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Protein Kinase A Regulates Gene-Specific Translational Adaptation in Differentiating Yeast

ABSTRACT: Cellular differentiation is driven by coordinately regulated changes in gene expression. Translational control of gene expression is increasingly recognized as pervasive and quantitatively significant, but the mechanisms responsible for widespread changes in gene-specific translation activity are largely unknown. Here we investigate the mechanisms responsible for translational reprogramming during cellular adaptation to the absence of glucose, a stimulus that induces invasive filamentous differentiation in yeast. We show that gene-specific translation efficiencies are highly adapted to cellular conditions and that glucose withdrawal is accompanied by widespread translational reprogramming at the level of translation initiation. We demonstrate that transcripts from <5% of genes make up the majority of translating mRNA in both rapidly dividing and starved cells. Moreover, the identities of these highly translated genes are growth-state specific, and they are subject to condition-dependent translational privilege. By comparing glucose starvation to other growth-attenuating stresses, we distinguish a glucose-specific translational response that regulates ribosomal protein and mitochondrial protein-coding genes. This response is mediated through signaling by protein kinase A (PKA). These findings reveal a high degree of growth-state specialization of the translatome and identify PKA as an important regulator of gene-specific translation activity. Examine translational adaptation in yeast in response to glucose starvation

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Pavanapuresan Vaidyanathan

PROVIDER: E-GEOD-51532 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Protein Kinase A Regulates Gene-Specific Translational Adaptation in Differentiating Yeast

Project description:Cellular differentiation is driven by coordinately regulated changes in gene expression. Translational control of gene expression is increasingly recognized as pervasive and quantitatively significant, but the mechanisms responsible for widespread changes in gene-specific translation activity are largely unknown. Here we investigate the mechanisms responsible for translational reprogramming during cellular adaptation to the absence of glucose, a stimulus that induces invasive filamentous differentiation in yeast. We show that gene-specific translation efficiencies are highly adapted to cellular conditions and that glucose withdrawal is accompanied by widespread translational reprogramming at the level of translation initiation. We demonstrate that transcripts from <5% of genes make up the majority of translating mRNA in both rapidly dividing and starved cells. Moreover, the identities of these highly translated genes are growth-state specific, and they are subject to condition-dependent translational privilege. By comparing glucose starvation to other growth-attenuating stresses, we distinguish a glucose-specific translational response that regulates ribosomal protein and mitochondrial protein-coding genes. This response is mediated through signaling by protein kinase A (PKA). These findings reveal a high degree of growth-state specialization of the translatome and identify PKA as an important regulator of gene-specific translation activity.

2014-04-25 | GSE51532 | GEO

Loss of a Conserved tRNA Anticodon Modification Perturbs Cellular Signaling

Project description:Transfer RNA (tRNA) modifications enhance the efficiency, specificity and fidelity of translation in all organisms. The anticodon modification mcm5s2U34 is required for normal growth and stress resistance in yeast; mutants lacking this modification have numerous phenotypes. Mutations in the homologous human genes are linked to neurological disease. The yeast phenotypes can be ameliorated by overexpression of specific tRNAs, suggesting that the modifications are necessary for efficient translation of specific codons. We determined the in vivo ribosome distributions at single codon resolution in yeast strains lacking mcm5s2U. We found accumulations at AAA, CAA, and GAA codons, suggesting that translation is slow when these codons are in the ribosomal A site, but these changes appeared too small to affect protein output. Instead, we observed activation of the GCN4-mediated stress response by a non- canonical pathway. Thus, loss of mcm5s2U causes global effects on gene expression due to perturbation of cellular signaling. WT yeast and mutants lacking anticodon tRNA modifications were grown in YPD, and subjected to ribosome footprint profiling (ribo-seq) and RNA-seq of poly-A selected RNA. Dataset contains biological replicates for WT, Δncs6 and Δuba4. Technical replicates were also performed for all RNA-seq datasets (using a different poly-A selection method).

2013-08-01 | E-GEOD-45366 | biostudies-arrayexpress

N6-Methyladenosine Guides mRNA Alternative Translation during Integrated Stress Response

Project description:The integrated stress response (ISR) facilitates cellular adaptation to a variety of stress conditions via phosphorylation of the common target eIF2α. During ISR, the translation of certain stress-related mRNAs is upregulated in spite of global suppression of protein synthesis.The selective translation often relieson alternative mechanisms, such as leaky scanning or reinitiation, but the underlying mechanism remains incompletely understood. Here we report that,in response to amino acid starvation, the reinitiation of ATF4 is not only governed by eIF2α-controlled ternary complex availability, but is also subjected to regulation by mRNA methylation in the form of N6-methyladenosine (m6A). We demonstrate that m6A in the 5' untranslated region (5’ UTR) controls ribosome scanning and subsequent start codonselection. Global profiling of initiating ribosomes reveals widespread alternative translation events influenced by mRNA methylation. Consistently, Fto-transgenic mice manifest enhanced ATF4 expression, highlighting the critical role of 5’ UTR methylation in translational regulation of ISR at cellular and organismal levels.

