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RNAPII interactome in INTS6 degron cells

ABSTRACT: In this study we show that acute INTS6 depletion remodels the RNAPII interactome and enhances ARMC5 binding, the same as what we observed in Ints6 KO cells. To investigate how acute INTS6 depletion affects RNAPII function, we determined the RNAPII interactome in the the Ints6 degron cells. Chromatin fractions from DMSO and dTAG treated Ints6 degron cells (treated for 1 hour) were analyzed by RNAPII IP-mass spectrometry. There are 12 samples in this MS run, samples 6-dTAG repeats1-3 and 6-DMSO repeats 1-3 are involved in the paper.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Flp-in-t-rex Cell

SUBMITTER: Michael Wierer

LAB HEAD: Jesper Svejstrup

PROVIDER: PXD053351 | Pride | 2025-12-22

REPOSITORIES: Pride

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Publications

Redundant pathways for removal of defective RNA polymerase II complexes at a promoter-proximal pause checkpoint.

Blears Daniel D Lou Jiangman J Fong Nova N Mitter Richard R Sheridan Ryan M RM He Dandan D Dirac-Svejstrup A Barbara AB Bentley David D Svejstrup Jesper Q JQ

Molecular cell 20241105 24

The biological purpose of Integrator and RNA polymerase II (RNAPII) promoter-proximal pausing remains uncertain. Here, we show that loss of INTS6 in human cells results in increased interaction of RNAPII with proteins that can mediate its dissociation from the DNA template, including the CRL3<sup>ARMC5</sup> E3 ligase, which ubiquitylates CTD serine<sub>5</sub>-phosphorylated RPB1 for degradation. ARMC5-dependent RNAPII ubiquitylation is activated by defects in factors acting at the promoter-pro ...[more]

PMID: 39504960

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RNAPII interactome in INTS6 KO and WT cells

Project description:In this study we show that INTS6 KO remodels the RNAPII interactome and enhances ARMC5 binding. To investigate how INTS6 affects RNAPII function, we determined the RNAPII interactome in the presence and absence of INTS6 using Ints6 KO cells. Chromatin fractions from WT and INTS6 KO cells, with or without DRB treatment, were analyzed by RNAPII IP-mass spectrometry.

2025-12-22 | PXD053346 | Pride

Distinct pathways for removal of defective RNA polymerase II transcription complexes at a promoter-proximal pause checkpoint

Project description:The Integrator complex interacts with RNA polymerase II (RNAPII) at promoter-proximal pause sites but its function there remains uncertain. Here, we show that when Integrator function is compromised by loss of INTS6, RNAPII interacts increasingly with proteins that mediate its dissociation from DNA, including CUL3-ARMC5, which ubiquitylates Ser5-phosphorylated Rpb1 and targets it for degradation. ARMC5-dependent RNAPII ubiquitylation is activated by defects in factors required for correctly regulated promoter-proximal pausing, including Integrator, DSIF and mRNA capping enzyme. This ARMC5 checkpoint curtails an appreciable fraction of RNAPII transcription, with ARMC5 knockout cells producing uncapped, nascent RNA transcripts that fail to mature into stable mRNA. Concomitant loss of INTS6 and ARMC5 greatly stabilizes RNAPII at the pause and has severe consequences for cell growth and viability. Our data support a model in which CRL3ARMC5 functions alongside Integrator in a checkpoint mechanism that removes faulty RNAPII complexes at promoter-proximal pause sites to safeguard transcription integrity.

2024-11-05 | GSE251941 | GEO

Distinct pathways for removal of defective RNA polymerase II transcription complexes at a promoter-proximal pause checkpoint [RN21114 Ttchemn-Seq]

Project description:The purpose and function of Integrator and RNA polymerase II (RNAPII) promoter-proximal pausing remain uncertain. Here, we show that when Integrator function is compromised by loss of INTS6, RNAPII interacts increasingly with proteins from alternative pathways for its DNA dissociation, including CUL3-ARMC5 (CRL3ARMC5), which ubiquitylates Ser5-phosphorylated Rpb1 and targets it for degradation. ARMC5-dependent RNAPII ubiquitylation is activated by defects in factors required for correctly regulated promoter-proximal pausing, including Integrator, DSIF and mRNA capping enzyme. This ARMC5 checkpoint curtails an appreciable fraction of RNAPII transcription, with ARMC5 knockout cells producing uncapped, nascent RNA transcripts that fail to mature into stable mRNA. Concomitant loss of INTS6 and ARMC5 greatly stabilizes RNAPII at the pause and has severe consequences for cell growth and viability. Our data support a model in which CRL3ARMC5 functions alongside Integrator in a checkpoint mechanism that removes faulty RNAPII complexes at promoter-proximal pause sites to safeguard transcription integrity.

