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Ubiquitylation of Pol II controls early stages of the transcription cycle [TT-seq]

ABSTRACT: Control of RNA Polymerase II (Pol II) through ubiquitylation is essential for the DNA-damage response. Here we reveal a distinct ubiquitylation pathway in human cells that targets excessive and defective Pol II molecules at the initial stages of the transcription cycle. This pathway, mediated by ARMC5CUL3 ubiquitin ligase, drives homeostatic Pol II turnover, and is further enhanced when early stages of transcription - initiation and pausing - are perturbed. Upon ARMC5 loss, Pol II accumulates in the free pool and in the promoter-proximal zone, but is not permitted into elongation. We identify Integrator subunit 8 (INTS8) as a gatekeeper preventing the release of excess Pol II molecules into gene bodies. Combined loss of ARMC5 and INTS8 has strong detrimental effects on cell growth, with ARMC5 loss exacerbating transcriptional defects seen in INTS8-depleted cells. These findings uncover that ARMC5CUL3 and INTS8 form a collaborative checkpoint, monitoring the quantity and quality of transcription complexes before they are licensed into elongation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE266973 | GEO | 2024/05/13

REPOSITORIES: GEO

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Ubiquitylation of Pol II controls early stages of the transcription cycle [ChIP-seq]

Project description:Control of RNA Polymerase II (Pol II) through ubiquitylation is essential for the DNA-damage response. Here we reveal a distinct ubiquitylation pathway in human cells that targets excessive and defective Pol II molecules at the initial stages of the transcription cycle. This pathway, mediated by ARMC5CUL3 ubiquitin ligase, drives homeostatic Pol II turnover, and is further enhanced when early stages of transcription - initiation and pausing - are perturbed. Upon ARMC5 loss, Pol II accumulates in the free pool and in the promoter-proximal zone, but is not permitted into elongation. We identify Integrator subunit 8 (INTS8) as a gatekeeper preventing the release of excess Pol II molecules into gene bodies. Combined loss of ARMC5 and INTS8 has strong detrimental effects on cell growth, with ARMC5 loss exacerbating transcriptional defects seen in INTS8-depleted cells. These findings uncover that ARMC5CUL3 and INTS8 form a collaborative checkpoint, monitoring the quantity and quality of transcription complexes before they are licensed into elongation.

2024-05-13 | GSE266972 | GEO

Ubiquitylation of Pol II controls early stages of the transcription cycle [dxChIP-seq]

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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.

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

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Ubiquitylation of Pol II controls early stages of the transcription cycle [TT-seq]

Project description:Ubiquitylation of Pol II controls early stages of the transcription cycle [TT-seq]

| PRJNA1108871 | ENA

ARMC5 controls the degradation of most Pol II subunits, and ARMC5 mutation increases neural tube defect risks in mice and humans

Project description:ARMC5 is an armadillo domain (ARM)-containing protein. We found that Armc5 KO mice had an increased incidence of neural tube defects (NTDs). ARMC5 is the substrate recognition component of a ubiquitin ligase that targets POLR2A, the largest subunit of RNA polymerase II (Pol II). Surprisingly, the absence of ARMC5 caused the accumulation of not only POLR2A, but most of the other 11 Pol II subunits associated with POLR2A, indicating that the degradation of the whole Pol II complex is compromised. This did not lead to generalized Pol II stalling or a generalized decrease in mRNA transcription. In neural progenitor cells, ARMC5 KO only dysregulated 106 genes, some of which are known to be involved in neural tube development. FOLH1, critical for folate metabolism and vital in neural tube development, was downregulated in the KO intestine, suggesting that it is a downstream effector gene for NTD. To assess whether ARMC5 gene mutation was associated with human NTD, we conducted whole-exome sequencing of 511 patients with myelomeningocele, a severe form of NTD. Nine deleterious single nucleotide variants were discovered in the ARMC5 coding sequence. Four of them were validated in that they weakened the interaction between ARMC5 and POLR2A; consequently, POLR2A ubiquitination was decreased. This proves that our findings in mice are relevant to human NTD; it also supports the role of Pol II in mediating NTD pathogenesis. Our findings indicate that mutations in ARMC5 increase the risk of NTD and support the role of Pol II in NTD pathogenesis. Further investigation is needed to determine the cause-and-effect relationship between an enlarged Pol II pool and NTD and to validate the potential role of downregulated FOLH1 expression in NTD pathogenesis.

2023-12-06 | PXD047572 | Pride

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