RNA-seq analysis in WT and rpb1-CTD-S2A strains
Ontology highlight
ABSTRACT:
ORGANISM(S): Schizosaccharomyces pombe
PROVIDER: GSE171307 | GEO | 2021/08/26
REPOSITORIES: GEO
Similar Datasets
Project description:RNA-seq analysis in WT and rpb1-CTD-S2A strains
| PRJNA719083 | ENA
Project description:The carboxy-terminal domain (CTD) of Rpb1, the largest component of the 12-subunit RNA polymerase II, consists of repeating Y1S2P3T4S5P6S7 heptapeptides (26 repeats in budding yeast). Each stage of transcription relies on the ordered recruitment and exchange of specific protein complexes that act on RNA polymerase II, its nascent transcripts, and the underlying chromatin. This dynamic process is orchestrated via patterned post-translational modifications of the CTD. To characterize the role of phosphorylation on Thr4, we examined the effect of Rpb1 alleles in which Thr4 was substituted with an alanine (T4A) or the phospho-mimic glutamate (T4E). Substitutions were made across all heptad repeats of the CTD.
We affinity purified HA-tagged Rpb1 from Saccharomyces cerevisiae strains bearing WT, T4A, and T4E CTDs. A control strain (Z26) lacking the HA-tagged Rpb1 was subjected to an identical affinity enrichment procedure. Three biological replicates were acquired for each type of affinity purification and analyzed independently. After TCA-precipitation, proteins were urea-denatured, reduced, alkylated, then digested with endoproteinase LysC followed by trypsin. The resulting peptide mixtures were analyzed by Multidimensional Protein Identification Technology (MudPIT). Label-free quantitative proteomics was used to identify and quantify the relative abundance of affinity-enriched complexes.
2017-02-24 | MSV000080573 | MassIVE
Project description:The carboxy-terminal domain of RPB1 subunit of RNA Polymerase II (CTD) plays an essential function in the regulation of gene expression and the coordination of co-transcriptional processes. CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast and its length is crucial for spatial organization of transcriptional machinery in the nucleus. We found that the proximal half of the CTD is sufficient to support RNA metabolism and co-transcriptional processing in steady state conditions in human cells. Signal induced transcription, however, is severely impaired upon CTD shortening. Our data suggest that CTD length increased in evolution to allow for spatio-temporal control of gene expression patterns at least in part by facilitating enhancer function.
2021-01-04 | GSE159092 | GEO
Project description:The carboxy-terminal domain (CTD) of RNA polymerase II (Pol II) consists of heptad repeats with the consensus motif Y1-S2-P3-T4-S5-P6-S7. Dynamic phosphorylation of the CTD coordinates Pol II progression through the transcription cycle. Monoclonal antibodies have been used to study in vivo the potentially phosphorylated CTD amino acids (Y1, S2, T4, S5 and S7). However, the epitopes detected by antibodies can be masked by proteins or modifications at neighbouring sites. Therefore, the effectiveness of antibodies in western blot or ChIP analysis reflects the number of accessible CTD phosphorylation marks, but not the total number of phosphorylations. Most importantly, CTD phospho-specific antibodies do not provide any heptad - (location) specific information of CTD phosphorylation. Due to these limitations, the principles and patterns of CTD phosphorylation remained elusive. Here, we use genetic and mass spectrometric approaches to directly detect and map phosphosites along the entire CTD. We confirm phosphorylation of CTD residues Y1, S2, T4, S5 and S7 in mammalian and yeast cells. Although specific phosphorylation signatures dominate, adjacent CTD repeats can be differently phosphorylated, leading to a high variation of coexisting phosphosites in mono- and di-heptad CTD repeats. Inhibition of CDK9 kinase specifically reduces S2 phosphorylation levels within the CTD.
2016-01-25 | PXD003159 | Pride
Project description:The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) orchestrates dynamic recruitment of specific cellular machines during different stages of transcription. Signature phosphorylation patterns of Y1S2P3T4S5P6S7 heptapeptide repeats of the CTD engage specific “readers.” While phospho-Ser5 and phospho-Ser2 marks are ubiquitous, phospho-Thr4 is reported to only impact specific genes. Here, we investigate the RNA expression profile in WT and CTD-mutant strains of S. cerevisiae.
2017-05-02 | GSE95417 | GEO
Project description:Transctriptome profiling of CTD-14 repeats, 2A, 5A mutants responding to 0.7N NaCl for 30mins. The study shows that phosphorylation at Ser5 sites plays a role in normal induction and repression of genes upon NaCl stress. The CTD14 strains harbors a plasmid expressing RPB1 with 14 wild-type CTD repeats. 5A strains carries a plasmid expressing a chimeric RPB1 in which the CTD was composed of 5 repeats of CTD-serine 5 substituted with Ala followed by 7 wild-type-sequenced repeats. The 2A strains carrys 8 repeats of CTD-serine 2 substituted with alanine followed by 7 wild-type-sequenced repeats.
