RNA PolyRNA Polymerase II CTD phosphatase Rtr1 prevents premature transcription termination
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ABSTRACT:
PROVIDER: PRJNA557233 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9856230_1.fastq.gz | Fastqsanger.gz | |||
| SRR9856230_2.fastq.gz | Fastqsanger.gz | |||
| SRR9856240_1.fastq.gz | Fastqsanger.gz | |||
| SRR9856240_2.fastq.gz | Fastqsanger.gz | |||
| SRR9856250_1.fastq.gz | Fastqsanger.gz |
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Project description:At the 3'-ends of genes, RNA polymerase (Pol) II is dephosphorylated at tyrosine 1 residues of its C-terminal domain (CTD), resulting in recruitment of transcription termination factors. We show that the multisubunit cleavage and polyadenylation factor (CPF) is a Pol II CTD phosphatase and its Glc7 subunit is required for Tyr1 dephosphorylation at the poly-adenylation site and Pol II termination in vivo. ChIP-chip was performed to examine the effect of Glc7 nuclear depletion on genome-wide Pol II occupancy [using ?-Rpb3 (1Y26, cat. no. W0012, neoclone) antibody] and CTD tyrosine 1 phosphorylation levels [using ?-TyrY1P (3D12, D. Eick) antibody].
2013-12-16 | E-MTAB-1528 | biostudies-arrayexpress
Project description:Transcription termination is key to gene regulation as it prevents transcription interference with neighboring genes. In Saccharomyces cerevisiae, termination at protein-coding genes is coupled to the cleavage of the nascent transcript, while most non-coding RNA transcription relies on a cleavage-independent termination pathway involving Nrd1, Nab3 and the helicase Sen1 (NNS pathway). In both pathways, the recruitment of termination factors involves phosphorylated forms of the RNA polymerase II C-terminal domain (CTD) but the contribution of individual CTD residues was never systematically investigated. Here, we investigated the impact of mutating phosphorylation sites in the CTD on termination. We observed widespread termination defects at protein-coding genes in mutants for Ser2 or Thr4 but rare defects in Tyr1 mutants for this class of genes. Instead, mutating Tyr1, or its phosphatase Glc7, led to widespread termination defects at non-coding genes known to terminate via the NNS pathway. These defects can be suppressed by slowing down transcription, suggesting that Tyr1 mediates termination via the regulation of elongation or pausing. Our work redefines the role of Tyr1 in termination at protein-coding genes in budding yeast and highlights its key role in termination by the NNS pathway.
2019-01-21 | GSE115027 | GEO
Project description:Transcription termination is key to gene regulation as it prevents transcription interference with neighboring genes. In Saccharomyces cerevisiae, termination at protein-coding genes is coupled to the cleavage of the nascent transcript, while most non-coding RNA transcription relies on a cleavage-independent termination pathway involving Nrd1, Nab3 and the helicase Sen1 (NNS pathway). In both pathways, the recruitment of termination factors involves phosphorylated forms of the RNA polymerase II C-terminal domain (CTD) but the contribution of individual CTD residues was never systematically investigated. Here, we investigated the impact of mutating phosphorylation sites in the CTD on termination. We observed widespread termination defects at protein-coding genes in mutants for Ser2 or Thr4 but rare defects in Tyr1 mutants for this class of genes. Instead, mutating Tyr1, or its phosphatase Glc7, led to widespread termination defects at non-coding genes known to terminate via the NNS pathway. These defects can be suppressed by slowing down transcription, suggesting that Tyr1 mediates termination via the regulation of elongation or pausing. Our work redefines the role of Tyr1 in termination at protein-coding genes in budding yeast and highlights its key role in termination by the NNS pathway.
2019-01-21 | GSE115026 | GEO
Project description:Efficient release of promoter-proximally paused Pol II into productive elongation is essential for gene expression. Recently, we reported that the Integrator complex can bind paused Pol II and drive premature transcription termination, potently attenuating the activity of target genes. Premature termination requires RNA cleavage by the endonuclease subunit of Integrator, but the roles of other Integrator subunits in gene regulation have yet to be elucidated. Here, we report that Integrator subunit 8 (IntS8) is critical for transcription repression through its association with Protein Phosphatase 2A (PP2A). We find that Integrator-bound PP2A dephosphorylates the Pol II C-terminal domain and Spt5, and prevents the transition to productive elongation. Blocking PP2A association with Integrator thus stimulates pause release and gene activation. These results reveal a second catalytic function associated with Integrator-mediated transcription termination, and suggest a new model for the control of productive elongation involving active competition between transcriptional kinases and phosphatases.
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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.
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Project description:RNA Seq and ChIP-exo experiments related to this project
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Project description:The pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11-mediated termination is essential for vertebrate development. A range of genomic analyses, including: mNET-seq, 3’ mRNA-seq, chromatin RNA-seq and ChIP-seq, reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome-wide. PCF11 binds preferentially between closely spaced genes, where it prevents transcriptional interference and downstream gene silencing. Notably, PCF11 is sub-stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto-regulatory mechanism involving premature termination of its own transcript, and are important for normal development. Both in human cell culture and during zebrafish development, PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination.
2019-02-25 | GSE123104 | GEO
Project description:The pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11-mediated termination is essential for vertebrate development. A range of genomic analyses, including: mNET-seq, 3’ mRNA-seq, chromatin RNA-seq and ChIP-seq, reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome-wide. PCF11 binds preferentially between closely spaced genes, where it prevents transcriptional interference and downstream gene silencing. Notably, PCF11 is sub-stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto-regulatory mechanism involving premature termination of its own transcript, and are important for normal development. Both in human cell culture and during zebrafish development, PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination.
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Project description:CDK9 is a kinase critical for the productive transcription of protein-coding genes by RNA polymerase II (pol II). As part of P-TEFb, CDK9 phosphorylates the carboxyl-terminal domain (CTD) of pol II and elongation factors, which allows pol II to elongate past the early elongation checkpoint (EEC) encountered soon after initiation. We show that, in addition to halting pol II at the EEC, loss of CDK9 activity causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, indicating that this kinase/phosphatase pair regulates transcription elongation and RNA processing at the end of protein-coding genes. We also confirm the splicing factor SF3B1 as a target of CDK9 and show that SF3B1 in complex with polyadenylation factors is lost from chromatin after CDK9 inhibition. These results emphasize the important roles that CDK9 plays in coupling transcription elongation and termination to RNA maturation downstream of the EEC.
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Project description:The largest subunit of RNA polymerase (Pol) II harbors a evolutionary conserved C-Terminal-Domain (CTD), composed of a repetition of heptapeptides, central in the transcriptional process. Here, we have analysed the transcriptional phenotypes of a CTD-∆5 mutant that carries a large CTD truncation. Unexpectedly, our results indicate that this mutant can widely transcribe genes in living cells but displays an extreme pervasive phenotype with severely impaired termination, similar to but more severely impaired than previously characterized mutations of CTD tyrosine residues. The CTD-∆5 mutant has also lost its interactions with the Mediator and Integrator complexes involved in activation of transcription and maturation of RNAs. Examination of long-distance interactions and CTCF binding pattern in CTD-∆5 did not indicate major changes in TAD domains or borders. Our data demonstrate that the CTD is largely dispensable for the act of transcription per se in living cells. We propose a model in which CTD-depleted Pol II has a lower entry rate into DNA but becomes extremely pervasive once engaged in transcription, resulting in a global loss of termination.
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