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Transcriptome analyses of dhh1 mutants in yeast

ABSTRACT: Previous experiments revealed that DHH1, a RNA helicase involved in the regulation of mRNA stability and translation, complemented the phenotype of a Saccharomyces cerevisiae mutant affected in the expression of genes coding for monocarboxylic-acids transporters, JEN1 and ADY2. In wild type cells, JEN1 expression had been shown to be undetectable in the presence of glucose or formic acid, and induced in the presence of lactate. In this work, we show that JEN1 mRNA accumulates in a dhh1 mutant, when formic acid was used as sole carbon source. Dhh1 interacts with the decapping activator Dcp1 and with the deadenylase complex. This led to the hypothesis that JEN1 expression is post-transcriptionally regulated by Dhh1 in formic acid. Analyses of JEN1 mRNAs decay in wild-type and dhh1 mutant strains confirmed this hypothesis. Microarray analyses of dhh1 mutant indicated that Dhh1 plays a large role in metabolic adaptation, suggesting that carbon source changes triggers a complex interplay between transcriptional and post-transcriptional effects.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE60983 | GEO | 2014/09/08

SECONDARY ACCESSION(S): PRJNA260037

REPOSITORIES: GEO

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Transcriptome analyses of dhh1 mutants in yeast

Project description:Previous experiments revealed that DHH1, a RNA helicase involved in the regulation of mRNA stability and translation, complemented the phenotype of a Saccharomyces cerevisiae mutant affected in the expression of genes coding for monocarboxylic-acids transporters, JEN1 and ADY2. In wild type cells, JEN1 expression had been shown to be undetectable in the presence of glucose or formic acid, and induced in the presence of lactate. In this work, we show that JEN1 mRNA accumulates in a dhh1 mutant, when formic acid was used as sole carbon source. Dhh1 interacts with the decapping activator Dcp1 and with the deadenylase complex. This led to the hypothesis that JEN1 expression is post-transcriptionally regulated by Dhh1 in formic acid. Analyses of JEN1 mRNAs decay in wild-type and dhh1 mutant strains confirmed this hypothesis. Microarray analyses of dhh1 mutant indicated that Dhh1 plays a large role in metabolic adaptation, suggesting that carbon source changes triggers a complex interplay between transcriptional and post-transcriptional effects. We compared gene expression between wild type and dhh1 deleted yeast strains grown either with formate or in glucose as sole carbon source. The experiments were replicated using biologically independant samples with dye swap. A total of four hybridizations were performed.

2014-09-08 | E-GEOD-60983 | biostudies-arrayexpress

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Ribosome profiling of dhh1∆ yeast

Project description:The impact of RNA structures in coding sequences (CDS) within mRNAs is poorly understood. Here  we identify a novel and highly conserved mechanism of translational control involving RNA structures within coding sequences and the DEAD-box helicase Dhh1. Using yeast genetics and  genome-wide ribosome profiling analyses we show that this mechanism, initially derived from studies  of the Brome Mosaic virus RNA genome, extends to yeast and human mRNAs highly enriched in  membrane and secreted proteins. All Dhh1-dependent mRNAs, viral and cellular, share key common  features. First, they contain long and highly structured CDSs, including a region located around 42 to  120 nucleotides after the translation initiation site, second, they are directly bound by Dhh1 with a  specific binding distribution and third, complementary experimental approaches suggest that they are  activated by Dhh1 at the translation initiation step. Our results show that ribosome translocation is not the only unwinding force of CDS and uncover a conserved layer of translational control that involve  RNA helicases and RNA folding within CDS providing novel opportunities for regulation of  membrane and secretome proteins.

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PSY3642_to_PSY3639_SC_aerobic

Project description:We designed and experimentally validated an in silico gene deletion strategy for engineering endogenous one-carbon (C1) metabolism in yeast. Specifically, a Flux Balance Analysis (FBA) model predicted that five genes ALT2, FDH1, FDH2, FUM1, and ZWF1, when deleted in combination, would cause Saccharomyces cerevisiae to overproduce and secrete formic acid under aerobic growth conditions. Once constructed, the quintuple mutant strain showed the predicted increase in formic acid secretion relative to a formate dehydrogenase mutant (fdh1 fdh2 ), while formic acid secretion in wild-type yeast was undetectable. Gene expression and physiological data generated post hoc identified a mitochondrial deficiency phenotype in the engineered strain and regulatory events that suggest a role for multisite modulation—the coordinated expression of multiple pathway enzymes—in controlling yeast C1 metabolism. Together, these results demonstrate that FBA-based modeling strategies can be useful tools for non-fermentative pathway design and discovery in eukaryotic microbes. In addition, the results indicate that seemingly unrelated mutations can be combined to modulate flux through biochemical reactions that interact at a systems level across subcellular compartments. Samples were processed in biological triplicate with one sample processed with dye orientations reversed to minimize the effects of incorporation bias.

2009-07-24 | E-GEOD-12240 | biostudies-arrayexpress

PSY3642_to_PSY3639_SC_aerobic

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Transcription profiling of Trypanosoma brucei DHH1 DEAD:DQAD mutant from pLEW100 for 24 hours

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2010-01-01 | E-MEXP-2026 | biostudies-arrayexpress

Decapping factor Dcp2 controls mRNA abundance and translation to adjust metabolism and filamentation to nutrient availability II

Project description:Degradation of most yeast mRNAs involves decapping by Dcp1/Dcp2. DEAD-box protein Dhh1 has been implicated as an activator of decapping, and as a translational repressor, but their functions in cells are incompletely understood. We have analyzed these questions by a combination of ribosome profiling, RNA-Seq, CAGE analysis of capped mRNAs.

2023-02-07 | GSE220578 | GEO

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Proteomic and transcriptomic analyses of Dhh1 complexes reveals mitochondrial RNP complex association with yeast P-bodies

Project description:P-bodies (PB) are cytoplasmic RNP complexes that aggregate into foci when cells are exposed to stress. While the core components and stress response of PB and related RNP granules are conserved, it remains unclear how and why cells assemble mRNP complexes into granule foci during stress. We use mass spectrometry and antibody-based microarray to analyze proteins and RNA, respectively, that are immunoisolated with the core PB protein Dhh1-GFP. Analysis of the RNA associated with Dhh1-GFP immunoisolate reveals an enrichment of mitochondrial catalytic RNPs complex, suggesting a role for PB in mitochondrial RNA processing. RNA from 7 anti-GFP immunoisolations as well as total (input) RNA for each experiment was prepared. The seven samples include one mock IP from a strain containing GFP alone as well as 6 from Dhh1-GFP strains, two replicates from a (+) glucose condition and four replicates from a (-) glucose condition in which Dhh1-GFP forms cytoplasmic foci. 5ug of total RNA and 200ng IP RNA was hybridized to cutsom Agilent microarrays and detected using the S9.6 monoclonal antibody to RNA:DNA hybrids (ATCC clone ) and a Cy3 labeled anti-mouse secondary antibody.

2015-09-16 | E-GEOD-65989 | biostudies-arrayexpress

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Project description:Induction of the expression of the DHH1 wild type from pLEW100 for 24 hours (M-1 24 hours Tetracycline). Non-induced cells were used as control.

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