Similar Datasets
Project description:Leo1 occupancy in presence of dengue virus NS5
| PRJNA433884 | ENA
Project description:Cyclin-dependent kinase 12 (CDK12) interacts with Cyclin K to form a functional nuclear kinase that promotes processive transcription elongation through phosphorylation of the RNA polymerase II (Pol II) C-terminal domain (CTD). To gain a broader understanding of CDK12 cellular function, we used chemical-genetic and phosphoproteomic screening to identify a landscape of nuclear human CDK12 substrates, including regulators of transcription, chromatin organization, and RNA splicing. We further validated LEO1, a subunit of the PAF1 complex (PAF1C), as a bona fide cellular substrate of CDK12. Acute depletion of LEO1, or substituting LEO1 phosphorylation sites with alanine, attenuated PAF1C association with elongating Pol II and impaired processive transcription elongation. We also found that LEO1 interacts with, and is dephosphorylated by, the Integrator-PP2A complex (INTAC) and that INTAC promotes the association of PAF1C with Pol II. Together, this study reveals a previously unknown role for CDK12 and INTAC in regulating LEO1 phosphorylation for transcriptional regulation, providing important insights into gene transcription and its regulation.
2023-06-08 | PXD038558 | Pride
Project description:Cyclin-dependent kinase 12 (CDK12) interacts with Cyclin K to form a a functional nuclear kinase complex, which has been reported to phosphorylate the carboxyl-terminal domain (CTD) of RNA polymerase II (Pol II) for transcriptional regulation and co-transcriptional RNA processing. However, the precise mechanisms and targets of CDK12 action remain largely unknown. Here, we combined a chemical genetic screen and phosphoproteomic strategies and identified a landscape of nuclear CDK12 substrates, which included proteins that regulate transcription, chromatin organization, and RNA splicing. Next, we confirmed that the LEO1 subunit of the transcription elongation factor PAF1 complex (PAF1C) is a bona fide substrate of CDK12. Acute depletion of LEO1 reduces Pol II occupancy on the chromatin, while mutations of LEO1 phosphorylation sites to non-phosphorylatable alanine residues attenuated the association of PAF1C with elongating Pol II and chromatin, resulting in impaired processive transcription elongation. Furthermore, LEO1 C-terminus could interact with and be dephosphorylated by the Integrator-PP2A complex (INTAC), while acute depletion of INTAC in cells promotes the association between PAF1C and elongating Pol II on the chromatin. Together, this study provides a novel transcriptional regulatory mechanism that the CDK12-INTAC axis fine-tunes LEO1 phosphorylation for processive transcription elongation.
2023-05-19 | GSE217544 | GEO
Project description:Project Abstract : Trimethylation of histone H3 lysine 4 (H3K4me3) is predominantly associated with transcriptional start sites (TSSs) and is believed to facilitate transcription initiation. Furthermore, H3K4me3 plays a role in defining cell fate and specific cellular functions. Nevertheless, the precise function of H3K4me3 in transcription activation remains a topic of ongoing debate. The Polymerase-associated factor 1 complex (Paf1C), which is integral to various transcription-related cellular processes, consists of five highly conserved subunits: Paf1, Ctr9, Rtf1, Cdc73, and Leo1. While all subunits of Paf1C are indispensable for the maintenance of H3K4 methylation levels, it is noteworthy that strains lacking Leo1 exhibited unaltered levels of H3K4me3. To elucidate the role of H3K4me3, we conducted a transcriptome analysis coupled with ChIP-sequencing of H3K4me3 in cells lacking Leo1. Our research uncovers a distinctive role of Leo1 in yeast, whereby it plays a pivotal role in maintaining sterol homeostasis through the suppression of Upc2 expression. Importantly, this role stands apart from the functions of other Paf1C subunits. H3K4me3 is essential for promoting the expression of sterol uptake genes that are Upc2-dependent when Leo1 is absent. Additionally, Set1 contributes to sterol homeostasis by regulating iron metabolism and mitochondrial functions rather than directly suppressing Upc2 expression. Therefore, our findings reveal a novel role for Leo1 in sterol homeostasis and highlight the importance of H3K4me3 in promoting transcription of response genes required for sterol uptake.
2024-08-01 | GSE244733 | GEO
Project description:The goal of this study was to compare the transcriptional profile (RNA-seq) of Dengue virus 2 and mock infected cells at 24 and 36 hours post infection. Dengue virus NS5 protein plays multiple functions in the cytoplasm of infected cells, enabling viral RNA replication and counteracting host antiviral responses. Here, we demonstrate a novel function of NS5 in the nucleus where it interferes with cellular splicing. Using global proteomic analysis of infected cells together with functional studies, we found that NS5 binds spliceosome complexes and modulates endogenous splicing. In particular, we show that NS5 alone, or in the context of viral infection, interacts with core components of the U5 snRNP particle, CD2BP2 and DDX23, alters the inclusion/exclusion ratio of alternative splicing events, and changes mRNA isoform abundance of known antiviral factors. Interestingly, a genome wide transcriptome analysis, using recently developed bioinformatics tools, revealed an increase of intron retention upon dengue virus infection, and viral replication was improved by silencing specific U5 components. Different mechanistic studies indicate that binding of NS5 to the spliceosome reduces the efficiency of pre-mRNA processing, independently of NS5 enzymatic activities. We propose that NS5 binding to U5 snRNP proteins hijacks the splicing machinery resulting in a less restrictive environment for viral replication.
