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Overexpression of transcriptional factors Kin28 and Pog1 suppresses the stress sensitivity caused by the rsp5 mutation

ABSTRACT: Rsp5 is an essential and multi-functional E3 ubiquitin ligase in Saccharomyces cerevisiae. We previously isolated the Ala401Glu rsp5 mutant, which is hypersensitive to various stresses. To understand the function of Rsp5 in stress responses, suppressor genes whose overexpression allows rsp5A401E cells to grow at high temperature were screened. The KIN28 and POG1 genes, encoding a subunit of the transcription factor TFIIH and a putative transcriptional activator, respectively, were identified as multicopy suppressors of not only high temperature but also LiCl stresses. The overexpression of Kin28 and Pog1 in rsp5A401E cells caused an increase in the transcriptional level of some stress proteins when exposed to temperature up-shift. DNA microarray analysis under LiCl stress revealed that the transcriptional level of some proteasome components was increased in rsp5A401E cells overexpressing Kin28 or Pog1. These results suggest that the overexpression of Kin28 and Pog1 enhances the protein refolding and degradation pathways in rsp5A401E cells. Keywords: mutant analysis, stress response

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE8729 | GEO | 2007/09/07

SECONDARY ACCESSION(S): PRJNA101985

REPOSITORIES: GEO

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Transcription profiling of S. cerevisiae overexpressing transcriptional factors Kin28 and Pog1 suppresses the stress sensitivity caused by the rsp5 mutation

Project description:Rsp5 is an essential and multi-functional E3 ubiquitin ligase in Saccharomyces cerevisiae. We previously isolated the Ala401Glu rsp5 mutant, which is hypersensitive to various stresses. To understand the function of Rsp5 in stress responses, suppressor genes whose overexpression allows rsp5A401E cells to grow at high temperature were screened. The KIN28 and POG1 genes, encoding a subunit of the transcription factor TFIIH and a putative transcriptional activator, respectively, were identified as multicopy suppressors of not only high temperature but also LiCl stresses. The overexpression of Kin28 and Pog1 in rsp5A401E cells caused an increase in the transcriptional level of some stress proteins when exposed to temperature up-shift. DNA microarray analysis under LiCl stress revealed that the transcriptional level of some proteasome components was increased in rsp5A401E cells overexpressing Kin28 or Pog1. These results suggest that the overexpression of Kin28 and Pog1 enhances the protein refolding and degradation pathways in rsp5A401E cells. Experiment Overall Design: Total RNA from S. cerevisiae was isolated by the method of KÃ¶hrer and Domdey (1991). Poly A mRNA was enriched from total RNA by Oligotex dT30 mRNA purification kit (Takara Bio). The Affimetrix yeast genome S98 arrays (YGS98 GeneChip, Affymetrix, Santa Clara, CA) were used as DNA microarray in this study. The biotinyated cRNA (15 Î¼g) probe was hybridized to DNA microarray at 45Â°C for 18 h according to Affymetrix userâs manual. Experiment Overall Design: The washing and staining of arrays were performed using the GeneChip Fluidics Station 400. Experiment Overall Design: The scanning of arrays was carried out using the GeneArray scanner (Agilent technologies, Palto Alto, CA).

2008-06-16 | E-GEOD-8729 | biostudies-arrayexpress

Transient transcriptome sequencing (TT-Seq) and 5'P-Seq of ATP-analog sensitive Kin28 budding yeast

Project description:Inhibition of Kin28/CDK7, the kinase subunit of TFIIH, leads to defects in transcription of protein-coding genes. Despite a severe reduction in nascent RNA synthesis, the majority of mRNAs retain their steady-state level upon inhibition. In this study, we examined the determinants of mRNA stability in cells experiencing transcriptional crisis via irreversible chemical inhibition of Kin28. We discovered that the inhibited Kin28 transcriptome resembles the transcriptome of cells treated with an inhibitor of protein synthesis. Indeed, inhibition of Kin28 induces a coordinated decrease in translation and an increase in P-body formation. Unexpectedly, integrated stress response effectors do not trigger the observed proteostasis and ribostasis. Rather, mRNAs that are buffered from degradation display a preference for Ski2 over Nab2 and are differentially sensitive to the 5′-exonuclease Xrn1. These findings reveal that a nuclear kinase, well-known for its role in early stages of RNA synthesis, orchestrates multiple molecular processes in different cellular compartments.

2022-01-18 | GSE125409 | GEO

Pol II and CTD Phosphorylation ChIP-chip of ATP-analog sensitive Kin28 budding yeast

2016-06-16 | GSE83412 | GEO

Comparative dynamic transcriptome analysis (cDTA-seq) and total RNA-seq of ATP-analog sensitive Kin28 budding yeast

2016-06-16 | GSE83416 | GEO

Comparative dynamic transcriptome analysis (cDTA-seq) and total RNA-seq of ATP-analog sensitive Kin28 budding yeast

