RNA Pol II CTD mutants
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ABSTRACT:
PROVIDER: PRJNA377084 | ENA |
REPOSITORIES: ENA
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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: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:We used RNA Pol II-CTD occupancy assay to identify mRNA isoform switch in mutant animals compared to wild type animals. Examination of RNA Pol II-CTD Ser5P recognized by 4H8 in wild type, sydn-1(0), ssup-72(0) and ssup-72(0);sydn-1(0)
2015-11-01 | E-GEOD-68203 | biostudies-arrayexpress
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:This RNA-Seq analysis compares gene expression of the RNA polymerase II CTD-mutants contrasting to the WT RNA polymerase II CTD (with isogenic 3' selection marker) in the fission yeast S. pombe
2021-02-25 | GSE160852 | GEO
Project description:Whole genome analysis of total RNA pol II, Ser2-, Ser5- and Ser7-phosphorylated RNA pol II, in WT and mutants of the C-terminal domain (CTD) kinases Ctk1 and Kin28, and localization of the termination factors Pcf11, Nrd1 and Rat1.
2010-09-15 | GSE23960 | GEO
Project description:We used RNA Pol II-CTD occupancy assay to identify mRNA isoform switch in mutant animals compared to wild type animals.
2015-11-01 | GSE68203 | GEO
Project description:To investigate the role of RNA Pol II CTD acetylation we performed comprehensive deacetylase inhibition using common small-molecule inhibitors and performed ChIP-seq from mouse cells using antibodies against RNA Polymerase II CTD modifications: (8WG16, unmodified heptads; K7ac, acetylated heptads; 4H8, S5-phosphorylated heptads). In addition, we tested the effect of knockdown of RPRD1B, a key transcription regulator that recruits RPAP2, a CTD S5-phosphatase.
2019-01-12 | GSE124996 | GEO
Project description:Spt6 is a multifunctional histone chaperone involved in the maintenance of chromatin structure during elongation by RNA polymerase II (Pol II). Spt6 has a tandem SH2 (tSH2) domain within its C-terminus that recognizes Pol II CTD peptides phosphorylated on Ser2, Ser5 or Try1 in vitro. Deleting the tSH2 domain, however, only has a partial effect on Spt6 occupancy in vivo, suggesting that more complex mechanisms are involved in the Spt6 recruitment. Our results show that the Ser2 kinases Bur1 and Ctk1, but not the Ser5 kinase Kin28, cooperate in recruiting Spt6, genome-wide. Interestingly, the Ser2 kinases promote the association of Spt6 in early transcribed regions and not toward the 3' end of genes, where phosphorylated Ser2 reaches its maximum level. Additionally, our results uncover an unexpected role for histone deacetylases (Rpd3 and Hos2) in promoting Spt6 interaction with elongating Pol II. Finally, our data suggest that phosphorylation of the Pol II CTD on Tyr1 promotes the association of Spt6 with the 3' end of transcribed genes, independently of Ser2 phosphorylation. Collectively, our results show that a complex network of interactions, involving the Spt6 tSH2 domain, CTD phosphorylation and histone deacetylases, coordinate the recruitment of Spt6 to transcribed genes in vivo. We examined the genome-wide distribution (using ChIP-chip) of Spt6. Spt6 occupancy was also assayed in mutants for CTD Serine 2 and Serine 5 kinases and in mutants for histone deacetylases. ChIPs were performed with a Myc-tagged version of Spt6. Most ChIPs (in Cy5) were hybridyzed against a control ChIP sample from an isogenic non-tagged strain (in Cy3). In the ChIP experiments with the spt6-202del mutant, non immunoprecipitated DNA (input) was used as the control. In addition to Spt6 ChIPs, the project includes RNAPII (Rpb3) ChIP-chip datasets, where an anti-Rpb3 antibody was used to ChIP RNAPII and non immunoprecipitated DNA (input) was used as the control. All ChIP-chip experiments were done in duplicates. Each microarray was normalized using the Lima Loess and replicates were combined using a weighted average method as previously described (Pokholok et al., 2005).
2014-09-25 | E-GEOD-61713 | biostudies-arrayexpress
Project description:Transcription termination in Saccharomyces cerevisiae can be performed by at least two distinct pathways and is directed by the phosphorylation status of the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II). Late termination of mRNAs is performed by the CPF/CF complex and requires CTD-Ser2 phosphorylation. Early termination of shorter cryptic unstable transcripts (CUTs) and small nucleolar RNAs (snoRNAs) is preformed by the Nrd1 complex, and requires CTD-Ser5 phosphorylation. In this study, mutants of the different termination pathways were compared by genome-wide expression analysis. Surprisingly, the expression changes observed upon loss of the CTD-Ser2 kinase Ctk1 are more similar to loss of a subunit of the Ser5P binding Nrd1-complex, than to loss of Ser2P binding factors. Tiling array analysis of ctk1Δ reveals readthrough at several hundred sites, including snoRNAs, as reported previously, but also many cryptic unstable transcripts, stable untranslated transcripts (SUTs) and other transcripts. Surprisingly, neither loss of CTK1 nor a Pol II CTD-Ser2 substitution mutant results in a global defect in termination of mRNAs, indicating that Ser2P is not essential for proper termination of most mRNAs. At snoRNA, Nrd1 location is shifted downstream in ctk1∆, indicating defective release rather than recruitment of Nrd1. Weakening the interaction between Nrd1 and Pol II rescues the readthrough in ctk1∆, likely by facilitating Nrd1 release. The termination defect is kinase activity dependent, but cannot be completely explained by loss of CTD-Ser2 phosphorylation , a major substrate of Ctk1, suggesting the involvement of an additional substrate. Mutant alleles of the elongation factor Spt5 rescue ctk1∆-dependent readthrough, indicating a role for Spt5 in this process, perhaps as a substrate of Ctk1. The results show that Ctk1 is more intimately involved in termination of small non-coding RNAs than was previously assumed and lead to a model in which Ctk1 influences Spt5 activity to achieve this.
2014-04-22 | GSE40254 | GEO