Integrator Recruits Protein Phosphatase 2A to Prevent Pause Release and Enable Transcription Termination
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ABSTRACT:
PROVIDER: PRJNA633845 | ENA |
REPOSITORIES: ENA
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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.
2020-10-06 | GSE150844 | GEO
Project description: Not available
| S-EPMC7660970 | biostudies-literature
Project description:RNAPII pausing/termination shortly after initiation is a hallmark of gene regulation. However, the molecular mechanisms involved are still to be uncovered. Here, we show that NELF interacts with Integrator complex subunits (INTScom) forming a stable complex with RNPII and Spt5. The interaction between NELF and INTScom subunits is RNA and DNA independent. Using both HIV-1 promoter and genome wide analyses, we demonstrate that Integrator subunits specifically control NELF-mediated RNAPII pause/release at coding genes. The strength of RNAPII pausing is determined by the nature of the NELF-associated complex. Interestingly, in addition to controlling RNAPII pause release INTS11 catalytic subunit of the INTScom is required for the synthesis of full length mRNA. Finally, INTScom-target genes are enriched in HIV-1 TAR/ NELF-binding element and in a 3’box sequence required for snRNA biogenesis. Revealing these unexpected functions of INTScom in regulating RNAPII pausing/release and completion of mRNA synthesis of NELF-target genes will contribute to our understanding of the gene expression cycle. Genome-wide expression in HeLa cells in the absence of Integrator 11, or NELF or mock (control) depleted by strand-specific RNASeq (Illumina)
2015-05-19 | E-GEOD-60585 | biostudies-arrayexpress
Project description:RNAPII pausing/termination shortly after initiation is a hallmark of gene regulation. However, the molecular mechanisms involved are still to be uncovered. Here, we show that NELF interacts with Integrator complex subunits (INTScom) forming a stable complex with RNPII and Spt5. The interaction between NELF and INTScom subunits is RNA and DNA independent. Using both HIV-1 promoter and genome wide analyses, we demonstrate that Integrator subunits specifically control NELF-mediated RNAPII pause/release at coding genes. The strength of RNAPII pausing is determined by the nature of the NELF-associated complex. Interestingly, in addition to controlling RNAPII pause release INTS11 catalytic subunit of the INTScom is required for the synthesis of full length mRNA. Finally, INTScom-target genes are enriched in HIV-1 TAR/ NELF-binding element and in a 3’box sequence required for snRNA biogenesis. Revealing these unexpected functions of INTScom in regulating RNAPII pausing/release and completion of mRNA synthesis of NELF-target genes will contribute to our understanding of the gene expression cycle. Binding profiles of Integrator subunits in HeLa cells by ChIP-Seq (Illumina) Please note that the MACS14*.tar.gz contains MACS output bed and xls files and the 'readme.txt' contains a detailed description of each file.
2015-05-19 | E-GEOD-60584 | biostudies-arrayexpress
Project description:RNAPII pausing/termination shortly after initiation is a hallmark of gene regulation. However, the molecular mechanisms involved are still to be uncovered. Here, we show that NELF interacts with Integrator complex subunits (INTScom) forming a stable complex with RNPII and Spt5. The interaction between NELF and INTScom subunits is RNA and DNA independent. Using both HIV-1 promoter and genome wide analyses, we demonstrate that Integrator subunits specifically control NELF-mediated RNAPII pause/release at coding genes. The strength of RNAPII pausing is determined by the nature of the NELF-associated complex. Interestingly, in addition to controlling RNAPII pause release INTS11 catalytic subunit of the INTScom is required for the synthesis of full length mRNA. Finally, INTScom-target genes are enriched in HIV-1 TAR/ NELF-binding element and in a 3’box sequence required for snRNA biogenesis. Revealing these unexpected functions of INTScom in regulating RNAPII pausing/release and completion of mRNA synthesis of NELF-target genes will contribute to our understanding of the gene expression cycle.
