Control of RNA pol II speed by PNUTS-PP1 and Spt5 dephosphorylation facilitates termination by a “sitting duck torpedo” mechanism
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ABSTRACT:
PROVIDER: PRJNA554298 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10322324_1.fastq.gz | Fastqsanger.gz | |||
| SRR10322324_2.fastq.gz | Fastqsanger.gz | |||
| SRR10322325_1.fastq.gz | Fastqsanger.gz | |||
| SRR10322325_2.fastq.gz | Fastqsanger.gz | |||
| SRR9674970.fastq.gz | Fastqsanger.gz |
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Project description:Control of transcription speed, which influences many co-transcriptional processes, is poorly understood. We report that PNUTS-PP1 phosphatase is a negative regulator of RNA pol II elongation rate. The PNUTS W401A mutation, which disrupts PP1 binding, causes genome-wide acceleration of transcription associated with hyper-phosphorylation of the Spt5 elongation factor. Immediately downstream of poly(A) sites, pol II decelerates from >2kb/min to <1 kb/min, which correlates with Spt5 dephosphorylation. Pol II deceleration and Spt5 dephosphorylation require poly(A) site recognition and the PNUTS-PP1 complex, which is in turn necessary for transcription termination. These results lead to a new model for termination, the “sitting duck torpedo” mechanism, where poly(A) site-dependent deceleration caused by PNUTS-PP1 and Spt5 dephosphorylation is required to convert pol II into a viable target for the Xrn2 terminator exonuclease. Spt5 and its bacterial homologue NusG therefore have related functions controlling kinetic competition between RNA polymerases and the termination factors that pursue them.
2019-10-30 | GSE134198 | GEO
Project description:Control of transcription speed, which influences many co-transcriptional processes, is poorly understood. We report that PNUTS-PP1 phosphatase is a negative regulator of RNA polymerase II (Pol II) elongation rate. The PNUTS W401A mutation, which disrupts PP1 binding, causes genome-wide acceleration of transcription associated with hyper-phosphorylation of the Spt5 elongation factor. Immediately downstream of poly(A) sites, Pol II decelerates from >2 kb/min to <1 kb/min, which correlates with Spt5 dephosphorylation. Pol II deceleration and Spt5 dephosphorylation require poly(A) site recognition and the PNUTS-PP1 complex, which is in turn necessary for transcription termination. These results lead to a model for termination, the "sitting duck torpedo" mechanism, where poly(A) site-dependent deceleration caused by PNUTS-PP1 and Spt5 dephosphorylation is required to convert Pol II into a viable target for the Xrn2 terminator exonuclease. Spt5 and its bacterial homolog NusG therefore have related functions controlling kinetic competition between RNA polymerases and the termination factors that pursue them.
| S-EPMC6927536 | biostudies-literature
Project description:Transcription termination pathways mitigate the detrimental consequences of unscheduled promiscuous initiation occurring at hundreds of thousands of genomic cis-regulatory elements. The Restrictor complex, composed of the Pol II-interacting protein WDR82 and the RNA-binding protein ZC3H4, suppresses processive transcription at thousands of extragenic sites in mammalian genomes. Restrictor-driven termination does not involve nascent RNA cleavage and its interplay with other termination machineries is unclear. Here we show that efficient termination at Restrictor-controlled extragenic transcription units involves the recruitment of the protein phosphatase 1 (PP1) regulatory subunit PNUTS, a negative regulator of the Spt5 elongation factor, and Symplekin, a protein associated with RNA cleavage complexes but also involved in cleavage-independent and phosphatase-dependent termination of non-coding RNAs in yeast. PNUTS and Symplekin act synergistically with, but independently from Restrictor to dampen processive extragenic transcription. Moreover, the presence of limiting nuclear levels of Symplekin imposes a competition for its recruitment among multiple transcription termination machineries, resulting in mutual regulatory interactions. Hence, by synergizing with Restrictor, Symplekin and PNUTS enable efficient termination of processive, long-range extragenic transcription.