2016-08-09 | GSE84285 | GEO
Project description:The goal of this study was to compare the transcriptional profile (RNA-seq) of Dengue virus 2 and mock infected cells at 24 and 36 hours post infection. Dengue virus NS5 protein plays multiple functions in the cytoplasm of infected cells, enabling viral RNA replication and counteracting host antiviral responses. Here, we demonstrate a novel function of NS5 in the nucleus where it interferes with cellular splicing. Using global proteomic analysis of infected cells together with functional studies, we found that NS5 binds spliceosome complexes and modulates endogenous splicing. In particular, we show that NS5 alone, or in the context of viral infection, interacts with core components of the U5 snRNP particle, CD2BP2 and DDX23, alters the inclusion/exclusion ratio of alternative splicing events, and changes mRNA isoform abundance of known antiviral factors. Interestingly, a genome wide transcriptome analysis, using recently developed bioinformatics tools, revealed an increase of intron retention upon dengue virus infection, and viral replication was improved by silencing specific U5 components. Different mechanistic studies indicate that binding of NS5 to the spliceosome reduces the efficiency of pre-mRNA processing, independently of NS5 enzymatic activities. We propose that NS5 binding to U5 snRNP proteins hijacks the splicing machinery resulting in a less restrictive environment for viral replication. A549 cells where infected with Dengue virus 2 or mock and after 24 and 36 hours post infection mRNA was purified. Then the transcriptional profile of these cells was analyzed using RNA-seq.
2016-08-09 | E-GEOD-84285 | biostudies-arrayexpress
Project description:Project Abstract : Trimethylation of histone H3 lysine 4 (H3K4me3) is predominantly associated with transcriptional start sites (TSSs) and is believed to facilitate transcription initiation. Furthermore, H3K4me3 plays a role in defining cell fate and specific cellular functions. Nevertheless, the precise function of H3K4me3 in transcription activation remains a topic of ongoing debate. The Polymerase-associated factor 1 complex (Paf1C), which is integral to various transcription-related cellular processes, consists of five highly conserved subunits: Paf1, Ctr9, Rtf1, Cdc73, and Leo1. While all subunits of Paf1C are indispensable for the maintenance of H3K4 methylation levels, it is noteworthy that strains lacking Leo1 exhibited unaltered levels of H3K4me3. To elucidate the role of H3K4me3, we conducted a transcriptome analysis coupled with ChIP-sequencing of H3K4me3 in cells lacking Leo1. Our research uncovers a distinctive role of Leo1 in yeast, whereby it plays a pivotal role in maintaining sterol homeostasis through the suppression of Upc2 expression. Importantly, this role stands apart from the functions of other Paf1C subunits. H3K4me3 is essential for promoting the expression of sterol uptake genes that are Upc2-dependent when Leo1 is absent. Additionally, Set1 contributes to sterol homeostasis by regulating iron metabolism and mitochondrial functions rather than directly suppressing Upc2 expression. Therefore, our findings reveal a novel role for Leo1 in sterol homeostasis and highlight the importance of H3K4me3 in promoting transcription of response genes required for sterol uptake.
2024-08-01 | GSE244788 | GEO
Project description:Maintenance of open and repressed chromatin states is crucial for regulation of gene expression. To study the genes involved in maintaining chromatin states we generated a random mutant library using the Hermes transposon mutagenesis system in fission yeast Schizosacchromyces pombe. The silencing of reporter genes inserted in the euchromatic region adjacent to the heterochromatic mating type locus was monitored. We identified Leo1-Paf1, a subcomplex of the RNA Polymerase II Associated Factor 1 Complex (Paf1C), required to prevent spreading of heterochromatin into euchromatin. Through high-resolution genome-wide ChIP (ChIP-exo) we mapped the heterochromatin mark H3K9me2 in leo1∆ cells. Loss of Leo1-Paf1 led to increased heterochromatin stability at several facultative heterochromatin loci. The RNAi machinery is the major pathway for heterochromatin formation in S. pombe. However, small RNA sequencing showed that heterochromatin assembly in leo1∆ cells was RNAi-independent. By examining histone turnover rate in leo1∆ cells, we showed that deletion of Leo1 decreased nucleosome turnover, which led to heterochromatin spreading. Our data revealed that Leo1-Paf1 promotes chromatin state fluctuations by enhancing histone turnover.
2015-10-18 | GSE66940 | GEO
Project description:Maintenance of open and repressed chromatin states is crucial for regulation of gene expression. To study the genes involved in maintaining chromatin states we generated a random mutant library using the Hermes transposon mutagenesis system in fission yeast Schizosacchromyces pombe. The silencing of reporter genes inserted in the euchromatic region adjacent to the heterochromatic mating type locus was monitored. We identified Leo1-Paf1, a subcomplex of the RNA Polymerase II Associated Factor 1 Complex (Paf1C), required to prevent spreading of heterochromatin into euchromatin. Through high-resolution genome-wide ChIP (ChIP-exo) we mapped the heterochromatin mark H3K9me2 in leo1∆ cells. Loss of Leo1-Paf1 led to increased heterochromatin stability at several facultative heterochromatin loci. The RNAi machinery is the major pathway for heterochromatin formation in S. pombe. However, small RNA sequencing showed that heterochromatin assembly in leo1∆ cells was RNAi-independent. By examining histone turnover rate in leo1∆ cells, we showed that deletion of Leo1 decreased nucleosome turnover, which led to heterochromatin spreading. Our data revealed that Leo1-Paf1 promotes chromatin state fluctuations by enhancing histone turnover.
2015-10-18 | GSE66939 | GEO
Project description:We compared polyIC stimulated cells in the presence of either GFP or NS5 protein
2018-08-18 | GSE118686 | GEO