2024-11-05 | GSE253353 | GEO

Distinct pathways for removal of defective RNA polymerase II transcription complexes at a promoter-proximal pause checkpointm [RN21236 dTAG TTchem-seq]

2024-11-07 | GSE253354 | GEO

A PP2A-Integrator complex fine-tunes transcription by opposing CDK9

Project description:Gene expression by RNA Polymerase II (RNAPII) is tightly controlled by Cyclindependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The RNAPII pausing checkpoint, engaged after transcription initiation, is controlled by CDK9 to regulate transcription in metazoans. We discovered that CDK9-mediated RNAPII pause-release is functionally opposed by a protein phosphatase 2A (PP2A) complex. PP2A dynamically competes for key CDK9 substrates, DSIF and RNAPIICTD, and is recruited to transcription pausing sites by the Integrator complex subunit INTS6. INTS6 depletion confers resistance to CDK9 inhibition in a variety of normal and tumor cell lines. Loss of INTS6 abolishes the Integrator-PP2A association leading to unrestrained CDK9 activity, which amplifies transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill MLLrearranged leukemias and solid tumors and provide therapeutic benefit in vivo. These data demonstrate that finely-tuned gene expression relies on the balance of kinase and phosphatase activity at the pausing checkpoint.

2022-08-12 | PXD022962 | Pride

ARMC5-mediated degradation of RNA polymerase II as a last resort in the promoter-proximal pausing checkpoint [TTchem-Seq]

Project description:RNA polymerase II (RNAPII) promoter-proximal pausing is an early step in the transcription cycle, which occurs alongside important events such as mRNA capping. Here, we identify a ubiquitin ligase complex, CUL3-ARMC5 (CRL3ARMC5) which is required for targeting promotor-proximally paused RNAPII for degradation. While a basal level of ARMC5-dependent RNAPII ubiquitylation/degradation occurs even in unperturbed cells, depletion of proteins regulating promoter-proximal pausing results in markedly increased ubiquitylation. We suggest that such RNAPII ubiquitylation/degradation serves as a “last resort” pathway for removing inept polymerases at a promoter-proximal pause checkpoint to avoid their release for unproductive transcript elongation. In agreement with this idea, ARMC5 knockout cells display increased levels of nascent transcription but with pre-mRNAs exhibiting capping defects. Together, our findings support a model in which promoter-proximal pausing functions as a transcription cycle checkpoint and indicate broad role for the CRL3ARMC5 ligase in RNAPII poly-ubiquitylation and degradation.

2024-11-05 | GSE219075 | GEO

ARMC5-mediated degradation of RNA polymerase II as a last resort in the promoter-proximal pausing checkpoint [RNA-Seq]

2024-11-05 | GSE219074 | GEO

ARMC5-mediated degradation of RNA polymerase II as a last resort in the promoter-proximal pausing checkpoint [PRO-seq]

2024-11-05 | GSE219073 | GEO

A PP2A-Integrator complex fine-tunes transcription by opposing CDK9 [ChIP-seq]

Project description:Gene expression by RNA Polymerase II (RNAPII) is tightly controlled by Cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The RNAPII pausing checkpoint, engaged after transcription initiation, is controlled by CDK9 to regulate transcription in metazoans. We discovered that CDK9-mediated RNAPII pause-release is functionally opposed by a protein phosphatase 2A (PP2A) complex. PP2A dynamically competes for key CDK9 substrates, DSIF and RNAPII-CTD, and is recruited to transcription pausing sites by the Integrator complex subunit INTS6. INTS6 depletion confers resistance to CDK9 inhibition in a variety of normal and tumor cell lines. Loss of INTS6 abolishes the Integrator-PP2A association leading to unrestrained CDK9 activity, which amplifies transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill MLL-rearranged leukemias and solid tumors and provide therapeutic benefit in vivo. These data demonstrate that f inely-tuned gene expression relies on the balance of kinase and phosphatase activity at the pausing checkpoint.

2021-05-23 | GSE163804 | GEO

A PP2A-Integrator complex fine-tunes transcription by opposing CDK9 [4sU-seq]

2021-05-23 | GSE163803 | GEO