Project description:During transcription initiation, the TFIIH-kinase Kin28/Cdk7 marks RNA polymerase II (Pol II) by phosphorylating the C-terminal domain (CTD) of its largest subunit. Here we describe a structure-guided chemical approach to covalently and specifically inactivate Kin28 kinase activity in vivo. This method of irreversible inactivation recapitulates both the lethal phenotype and the key molecular signatures that result from genetically disrupting Kin28 function in vivo. Inactivating Kin28 impacts promoter release to differing degrees and reveals a â??checkpointâ?? during the transition to productive elongation. While promoter-proximal pausing is not observed in budding yeast, inhibition of Kin28 attenuates elongation-licensing signals, resulting in Pol II accumulation at the +2 nucleosome and reduced transition to productive elongation. Furthermore, upon inhibition, global stabilization of mRNA masks different degrees of reduction in nascent transcription. This study resolves long-standing controversies on the role of Kin28 in transcription and provides a rational approach to irreversibly inhibit other kinases in vivo. Total RNA was collected from wild-type and analog-sensitive Kin28 strains treated with reversible inhibitor 1-NAPP-1, irreversible inhibitor CMK, and solvent control DMSO. Equivalent ratios of S. pombe : S. cerevisiae cells were added to each sample before RNA extraction for normalization of read counts after sequencing. Nascent RNA was purified from total RNA by 4-thiouracil labeling, biotinylation, and streptavidin-pulldown. As a negative control, nascent RNA was also extracted from total RNA from cells that had not been treated with 4-thiouracil.

2016-06-16 | E-GEOD-83416 | biostudies-arrayexpress

ZmNF-YA1 contributes to maize thermotolerance by regulating heat shock response

Project description:Maize is a staple food, feed, and industrial crop. Heat stress (HS) is one of the major stresses affecting maize production and is usually accompanied by other stresses, such as drought. Our previous study identified a heterotrimer complex, ZmNF-YA1-YB16-YC17, in maize. ZmNF-YA1 and ZmNF-YB16 were positive regulators of the drought stress response and were involved in maize root development. In this study, we investigated whether ZmNF-YA1 confers HS tolerance in maize. The ZmNF-YA1 mutant and overexpression lines were used to test the role of ZmNF-YA1 in maize thermotolerance. The ZmNF-YA1 mutant was more temperature-sensitive than the WT, while the ZmNF-YA1 overexpression lines showed a thermotolerant phenotype. Higher malondialdehyde content and reactive oxygen species accumulation were observed in the mutant, followed by WT and overexpression lines after HS treatment, while an opposite trend was observed for chlorophyll content. RNA-seq was used to analyze transcriptome changes in W22 and nf-ya1 plants in response to HS at great depths. Based on their expression profiles, the HS response-related differentially expressed genes in nf-ya1 and W22 were grouped into seven clusters via k-means clustering. Gene Ontology (GO) enrichment analysis of Clade 1, in which ZmNF-YA1 and HS induced, GO terms for protein refolding and protein stabilization, and terms for various stress responses were considerably enriched. Thus, the contribution of ZmNF-YA1 to protein stabilization, refolding, and regulation of ABA, ROS, and heat/temperature signaling may be the major reason why ZmNF-YA1 overexpression enhanced heat tolerance, and the mutant showed a heat-sensitive phenotype.

2024-06-02 | GSE268429 | GEO

Kin28 Regulates the Transient Association of Mediator with Core Promoters

Project description:Mediator is an essential, broadly utilized eukaryotic transcriptional co-activator. How and what it communicates from activators to RNA polymerase II remains an open question. Here we performed genome-wide location profiling of yeast Mediator subunits. Mediator is not found at core promoters but rather occupies the upstream activating sequence (UAS), upstream of the pre-initiation complex. In the absence of Kin28/CDK7 kinase activity, or in cells where the CTD is mutated to replace Ser5 with alanines, however, Mediator accumulates at core promoters together with RNAPII. We propose that Mediator is quickly released from promoters upon Ser5 phosphorylation by Kin28/CDK7, which also allows for RNAPII to escape from the promoter.

2014-03-24 | GSE55402 | GEO

Saccharomyces cerevisiae

Project description:Functional analysis of TFIIH (Kin28) in Saccharomyces cerevisiae

| PRJNA237486 | ENA

Structure of TFIIK for phosphorylation of CTD of RNA polymerase II

Project description:TFIIH is a 10-protein complex that is conserved through out eukaryotes. TFIIH has two primary cellular functions: transcription initiation and nucleotide excision repair (NER). In transcription initiation, TFIIH acts as a structural scaffold, phosphorylates the RNA polymerase II (pol II) C-terminal domain (CTD) and translocates promoter DNA through the pol II active site to facilitate start site selection. In NER, again is a structural scaffold, opens a bubble around damaged DNA and scans the damaged strand for bulky lesions. In yeast (Saccharomyces cerevisiae), TFIIH is composed of the two helicases Ssl2 and Rad3, the scaffolding subunits Tfb1, Tfb2, Tfb4 and Ssl1 and the kinase subunits Kin28, Ccl1 and Tfb3.

2021-07-07 | PXD021211 | Pride

Structure of TFIIH/Rad4-Rad23-Rad33 in damaged DNA opening in Nucleotide Excision Repair

Project description:TFIIH is a 10-protein complex that is conserved throughout eukaryotes. TFIIH has two primary cellular functions: transcription initiation and nucleotide excision repair (NER). NER in eukaryotes begins by recognition of a bulky lesion by the obligate dimer Rad4-Rad23. This is followed closely by the recruitment of TFIIH which is a structural scaffold, opens a bubble around damaged DNA and scans the damaged strand for bulky lesions. This facilitates the recruitment of two exonucleases which excise the damages strand before an undamaged complement is synthesize. In yeast (Saccharomyces cerevisiae), TFIIH is composed of the two helicases Ssl2 and Rad3, the scaffolding subunits Tfb1, Tfb2, Tfb4 and Ssl1 and the kinase subunits Kin28, Ccl1 and Tfb3, though these later 3 are dispensable for NER.

2021-07-07 | PXD021212 | Pride

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