Similar Datasets
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
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 | GSE200991 | 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 | GSE201000 | 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 | GSE200992 | GEO
Project description:In this study, we found that the host protein PNUTS suppressed Kaposi's sarcoma-associated herpesvirus (KSHV) gene expression during lytic reactivation and from heterologous viral reporters. To gain insights into the mechanism, we performed ChIP-seq of the pol II subunit RPB3 in cells with an integrated reporter containing the KSHV ORF59 gene. Our results were consistent with previous reports showing that depletion of PNUTS leads to global loss of pol II slow down and termination after polyadenylation sites. In contrast, PNUTS depletion appeared to decrease promoter-proximal pausing on our integrated reporter suggesting a distinct mode of gene regulation on viral genes.
2022-11-22 | GSE215345 | GEO
Project description:The c-MYC (MYC) oncoprotein is deregulated in over 50% of cancers, yet regulatory mechanisms controlling MYC remain unclear. To this end, we interrogated the MYC interactome using BioID mass spectrometry (MS) and identified PP1 (protein phosphatase 1) and its regulatory subunit PNUTS (protein phosphatase-1 nuclear-targeting subunit) as MYC interactors. We demonstrate that endogenous MYC and PNUTS interact across multiple cell types and that they co-occupy MYC target gene promoters. Inhibiting PP1 by RNAi or pharmacological inhibition results in MYC hyperphosphorylation at multiple serine and threonine residues, leading to a decrease in MYC protein levels due to proteasomal degradation through the canonical SCFFBXW7 pathway. MYC hyperphosphorylation can be rescued specifically with exogenous PP1, but not other phosphatases. Hyperphosphorylated MYC retained interaction with its transcriptional partner MAX, but binding to chromatin is significantly compromised. Our work demonstrates that PP1/PNUTS stabilizes chromatin-bound MYC in proliferating cells.
| S-EPMC6115416 | biostudies-literature
Project description:The function of protein phosphatase 1 nuclear-targeting subunit (PNUTS)--one of the most abundant nuclear-targeting subunits of protein phosphatase 1 (PP1c)--remains largely uncharacterized. We show that PNUTS depletion by small interfering RNA activates a G2 checkpoint in unperturbed cells and prolongs G2 checkpoint and Chk1 activation after ionizing-radiation-induced DNA damage. Overexpression of PNUTS-enhanced green fluorescent protein (EGFP)--which is rapidly and transiently recruited at DNA damage sites--inhibits G2 arrest. Finally, γH2AX, p53-binding protein 1, replication protein A and Rad51 foci are present for a prolonged period and clonogenic survival is decreased in PNUTS-depleted cells after ionizing radiation treatment. We identify the PP1c regulatory subunit PNUTS as a new and integral component of the DNA damage response involved in DNA repair.
| S-EPMC2966950 | biostudies-literature
Project description:In S phase, the replication and transcription of genomic DNA need to accommodate each other, otherwise their machineries collide, with chromosomal instability as a possible consequence. Here, we characterized the human replication fork barrier (RFB) that is present downstream from the 47S pre-rRNA gene (ribosomal DNA [rDNA]). We found that the most proximal transcription terminator, Sal box T1, acts as a polar RFB, while the other, Sal box T4/T5, arrests replication forks bidirectionally. The fork-arresting activity at these sites depends on polymerase I (Pol I) transcription termination factor 1 (TTF-1) and a replisome component, TIMELESS (TIM). We also found that the RFB activity was linked to rDNA copies with hypomethylated CpG and coincided with the time that actively transcribed rRNA genes are replicated. Failed fork arrest at RFB sites led to a slowdown of fork progression moving in the opposite direction to rRNA transcription. Chemical inhibition of transcription counteracted this deceleration of forks, indicating that rRNA transcription impedes replication in the absence of RFB activity. Thus, our results reveal a role of RFB for coordinating the progression of replication and transcription activity in highly transcribed rRNA genes.
| S-EPMC4405639 | biostudies-literature
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:The Rif1 protein negatively regulates telomeric TG repeat length in the budding yeast Saccharomyces cerevisiae, but how it prevents telomere over-extension is unknown. Rif1 was recently shown to control DNA replication by acting as a Protein Phosphatase 1 (PP1)-targeting subunit. Therefore, we investigated whether Rif1 controls telomere length by targeting PP1 activity. We find that a Rif1 mutant defective for PP1 interaction causes a long-telomere phenotype, similar to that of rif1? cells. Tethering PP1 at a specific telomere partially substitutes for Rif1 in limiting TG repeat length, confirming the importance of PP1 in telomere length control. Ablating Rif1-PP1 interaction is known to cause precocious activation of telomere-proximal replication origins and aberrantly early telomere replication. However, we find that Rif1 still limits telomere length even if late replication is forced through deletion of nearby replication origins, indicating that Rif1 can control telomere length independent of replication timing. Moreover we find that, even at a de novo telomere created after DNA synthesis during a mitotic block, Rif1-PP1 interaction is required to suppress telomere lengthening and prevent inappropriate recruitment of Tel1 kinase. Overall, our results show that Rif1 controls telomere length by recruiting PP1 to directly suppress telomerase-mediated TG repeat lengthening.
| S-EPMC5934629 | biostudies-literature