isolated from a large amount of wt yeast from a single culture was used as a common reference. This common reference was used for each separate hybridization and used in the statistical analysis to obtain an average expression-profile for each deletion mutant relative to the wt. Two independent cultures were hybridized on two separate microarrays. For the first hybridization the Cy5 (red) labeled cRNA from the deletion mutant is hybridized together with the Cy3 (green) labeled cRNA from the common reference. For the replicate hybridization, the labels are swapped. Each gene is represented twice on the microarray, resulting in four measurements per mutant. Two overnight WT cultures were used to inoculate 50 ml cultures at an OD600 of 0.15. These were depleted of glucose by growing for 24 hours and were used the next day to inoculate 500 ml cultures in fresh medium to an OD600 of 0.15. Samples for expression profiling were taken immediately after, as well as 3, 7.5, 15, 30, 60, 110, 150, and 300 minutes after inoculation into fresh medium.
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Project description:Ribosome profiling provides an opportunity to not only assess how the relative abundance of ribosome association with mRNAs changes in different conditions, but to look more closely at where along mRNAs those ribosomes bind. Here, we used ribosome profiling to calculate the ribosome polarity scores and changes in ribosome footprint read density in both aggregate and gene-specific ways. We profiled a time course of acute glucose starvation followed by glucose readdition and a multi-day growth course through the diauxic shift into stationary phase. We found that ribosome polarity became positive in postdiauxic shift conditions relative to log phase. In acute starvation, polarity shifted positive at our earliest time points but did not continue to do so at later time points. This is consistent with a read density analysis which demonstrated increased density on the 3’ half of genes after glucose starvation. Additionally, we performed ribosome profiling in samples that had glucose added back following acute starvation and observed a wave of new ribosome movement near the start codon and approximately 2,000 nucleotides downstream on open reading frames after one and five minutes of readdition, respectively. Our ribosome profiling analysis suggested that elongation slows during starvation which leads to a buildup of ribosomes on the 3’ halves of mRNAs. Further, it also indicated ribosomes previously built up can resume translation upon glucose readdition. We used reporter assays to corroborate these findings in vivo. Together, these results demonstrate how yeast regulate translation in response to glucose starvation.
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Project description:Pancreatic beta-cells are specialized for coupling glucose metabolism to insulin peptide production and secretion. Acute glucose exposure robustly and coordinately increases translation of proinsulin and proteins required for secretion of mature insulin peptide. By contrast, chronically elevated glucose levels that occur during diabetes impair beta-cell insulin secretion and have been shown experimentally to suppress insulin translation. Whether translation of other genes critical for insulin secretion are similarly downregulated by chronic high glucose is unknown. Here, we used high-throughput ribosome profiling and nascent proteomics in MIN6 insulinoma cells to elucidate the genome-wide impact of sustained high glucose on beta-cell mRNA translation. Prior to induction of ER stress or suppression of global translation, sustained high glucose suppressed glucose-stimulated insulin secretion and downregulated translation of not only insulin, but also of mRNAs related to insulin secretory granule formation, exocytosis, and metabolism-coupled insulin secretion. Translation of these mRNAs was also downregulated in primary rat and human islets following ex-vivo incubation with sustained high glucose and in an in vivo model of chronic mild hyperglycemia. Furthermore, translational downregulation decreased cellular abundance of these proteins. Our study uncovered a translational regulatory circuit during beta-cell glucose toxicity that impairs expression of proteins with critical roles in beta-cell function.
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Project description:We previously reported that ribosome stalling at AUG-stop sequences in the 5'-UTR plays a critical role in regulating the expression of Arabidopsis thaliana NIP5;1, which encodes a boron uptake transporter, in response to boron conditions in media. Here, we conducted ribosome profiling analysis to reveal the genome-wide regulation of translation in response to boron conditions in A. thaliana. We identified 460 translationally regulated genes. Transcripts with reduced translation efficiency were rich in upstream open reading frames (uORFs), highlighting the importance of uORF-mediated translational regulation. We found that 148 uORF instances had greater ribosome density under high boron conditions. Moreover, translationally downregulated transcripts were rich in minimum uORFs (AUG-stops), suggesting that AUG-stops play a global role in the boron response. Boron increased the ribosome occupancy of stop codons, indicating that this element is involved in global translational termination processes.
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Project description:Cell survival in changing environments requires appropriate regulation of gene expression, including post-transcriptional gene regulatory mechanisms. Based on reporter gene studies in glucose-starved yeast, it was proposed that translationally silenced eukaryotic mRNAs accumulate in P-bodies and can return to active translation. We present evidence contradicting the notion that this model is a widespread and general phenomenon. First, genome-wide measurements of mRNA abundance, translation, and ribosome occupancy following glucose withdrawal show that most mRNAs are lost from the cell coincident with their loss from polysomes. Second, only a very limited sub-population of translationally repressed transcripts, comprising fewer than 400 genes, can be reactivated for translation upon glucose re-addition in the absence of new transcription. This highly selective post-transcriptional regulation could be a mechanism for cells to minimize the energetic costs of reversing gene-regulatory decisions in rapidly changing environments by transiently preserving a pool of transcripts whose translation is rate-limiting for growth. Sigma 1278b yeast were grown to OD600=1.0-1.1 in YPD in baffled flasks at 30C with vigorous shaking. Cells were harvested by centrifugation and resuspended in pre-warmed YPA medium lacking glucose. Cells were returned to 30C for shaking for 10,20,30,60, or 120 minutes.
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Project description:Cell survival in changing environments requires appropriate regulation of gene expression, including post-transcriptional gene regulatory mechanisms. Based on reporter gene studies in glucose-starved yeast, it was proposed that translationally silenced eukaryotic mRNAs accumulate in P-bodies and can return to active translation. We present evidence contradicting the notion that this model is a widespread and general phenomenon. First, genome-wide measurements of mRNA abundance, translation, and ribosome occupancy following glucose withdrawal show that most mRNAs are lost from the cell coincident with their loss from polysomes. Second, only a very limited sub-population of translationally repressed transcripts, comprising fewer than 400 genes, can be reactivated for translation upon glucose re-addition in the absence of new transcription. This highly selective post-transcriptional regulation could be a mechanism for cells to minimize the energetic costs of reversing gene-regulatory decisions in rapidly changing environments by transiently preserving a pool of transcripts whose translation is rate-limiting for growth.
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