RNA-seq of WT, hrr25as, and hrr25is treated with inhibitors
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE107165 | GEO | 2018/12/02
REPOSITORIES: GEO
Similar Datasets
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 transcrption. 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 genome-wide occupancy of Pol II, phospho-Thr4, Hrr25, and key reader Rtt103 in WT and irreversibly sensitive Hrr25 (hrr25is) mutant strains of S. cerevisiae.
2018-12-02 | GSE107164 | GEO
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 genome-wide occupancy of Pol II, phospho-Thr4, and key reader Rtt103 in WT and CTD-mutant strains of S. cerevisiae.
2017-05-02 | GSE95418 | GEO
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:ChIP-chip was performed to identify the genomic binding locations for the termination factors Nrd1, and Rtt103, and for RNA polymerase (Pol) II phosphorylated at the tyrosine 1 and threonine 4 position of its C-terminal domain (CTD). In different phases of the transcription cycle, Pol II recruits different factors via its CTD, which consists of heptapeptide repeats with the sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Here we show that the CTD of transcribing yeast Pol II is phosphorylated at Tyr1, and that this impairs recruitment of termination factors. Tyr1 phosphorylation levels rise downstream of the transcription start site (TSS), and decrease before the polyadenylation (pA) site. Tyr1-phosphorylated gene bodies are depleted of CTD-binding termination factors Nrd1, Pcf11, and Rtt103. Tyr1 phosphorylation blocks CTD binding by these termination factors, but stimulates binding of elongation factor Spt6. These results show that CTD modifications can not only stimulate but also block factor recruitment, and lead to an extended CTD code for transcription cycle coordination.
2012-06-29 | E-MTAB-1060 | 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:The production of a mature mRNA in eukaryotes requires both transcription by RNA polymerase (pol) II and co-transcriptional processes, including mRNA capping, splicing, and cleavage and polyadenylation. The pol II complex serves as a hub to coordinate transcription and co-transcriptional processes, especially via the carboxyl terminal domain (CTD) of the large subunit of pol II that is composed in human of 52 repeats of the heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 and that can be modified by several post-translational modifications. To understand better the effects of pre-mRNA splicing on pol II transcription, we investigated the transcriptional effects of two small molecule inhibitors of splicing: Madrasin and Isoginkgetin. We found that while Madrasin can quickly inhibit pre-mRNA splicing, it is not the case of Isoginkgetin, which affects transcription before any visible effect on pre-mRNA splicing. Interestingly, we found that these two small molecules promote a global transcriptional readthrough, including intronless protein-coding genes and histone genes, which is not the case of the splicing inhibitors targeting the splicing factor SF3B1.
2024-08-20 | GSE221279 | GEO
Project description:In mammals, the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II consists of 52 conserved heptapeptide repeats containing the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Post-translational modifications of the CTD coordinate the transcription cycle and various steps of mRNA maturation. Here we describe Tyr1 phosphorylation (Tyr1P) as a hallmark of initiating Pol II in mammalian cells, in contrast to what was described in yeast. Tyr1P is predominantly found in antisense orientation at promoters but is also specifically enriched at active enhancers. Mutation of Tyr1 to phenylalanine (Y1F) prevents the formation of the hyper-phosphorylated Pol IIO form, induces degradation of Pol II to the Pol IIB form and results in a lethal phenotype. Our results suggest that Tyr1P has evolved specialized and essential functions in higher eukaryotes associated with antisense promoter and enhancer transcription, and Pol II stability.
2014-06-01 | GSE52914 | GEO
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 5M-bM-^@M-^Y and 3M-bM-^@M-^Y 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 3M-bM-^@M-^Y 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. In this study, we investigated the function and ChIPseq genome-wide profiling of Thr4P residue (using the 6D7 antibody) of the Pol II CTD in Raji human B cells in comparison with either total Pol II profiling (N20 antibody, santa-cruz sc-899x), Ser5P CTD (3E8) or Ser2P (3E10) profiling in WT Raji cells. In another set of experiments, we also analysed total Pol II profiling (using an HA tag at the N-terminus of RPB1 and HA antibody Abcam ab9110) when endogenous enzyme is shut down by alpha-amanitin and replaced by either a recominant Pol II with 48 consensus repeats of the CTD (con48) or a mutated version where Thr4 residues were replaced by Ala (Thr4-Ala).In total 6 experimental sets (Pol IIt, Ser5P, Ser2P, Thr4P, con48, Thr4-Ala) were generated for our analysis and for each a biological replicate was performed. Biological replicates were merged when the data showed comparable signal noise ratio. Otherwise a unique replicate, showing the best noise ratio, was chosen for further analysis although the second replicate (for Ser2P and Thr4-Ala experiments). An input control (genomic DNA extracted after reverse crosslinking of the nuclear chip extracts) was performred and used for substraction to the ChIP experiments. One specific input material was used for wt cells, one for con48 and one for Thr4-Ala. Our data were processed to generate final wig files using our in house analysis pipeline essentially as described in Koch et al, (2011) NSMB 18 (8) p956.In brief, after alignment, sequence tags are: (i) artefact removed, (ii) elongated to an in silico optimized actual size of the initial fragments , (iii) input substracted, (iv) merged if applicable, (v) scaled for all experiments to correct for variation of tag number in between experiments. Several of the raw data files were no longer available.
2012-06-04 | E-GEOD-37519 | biostudies-arrayexpress
Project description:RNA polymerase II relies on a repetitive sequence domain (YSPTSPS) within its largest subunit to orchestrate transcription. While phosphorylation on Ser2/Ser5 of the C-terminal heptad repeats is well-established, Thr4’s role remains enigmatic. Paradoxically, Thr4 phosphorylation was only detected after transcription end sites despite functionally implicated in pausing, elongation, termination, and mRNA processing. Our investigation revealed that Thr4 phosphorylation detection was obstructed by flanking Ser5 phosphorylation at the onset of transcription, which can be removed selectively. Subsequent proteomic analyses identified many proteins recruited to transcription via Thr4 phosphorylation, which previously were attributed to Ser2. Importantly, loss of Thr4 phosphorylation greatly reduces Ser2 phosphorylation, revealing a crosstalk between the two marks. Finally, the function analysis of the Thr4 phosphorylation highlighted its role in alternative 3’-end processing within pro-proliferative genes. Our findings unveil the true genomic location of this evolutionarily conserved phosphorylation mark and prompt a reassessment of functional assignments of the CTD.
2024-08-01 | GSE262826 | GEO
Project description:RNA polymerase II relies on a repetitive sequence domain (YSPTSPS) within its largest subunit to orchestrate transcription. While phosphorylation on Ser2/Ser5 of the C-terminal heptad repeats is well-established, Thr4’s role remains enigmatic. Paradoxically, Thr4 phosphorylation was only detected after transcription end sites despite functionally implicated in pausing, elongation, termination, and mRNA processing. Our investigation revealed that Thr4 phosphorylation detection was obstructed by flanking Ser5 phosphorylation at the onset of transcription, which can be removed selectively. Subsequent proteomic analyses identified many proteins recruited to transcription via Thr4 phosphorylation, which previously were attributed to Ser2. Importantly, loss of Thr4 phosphorylation greatly reduces Ser2 phosphorylation, revealing a crosstalk between the two marks. Finally, the function analysis of the Thr4 phosphorylation highlighted its role in alternative 3’-end processing within pro-proliferative genes. Our findings unveil the true genomic location of this evolutionarily conserved phosphorylation mark and prompt a reassessment of functional assignments of the CTD.
2024-07-31 | GSE262702 | GEO