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RPB7/CTDP1 stabilizes and licenses Pol II termination to reinitiation [RNA-Seq]

ABSTRACT: The regulations of transcription process have been extensively investigated, but how RNA Pol II terminates and reinitiates a new round of transcription remains elusive. Here, we found that RPB7 loss triggers the degradation of RNA Pol II. RPB7 degradation combing knock out of E3 ligase Cullin 3 led to the accumulation of phosphorylated Pol II in the soluble fractions and Pol II reinitiation failure. The stability of recycled Pol II is dependent on the loop-region and dimerization of RPB7 and the phosphorylation state of Pol II CTD. Site-specific cancer mutations in the RPB7 loop-region caused the dysregulations of specific gene expression. Moreover, the loss of RPB7 or CTD phosphatase subunit 1 (CTDP1) result in the defects in Pol II termination-reinitiation recycle. Together, our study demonstrates that RPB7/CTDP1 controls the dephosphorylation and stabilization of RNA Pol II to permit RNA Pol II termination to reinitiation recycle in mammalian cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE223472 | GEO | 2025/02/05

REPOSITORIES: GEO

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RPB7/CTDP1 stabilizes and licenses Pol II termination to reinitiation [ChAR-Seq]

Project description:The regulations of transcription process have been extensively investigated, but how RNA Pol II terminates and reinitiates a new round of transcription remains elusive. Here, we found that RPB7 loss triggers the degradation of RNA Pol II. RPB7 degradation combing knock out of E3 ligase Cullin 3 led to the accumulation of phosphorylated Pol II in the soluble fractions and Pol II reinitiation failure. The stability of recycled Pol II is dependent on the loop-region and dimerization of RPB7 and the phosphorylation state of Pol II CTD. Site-specific cancer mutations in the RPB7 loop-region caused the dysregulations of specific gene expression. Moreover, the loss of RPB7 or CTD phosphatase subunit 1 (CTDP1) result in the defects in Pol II termination-reinitiation recycle. Together, our study demonstrates that RPB7/CTDP1 controls the dephosphorylation and stabilization of RNA Pol II to permit RNA Pol II termination to reinitiation recycle in mammalian cells.

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RPB7/CTDP1 stabilizes and licenses Pol II termination to reinitiation [ChIP-Seq]

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CTDP1 and RPB7 stabilize and license Pol II termination to reinitiation [Micro-C]

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CTDP1 and RPB7 stabilize and license Pol II termination to reinitiation [PRO-seq]

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CTD tyrosine phosphorylation impairs termination factor recruitment to transcribing RNA polymerase II

Project description:ChIP-chip was performed to identify the genomic binding locations for the termination factors Nrd1, and Rtt103, and for RNA polymerase (Pol) II phosphorylated at the tyrosine 1 and threonine 4 position of its C-terminal domain (CTD). In different phases of the transcription cycle, Pol II recruits different factors via its CTD, which consists of heptapeptide repeats with the sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Here we show that the CTD of transcribing yeast Pol II is phosphorylated at Tyr1, and that this impairs recruitment of termination factors. Tyr1 phosphorylation levels rise downstream of the transcription start site (TSS), and decrease before the polyadenylation (pA) site. Tyr1-phosphorylated gene bodies are depleted of CTD-binding termination factors Nrd1, Pcf11, and Rtt103. Tyr1 phosphorylation blocks CTD binding by these termination factors, but stimulates binding of elongation factor Spt6. These results show that CTD modifications can not only stimulate but also block factor recruitment, and lead to an extended CTD code for transcription cycle coordination.

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RNA polymerase II termination involves CTD tyrosine dephosphorylation by CPF subunit Glc7

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Mammalian NET-seq reveals genome-wide nascent transcription coupled to RNA processing

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2015-04-23 | E-GEOD-60358 | biostudies-arrayexpress

TOX4 Facilitates Promoter-Proximal Pausing and C-Terminal Domain Dephosphorylation of RNA Polymerase II in Human Cells

Project description:TOX4 is one of the regulatory proteins of PP1 phosphatases with poorly understood functions. Here we show that chromatin occupancy pattern of TOX4 resembles that of RNA polymerase II (Pol II), and its loss increases cellular level of C-terminal domain (CTD) phosphorylated Pol II but mainly decreases Pol II occupancy on promoters. In addition, elongation rate analyses by 4sUDRB-seq suggest that TOX4 restricts pause release and early elongation but promotes late elongation. Moreover, TT-seq analyses indicate that TOX4 loss mainly decreases transcriptional output. Mechanistically, TOX4 may restrict pause release through facilitating CTD serine 2 and DSIF dephosphorylation, and promote Pol II recycling and reinitiation through facilitating CTD serines 2 and 5 dephosphorylation. Furthermore, among the PP1 phosphatases, TOX4 preferentially binds PP1α and is capable of facilitating Pol II CTD dephosphorylation in vitro. These results lay the foundation for a better understanding of the role of TOX4 in transcriptional regulation.

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Mammalian NET-seq reveals genome-wide nascent transcription coupled to RNA processing

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RNA polymerase II activity revealed by GRO-seq and pNET-seq in Arabidopsis

Project description:RNA polymerase II (Pol II) play an essential role in gene expression. Here, we adapted mammalian Native Elongation Transcript sequencing and Global Run On sequencing to profile nascent RNA genome wide in Arabidopsis. We found Pol II tends to accumulate downstream of transcription start site and pausing at proximal promoter is an important regulatory step for Pol II transcription although loosely controlled. Furthermore, the Pol II with unphosphorylated carboxyl-terminal domain (CTD) mainly accumulates downstream the TSS, while the Ser5P CTD Pol II associates with spliceosome, and the Ser2P CTD Pol II presents a sharp peak 250 base pair downstream of polyadenylation site indicating a stringent control of termination for protein coding genes; whilst the termination of noncoding genes is not. Active expressed genes can be classified into three clusters according to the distribution patterns of different Pol II isoforms. In summary, we demonstrated the modified plant GRO-seq and mNET-seq are suitable to study RNA Pol II dynamics in planta. Although transcription is conserved among high eukaryotes, Pol II has its feature in Arabidopsis.

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