2017-08-15 | GSE102659 | GEO

Stress-induced translation inhibition through release of 40S scanning initiation factors

Project description:Cellular responses to environmental stress are frequently mediated by RNA-binding proteins (RBPs). Here, we examined global RBP dynamics in Saccharomyces cerevisiae in response to glucose starvation and heat shock. Each stress induced rapid remodeling of the RNA-protein interactome, without corresponding changes in RBP abundance. Consistent with general translation shutdown, ribosomal proteins contacting the mRNA showed decreased RNA-association. Among translation components, RNA-association was most reduced for initiation factors involved in 40S scanning (eIF4A, eIF4B, and Ded1), indicating a common mechanism of translational repression. In unstressed cells, eIF4A, eIF4B, and Ded1 primarily targeted the 5′-ends of mRNAs. Following glucose withdrawal, 5’-binding was abolished within 30sec, explaining the rapid translation shutdown, but mRNAs remained stable. Heat shock induced progressive loss of 5’ RNA-binding by initiation factors over ~16min. Translation shutoff provoked selective 5′-degradation of mRNAs encoding translation-related factors, mediated by Xrn1. These results reveal mechanisms underlying translational control of gene expression during stress.

2020-06-08 | GSE148166 | GEO

Gcn2 eIF2a kinase mediates combinatorial translational regulation through nucleotide motifs and uORFs in the mRNA 5’ leader regions

Project description:Cellular signaling controls translation through cis regulatory elements found in mRNAs. Gcn2 is the master regulator of translation during nutrient limitation in normal and cancer cells. Activated Gcn2 phosphorylates eIF2a, thereby repressing general translation while activating translation of specific mRNAs with upstream ORFs (uORFs) in its leader regions. Here we performed genome-wide measurement of mRNA translation during histidine starvation in fission yeast Schizosaccharomyces pombe. Polysomal microarray hybridization experiments identified a group of 1779 genes whose translation is up-regulated in Gcn2-dependent manner. We find that translation is reprogrammed to enhance organellar synthesis and transcription and repress ribosome synthesis. The 1779 genes included gcn5 and hri2 shown to promote growth under histidine starvation. They encode histone acetyl transferase and heme-binding eIF2a kinase activated by oxidative stress, and their 5’-leader contains 3 or 4 uORFs, respectively. Our reporter studies show that uORFs in gcn5 and hri2 operate similarly to those found in S. cerevisiae GCN4, the founding case for uORF-dependent regulation. Moreover, motif analysis identified 5’-UGA(C/G)GG-3’ as a motif promoting translation during starvation. Using hrd1 5’UTR with such a motif, we demonstrated its requirement in Gcn2-dependent translational control. To our knowledge, this is the first nucleotide motif found to be responsible for translational control by Gcn2. We propose that Gcn2 mediates translational control of more specific mRNAs than previously anticipated

2020-09-23 | GSE143299 | GEO

Ribosome profiling upon glucose starvation in S. cerevisiae

Project description:A universal feature of the response to stress and nutrient limitation is transcriptional upregulation of genes encoding proteins important for survival. Interestingly, under many of these conditions overall protein synthesis levels are reduced, thereby dampening the stress response at the level of protein expression. For example, during glucose starvation in yeast, translation is rapidly and reversibly repressed, yet transcription of many stress- and glucose-repressed genes is increased. Using ribosome profiling and microscopy, we found that this transcriptionally upregulated gene set consists of two classes: (1) one producing mRNAs that are preferentially translated during glucose limitation and are diffusely localized in the cytoplasm – this class includes many heat shock protein mRNAs; and (2) another producing mRNAs that are poorly translated during glucose limitation, have high rates of translation initiation, and are concentrated in foci that co-localize with P bodies and stress granules – this class is enriched for glucose metabolism mRNAs. Remarkably, the information specifying differential localization and translation of these two classes of mRNAs is encoded in the promoter sequence – promoter responsiveness to heat shock factor (Hsf1) specifies diffuse cytoplasmic localization and preferential translation upon glucose starvation, whereas different promoter elements upstream of genes encoding poorly translated glucose metabolism mRNAs direct these mRNAs to RNA granules under glucose starvation. Thus, promoter sequences and transcription factor binding can influence not only mRNA levels, but also subcellular localization of mRNAs and the efficiency with which they are translated, enabling cells to tailor protein production to environmental conditions. Examination of mRNA translation in S. cerevisiae upon glucose starvation.