2024-05-23 | PXD043638 | Pride
Project description:The Pol II transcription cycle is ordered by CDKs and phosphatases. In fission yeast, Cdk9 phosphorylates carboxy-terminal repeats (CTRs) of Spt5 while inhibiting PP1 during elongation. Transcription past the cleavage and polyadenylation signal (CPS) coincides with PP1-dependent Spt5 dephosphorylation and leads to Pol II pausing with phosphorylated CTD-Ser2 (pSer2). Here we show this switch is conserved in humans: Cdk9 inhibition decreases phosphorylation of both PP1g and Spt5-Thr806 (pThr806), and induces pSer2 upstream of the CPS, whereas PP1 depletion increases pThr806. Moreover, in unperturbed cells, pThr806 is diminished in 3’-paused complexes where pSer2 is maximal. Cdk9 also phosphorylates Spt5 on Ser666, a PP1-refractory site between conserved KOW4 and KOW5 motifs; pSer666 increases upon promoter-proximal pause release and, in contrast to pThr806, persists beyond the CPS. We identify PP4—another target of inhibitory phosphorylation by Cdk9—as a pSer666 phosphatase. PP4 and PP1g are enriched at 5’ and 3’ ends of genes, respectively, consistent with pSer666 and pThr806 distributions. Therefore, distinct Cdk9-phosphatase switches operate on Spt5 at different steps in transcription.
2020-08-04 | GSE138548 | GEO
Project description:The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to ensure proper mRNA maturation and prevent interference between neighboring genes. Pol II slowing downstream of the cleavage and polyadenylation signal (CPS) leads to recruitment of cleavage and polyadenylation factors and termination, but how this chain of events is initiated remains unclear. In a chemical-genetic screen we identified protein phosphatase 1 (PP1) isoforms as substrates of human positive transcription elongation factor b (P-TEFb), the cyclin-dependent kinase 9 (Cdk9)-cyclin T1 complex. Here we show that Cdk9 and PP1 govern phosphorylation of the conserved transcription factor Spt5 in the fission yeast Schizosaccharomyces pombe. Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis2. Sites phosphorylated by Cdk9 in the Spt5 carboxy-terminal domain (CTD) are dephosphorylated by Dis2 in vitro, and Cdk9 inhibition in vivo leads to rapid Spt5 dephosphorylation that is retarded by concurrent Dis2 inactivation. Chromatin immunoprecipitation and sequencing (ChIP-seq) analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the CPS, prior to or concomitant with Pol II pausing. A Dis2-inactivating mutation stabilizes Spt5 phosphorylation (pSpt5) on chromatin, promotes transcription beyond the normal termination zone detected by precision run-on transcription and sequencing (PRO-seq), and is suppressed by ablation of Cdk9 target sites in Spt5. These results support a model whereby the transition of Pol II from elongation to termination is regulated by opposing activities of Cdk9 and Dis2 towards their common substrate Spt5—a bistable switch analogous to a Cdk1-PP1 module that controls exit from mitosis.
2018-06-07 | GSE102590 | GEO
Project description:RNA polymerase II progression from initiation to elongation is driven in part by a cascade of protein kinases acting on the core transcription machinery. Conversely, the corresponding phosphatases, notably PP2A and PP1—the most abundant serine-threonine phosphatases in cells—are thought to mainly impede polymerase progression, respectively restraining pause release at promoters and polymerase elongation at terminators. Here we reveal an unexpected role of PP1, within the PNUTS-PP1 complex, in sustaining global transcriptional activation. Acute disruption of PNUTS-PP1 leads to severe defects in the release of paused polymerase and subsequent downregulation for the majority of transcribed genes. Mechanistically, PNUTS-PP1 promotes pause release by dephosphorylating multiple substrates, including the 7SK snRNP subunit MEPCE, a known regulator of pause release. PNUTS-PP1 exhibits antagonistic functions compared to INTAC phosphatase, which generally inhibits pause release. Our research thus highlights the opposing roles of PP1 and PP2A in modulating genome-wide transcriptional pausing and gene expression.