2015-05-19 | GSE60585 | GEO
Project description:RNAPII pausing/termination shortly after initiation is a hallmark of gene regulation. However, the molecular mechanisms involved are still to be uncovered. Here, we show that NELF interacts with Integrator complex subunits (INTScom) forming a stable complex with RNPII and Spt5. The interaction between NELF and INTScom subunits is RNA and DNA independent. Using both HIV-1 promoter and genome wide analyses, we demonstrate that Integrator subunits specifically control NELF-mediated RNAPII pause/release at coding genes. The strength of RNAPII pausing is determined by the nature of the NELF-associated complex. Interestingly, in addition to controlling RNAPII pause release INTS11 catalytic subunit of the INTScom is required for the synthesis of full length mRNA. Finally, INTScom-target genes are enriched in HIV-1 TAR/ NELF-binding element and in a 3’box sequence required for snRNA biogenesis. Revealing these unexpected functions of INTScom in regulating RNAPII pausing/release and completion of mRNA synthesis of NELF-target genes will contribute to our understanding of the gene expression cycle.
2015-05-19 | GSE60584 | GEO
Project description:A PP2A-Integrator complex regulates transcriptional pause-release by functionally opposing CDK9
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Project description:Complexes containing INTS3 and either NABP1 or NABP2 were initially characterized in DNA damage responses, but their biochemical function remained unknown. Using affinity purifications and HIV Integration Targeting-Sequencing (HIT-Seq), we find that these complexes are part of the Integrator complex, which binds RNA Polymerase II and regulates specific target genes. Integrator cleaves snRNAs as part of their processing to their mature form in a mechanism that is intimately coupled with transcription termination. However, HIT-Seq reveals that Integrator also binds to the 3’ end of replication-dependent histones and promoter proximal regions of genes with polyadenylated transcripts. Depletion of Integrator subunits results in transcription termination failure, disrupting histone mRNA processing, and leading to polyadenylation of snRNAs and histone mRNAs. Furthermore, promoter proximal binding of Integrator negatively regulates expression of genes whose transcripts are normally polyadenylated. Integrator recruitment to all three gene classes is DSIF-dependent, suggesting that Integrator functions as a termination complex at DSIF-dependent RNA Polymerase II pause sites. HITseq was used to interrogate chromatin binding sites of of proteins in the complex (including INIP, INTS3, INTS9, INTS11, NABP1, NABP2, NELFA, NELFB, NELFCD, NELFE, SPT5). These proteins were fused to LEDGF-IBD and expressed in mouse LEDGF-/-MEF cells. The fusion proteins then target HIV integration to the chromatin binding sites. The HIV integration sites were identified using linker-mediated PCR and highthroughput sequencing and serve as surrogate for chromatin binding sites. Mapping was done using mouse genome mm9.
2015-01-20 | E-GEOD-65090 | biostudies-arrayexpress
Project description:We investigated the function of the Integrator Complex and PP2A during transcription.
2021-05-23 | GSE145379 | GEO
Project description:MicroRNA (miRNA) homeostasis is crucial for the post-transcriptional regulation of their target genes and miRNA dysregulation has been linked to multiple diseases, including cancer. The mechanistic steps of miRNA biogenesis are well understood at molecular level1,2. Subsequent miRNA duplex loading into members of the Argonaute (Ago) protein family, representing the core of the RNA-induced silencing complex (RISC), is pivotal to miRNA-mediated gene silencing3-5. The Integrator complex has been previously identified as important regulator of RNA maturation, RNA polymerase II pause-release, and premature transcriptional termination, processes dependent on Integrator’s catalytical activity6-9. Here we report that absence of the Integrator complex leads to a global loss of mature miRNAs. By incorporating 4-Thiouridine (s4U) in nascent transcripts, we traced miRNA fate from biogenesis to stabilization and identified Integrator to be essential for proper miRNA assembly into RISC. Indeed, Integrator enhances miRNA-Ago2 interaction in vitro, which functionally translates to amplified miRNA target cleavage. These findings demonstrate for the first time cytoplasmic Integrator complex functions and stress an indirect role of Integrator in transcription regulation through modulation of miRNA abundance and stability.
2023-02-14 | GSE178127 | GEO