2014-12-03 | GSE60700 | GEO
Project description:In fission yeast, the nuclear-localized Lsk1p-Lsc1p-Lsg1p cyclin dependent kinase complex is required for the reliable execution of cytokinesis and is also required for Ser-2 phosphorylation RNA pol II carboxy terminal domain. To address whether alterations in CTD phosphorylation might selectively alter expression of cytokinesis genes, expression profiling of site-directed CTD mutants was performed. Strains bearing the rpb1-12XCTD and rpb1-12XS2ACTD mutations were grown to mid-log phase in YES media and treated with 0.5uM LatA (or the solvent control, DMSO) for three hours at 30C. Three biological replicates were performed.
2011-08-31 | E-GEOD-31369 | biostudies-arrayexpress
Project description:Transctriptome profiling of CTD-14 repeats, 2A, 5A mutants responding to 0.7N NaCl for 30mins. The study shows that phosphorylation at Ser5 sites plays a role in normal induction and repression of genes upon NaCl stress. The CTD14 strains harbors a plasmid expressing RPB1 with 14 wild-type CTD repeats. 5A strains carries a plasmid expressing a chimeric RPB1 in which the CTD was composed of 5 repeats of CTD-serine 5 substituted with Ala followed by 7 wild-type-sequenced repeats. The 2A strains carrys 8 repeats of CTD-serine 2 substituted with alanine followed by 7 wild-type-sequenced repeats. Two-color fluorescence arrays reporting on mRNA abunance in strains before and after 30 min with 0.7M NaCl treatment
2014-12-03 | E-GEOD-60700 | biostudies-arrayexpress
Project description:Eukaryotic RNA polymerase II (Pol II) has evolved an array of heptad repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 at the carboxy-terminal domain (CTD) of the large subunit (Rpb1). Differential phosphorylation of Ser2, Ser5, and Ser7 in the 5’ and 3’ regions of genes coordinates the binding of transcription and RNA processing factors to the initiating and elongating polymerase complexes. Here, we report phosphorylation of Thr4 by Polo-like-kinase-3 in mammalian cells. ChIPseq analyses indicate an increase of Thr4-P levels in the 3’ region of genes occurring subsequently to an increase of Ser2-P levels. A Thr4/Ala mutant of Pol II displays a lethal phenotype. This mutant reveals a global defect in RNA elongation, while initiation is largely unaffected. Since Thr4 replacement mutants are viable in yeast we conclude that this amino acid has evolved an essential function(s) in the CTD of Pol II for gene transcription in mammalian cells.
2012-06-04 | GSE37519 | GEO
Project description:RPB1, the largest subunit of RNA polymerase II, contains a highly modifiable C-terminal domain (CTD) that consists of variations of a consensus heptad repeat sequence (Y1S2P3T4S5P6S7). The consensus CTD repeat motif and tandem organization represent the ancestral state of eukaryotic RPB1, but across eukaryotes CTDs show considerable diversity in repeat organization and sequence content. These differences may reflect lineage-specific CTD functions mediated by protein interactions. Mammalian CTDs contain eight non-consensus repeats with a lysine in the seventh position (K7). Posttranslational acetylation of these sites was recently shown to be required for proper polymerase pausing and regulation of two growth factor-regulated genes. To investigate the origins and function of RPB1 CTD acetylation (acRPB1), we computationally reconstructed the evolution of the CTD repeat sequence across eukaryotes and analyzed the evolution and function of genes dysregulated when acRPB1 is disrupted. Modeling the evolutionary dynamics of CTD repeat count and sequence content across diverse eukaryotes revealed an expansion of the CTD in the ancestors of Metazoa. The new CTD repeats introduced the potential for acRPB1 due to the appearance of distal repeats with lysine at position seven. This was followed by a further increase in the number of lysine-containing repeats in developmentally complex clades like Deuterostomia. Mouse genes enriched for acRPB1 occupancy at their promoters and genes with significant expression changes when acRPB1 is disrupted are enriched for several functions, such as growth factor response, gene regulation, cellular adhesion, and vascular development. Genes occupied and regulated by acRPB1 show significant enrichment for evolutionary origins in the early history of eukaryotes through early vertebrates. Our combined functional and evolutionary analyses show that RPB1 CTD acetylation was possible in the early history of animals, and that the K7 content of the CTD expanded in specific developmentally complex metazoan lineages. The functional analysis of genes regulated by acRPB1 highlight functions involved in the origin of and diversification of complex Metazoa. This suggests that acRPB1 may have played a role in the success of animals. We used a HA-tagged mouse RPB1 construct in which all K7 residues were substituted with arginines (8KR). This mutation resembles unacetylated lysines by conserving the positive charge at these positions, but preventing acetylation. To examine the potential functions of K7 acetylation in regulating gene expression, we stably expressed wildtype or 8KR HA-RPB1 in murine NIH/3T3 fibroblasts and cultured these cells in media containing α-amanitin. Both were expressed at equivalent levels, but acetylation was present only in wildtype, and not mutant HA-RPB1. We then performed gene expression profiling using the Affymetrix Mouse Gene 1.0 ST microarray with three biological replicates.
2015-02-19 | E-GEOD-66088 | biostudies-arrayexpress