2024-12-02 | GSE279151 | GEO
Project description:RNA polymerase II progression from initiation to elongation is driven in part by a cascade of protein kinases acting on the core transcription machinery. Conversely, the corresponding phosphatases, notably PP2A and PP1—the most abundant serine-threonine phosphatases in cells—are thought to mainly impede polymerase progression, respectively restraining pause release at promoters and polymerase elongation at terminators. Here we reveal an unexpected role of PP1, within the PNUTS-PP1 complex, in sustaining global transcriptional activation. Acute disruption of PNUTS-PP1 leads to severe defects in the release of paused polymerase and subsequent downregulation for the majority of transcribed genes. Mechanistically, PNUTS-PP1 promotes pause release by dephosphorylating multiple substrates, including the 7SK snRNP subunit MEPCE, a known regulator of pause release. PNUTS-PP1 exhibits antagonistic functions compared to INTAC phosphatase, which generally inhibits pause release. Our research thus highlights the opposing roles of PP1 and PP2A in modulating genome-wide transcriptional pausing and gene expression.
2024-12-02 | GSE279150 | GEO
Project description:Base J is a modified DNA base that is enriched at RNA polymerase II termination sites and telomeres. We previously found that base J functions to prevent readthrough transcription within gene clusters in T. brucei. We now identify a complex in Leishmania and T. brucei composed of PNUTS, PP1, Wdr82 and a J-binding protein (JBP3). We show the RNAi ablation of PNUTS in T. brucei leads to readthrough transcription and expression of downstream genes.
2020-01-14 | GSE135708 | GEO
Project description:Age-related diseases pose great challenges to health care systems worldwide. During aging, endothelial senescence increases the risk for cardiovascular disease. Recently, it was described that Phosphatase 1 Nuclear Targeting Subunit (PNUTS) has a central role in cardiomyocyte aging and homeostasis. Here, we determined the role of PNUTS in endothelial cell aging. We confirmed that PNUTS is repressed in senescent endothelial cells (ECs). Moreover, PNUTS silencing elicits several of the hallmarks of endothelial aging: senescence, reduced angiogenesis and loss of barrier function. To validate our findings in vivo, we generated an endothelial-specific inducible PNUTS-deficient mouse line (Cdh5-CreERT2;PNUTSfl/fl), termed PNUTSEC-KO. Two weeks after PNUTS deletion, PNUTSEC-KO mice presented severe multiorgan failure and vascular leakage. Transcriptomic analysis of PNUTS-silenced HUVECs and lungs of PNUTSEC-KO mice revealed that the PNUTS-PP1 axis tightly regulates the expression of semaphorin 3B (SEMA3B). Indeed, silencing of SEMA3B completely restored barrier function after PNUTS loss-of-function. These results reveal a pivotal role for PNUTS in endothelial homeostasis through a SEMA3B downstream pathway that provides a potential target against the effects of aging in ECs.
2024-04-15 | GSE263742 | GEO
Project description:Control of RNA Polymerase II (pol II) elongation is a critical component of gene expression in mammalian cells. The PNUTS-protein phosphatase 1 (PP1) complex controls elongation rates, slowing pol II after polyadenylation sites to promote termination. The Kaposi's sarcoma-associated herpesvirus (KSHV) co-opts pol II to express its genes, but little is known about its regulation of pol II elongation. We identified PNUTS as a suppressor of a KSHV reporter gene in a genome-wide CRISPR screen. PNUTS depletion also enhances global KSHV gene expression and overall viral replication. Reflecting its host gene activities, PNUTS binds viral RNAs downstream of polyadenylation sites, restricts transcription readthrough of viral genes, and requires PP1 interaction. Surprisingly, PNUTS represses the KSHV reporter by decreasing productive elongation at the 5´-end of the gene. From these data, we conclude that PNUTS' activity forms an intrinsic barrier to KSHV replication likely by suppressing pol II elongation at promoter-proximal regions.
2022-11-19 | GSE201045 | GEO