2014-07-28 | E-GEOD-56622 | biostudies-arrayexpress

The Small Molecule ISRIB Reverses the Effects of eIF2α Phosphorylation on Translation and Stress Granule Assembly

Project description:We recently identified ISRIB as a potent inhibitor of the integrated stress response (ISR). ISRIB renders cells resistant to the effects of eIF2α phosphorylation and enhances long-term memory in rodents (10.7554/eLife.00498). Here we show by genome-wide in vivo ribosome profiling that translation of a restricted subset of mRNAs is induced upon ISR activation. ISRIB substantially reversed the translational effects elicited by phosphorylation of eIF2α and induced no major changes in translation or mRNA levels in unstressed cells. eIF2α phosphorylation-induced stress granule (SG) formation was blocked by ISRIB. Strikingly, ISRIB addition to stressed cells with pre-formed SGs induced their rapid disassembly, liberating mRNAs into the actively translating pool. Restoration of mRNA translation and modulation of SG dynamics may be an effective treatment of neurodegenerative diseases characterized by eIF2α phosphorylation, SG formation and cognitive loss. Ribosome profiling with paired RNA-seq

2015-02-27 | E-GEOD-65778 | biostudies-arrayexpress

Ribosome profiling and RNA-seq of an acute glucose starvation timecourse and 5 day growth course in S. cerevisiae

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.

2022-04-11 | GSE200491 | GEO

The Rim15p homolog RimO mediates starvation response and is required for secondary metabolite production in Aspergillus nidulans

Project description:Depending on the prevailing environmental, developmental and nutritional conditions Aspergillus nidulans produces different combinations of secondary metabolites (SMs). To activate the biosynthetic gene cluster (BGC) for a given SM global, chromatin-based de-repression must be integrated with pathway-specific induction signals. Here we describe a new global regulator affecting the set of starvation-induced SMs. We found a hitherto uncharacterized putative PAS-domain containing kinase to be upregulated in SM-producing cultures. The predicted protein is highly similar to yeast Rim15 which transmits nutritional and stress signals to downstream effectors involved in chromatin regulation, stress and starvation response. The putative kinase – termed RimO – is required for the transcription of a number of starvation-induced SMs including sterigmatocystin (ST). RimO regulates the pathway-specific transcription factor AflR both at the transcriptional and post-translational level. In the absence of RimO aflR is barely transcribed under SM conditions and the protein is not retained in the nucleus. Genome-wide transcriptional profiling showed that cells lacking rimO fail to correctly respond to carbon and nitrogen starvation and continue to transcribe genes of the basic metabolism to a high level. Conversely, overexpression of the kinase made cells largely insensitive to glucose repression and led to overproduction of several secondary metabolites. We propose a model that positions RimO downstream of PKA being necessary to transmit the starvation signal to downstream targets, including chromatin and transcriptional regulators of SM gene clusters. Additionally, it may also regulate signaling modules required for recognizing the starvation response.

2022-03-01 | GSE184618 | GEO

Widespread and dynamic translational control of red blood cell development

Project description:Cell development requires tight yet dynamic control of protein production. Here, we use murine erythropoiesis as a model to study translational regulatory dynamics during mammalian cell differentiation. We uncover pervasive translational control of protein synthesis, including widespread alternative translation initiation and termination, stoichiometric synthesis, and dynamic use of upstream reading frames. We thus unravel translational regulatory programs during erythropoiesis, comprising hundreds of mRNAs with dynamic translation efficiencies and their cell type-specific regulators. A major such program involves enhanced decoding of specific mRNAs depleted in terminally differentiating/enucleating cells with decreasing transcriptional capacity. Rbm38, an erythroid-specific RNA-binding protein, promotes translation of many such genes by recruiting the translation initiation factor eIF4G. Inhibiting Rbm38 blocks red cell production, and mice lacking Rbm38 develop anemia, demonstrating an essential role for Rbm38-mediated enhanced translation of irreplaceable mRNAs. These findings reveal critical roles for dynamic translational control in supporting specialized mammalian cell formation.

2016-12-01 | GSE83823 | GEO

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