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SPT5 regulates RNA polymerase II stability via Cullin 3–ARMC5 recognition [RNA-Seq]

ABSTRACT: The stability of RNA polymerase II (Pol II) is tightly regulated during transcriptional elongation for proper control of gene expression. Our recent studies revealed that promoter-proximal Pol II is destabilized via the ubiquitin E3 ligase cullin 3 (CUL3) upon loss of transcription elongation factor SPT5. Here, we investigate how CUL3 recognizes chromatin-bound Pol II as a substrate. Using an unbiased proteomic screening approach, we identify armadillo repeat-containing 5 (ARMC5) as a CUL3 adaptor required for VCP/p97-dependent degradation of SPT5-depleted, chromatin-bound Pol II. Genome-wide analyses indicate that ARMC5 targets promoter-proximal Pol II in a BTB domain–dependent manner. Further biochemical analysis demonstrates that interaction between ARMC5 and Pol II requires the transcriptional cyclin-dependent kinase 9 (CDK9), supporting a phospho-dependent degradation model. We propose that defective, promoter-proximal Pol II that lacks SPT5 is rapidly eliminated from chromatin in a noncanonical early termination pathway that requires CDK9-dependent interaction with the CUL3-ARMC5 ubiquitin ligase complex.

ORGANISM(S): Homo sapiens

PROVIDER: GSE277370 | GEO | 2025/01/24

REPOSITORIES: GEO

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SPT5 regulates RNA polymerase II stability via Cullin 3–ARMC5 recognition [ChIP-seq]

Project description:The stability of RNA polymerase II (Pol II) is tightly regulated during transcriptional elongation for proper control of gene expression. Our recent studies revealed that promoter-proximal Pol II is destabilized via the ubiquitin E3 ligase cullin 3 (CUL3) upon loss of transcription elongation factor SPT5. Here, we investigate how CUL3 recognizes chromatin-bound Pol II as a substrate. Using an unbiased proteomic screening approach, we identify armadillo repeat-containing 5 (ARMC5) as a CUL3 adaptor required for VCP/p97-dependent degradation of SPT5-depleted, chromatin-bound Pol II. Genome-wide analyses indicate that ARMC5 targets promoter-proximal Pol II in a BTB domain–dependent manner. Further biochemical analysis demonstrates that interaction between ARMC5 and Pol II requires the transcriptional cyclin-dependent kinase 9 (CDK9), supporting a phospho-dependent degradation model. We propose that defective, promoter-proximal Pol II that lacks SPT5 is rapidly eliminated from chromatin in a noncanonical early termination pathway that requires CDK9-dependent interaction with the CUL3-ARMC5 ubiquitin ligase complex.

2025-01-24 | GSE277372 | GEO

SPT5 stabilizes promoter-proximal RNA polymerase II prior to release into gene bodies

Project description:The regulation of gene expression by RNA polymerase II (Pol II) is a multistep process requiring the concerted action of diverse transcription factors. Very little is known about in vivo function of transcription factor SPT5 as its deletion results in loss of viability. To circumvent this issue and define in vivo mechanism of action for SPT5, we employed acute degradation of SPT5 and studied its consequence on transcription. We find that SPT5 loss triggers degradation of the core Pol II subunit RPB1, a process which we show to be evolutionarily conserved from yeast to human. This RPB1 degradation requires the E3 ubiquitin ligase Cullin 3, the unfoldase VCP/p97 and a novel form of CDK9 kinase complex. SPT5 specifically stabilizes Pol II at promoter proximal regions, permitting Pol II release from promoters to gene bodies. Our findings provide mechanistic insight into the in vivo function of SPT5 in stabilization of promoter-proximal Pol II prior to release into gene bodies for safeguarding accurate gene expression.

2021-08-31 | GSE168827 | GEO

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

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

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RPB1 ChIP-Seq of adrenal glands tissue from Wild Type (WT) and Armc5 knockout mice

Project description:RPB1-specific ubiquitin ligase (E3) controls the degradation of this largest subunit of Pol II and consequently the Pol II pool size. Although several RPB1-specific E3s have been documented, their function is only revealed in cells with massive DNA damage. We demonstrated that ARMC5 physically interacted with cullin3 (CUL3) and RPB1. ARMC5 is an armadillo domain-containing protein with unknown mechanisms of action. ARMC5 deletion caused significant RPB1 accumulation in all the major organs under a physiological condition in the absence of artificially induced DNA damage. This is accompanied by reduced RPB1 ubiquitination. In vitro ubiquitination assay proved that ARMC5, CUL3, and RBX1 formed an effective E3 for RPB1. RPB1 was confirmed as being highly accumulated in the adrenal gland nodules from PBMAH patients with mutations in the ARMC5 coding sequence. Surprisingly, the compromised RPB1 degradation did not lead to increased Pol II stalling according to ChIP-Seq, or a general decrease of gene transcription according to RNA-seq. On the contrary, among 1486 differentially expressed genes in the KO adrenal glands, 93.5% of them were upregulated, probably due to the enlarged Pol II pool size. Our results showed that the ARMC3-CUL3 E3 was the dominant E3 in normal cells and organs and controlled Pol II pool size. The abnormally large Pol II pool size due to ARMC5 mutation dysregulated a large number of genes that in turn led to many phenotypes, including PBMAH in humans.

2022-05-20 | GSE169578 | GEO

RNA-Seq of adrenal glands tissue from Wild Type (WT) and Armc5 knockout mice

2022-05-20 | GSE169263 | GEO

Distinct Cdk9-phosphatase switches act on elongation factor Spt5 at the beginning and end of the RNA polymerase II transcription cycle

Project description:The Pol II transcription cycle is ordered by CDKs and phosphatases. In fission yeast, Cdk9 phosphorylates carboxy-terminal repeats (CTRs) of Spt5 while inhibiting PP1 during elongation. Transcription past the cleavage and polyadenylation signal (CPS) coincides with PP1-dependent Spt5 dephosphorylation and leads to Pol II pausing with phosphorylated CTD-Ser2 (pSer2). Here we show this switch is conserved in humans: Cdk9 inhibition decreases phosphorylation of both PP1g and Spt5-Thr806 (pThr806), and induces pSer2 upstream of the CPS, whereas PP1 depletion increases pThr806. Moreover, in unperturbed cells, pThr806 is diminished in 3’-paused complexes where pSer2 is maximal. Cdk9 also phosphorylates Spt5 on Ser666, a PP1-refractory site between conserved KOW4 and KOW5 motifs; pSer666 increases upon promoter-proximal pause release and, in contrast to pThr806, persists beyond the CPS. We identify PP4—another target of inhibitory phosphorylation by Cdk9—as a pSer666 phosphatase. PP4 and PP1g are enriched at 5’ and 3’ ends of genes, respectively, consistent with pSer666 and pThr806 distributions. Therefore, distinct Cdk9-phosphatase switches operate on Spt5 at different steps in transcription.

2020-08-04 | GSE138548 | GEO

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

2024-11-07 | GSE253354 | GEO

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.

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