Combinatorial phosphorylation on CTD of RNA polymerase II selectively controls transcription and export of protein-coding mRNAs
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ABSTRACT:
PROVIDER: PRJNA1208980 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR31952554.fastq.gz | Fastqsanger.gz | |||
| SRR31952555.fastq.gz | Fastqsanger.gz | |||
| SRR31952556.fastq.gz | Fastqsanger.gz |
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Project description:C-terminal domain (CTD) of RNA polymerase II is crucial for recruiting transcription regulators via specific post-translational modifications (PTM), especially phosphorylation. The hypothesis of combination of PTMs, or ‘CTD code’, that can allow precise and dynamic recruitment of transcription machinery is highly attractive, yet the experimental evidence to support this hypothesis has been scarce. Here, despite lacking specific antibodies for combinatorial CTD phosphorylation, we developed an innovative approach that detects double phosphorylation patterns on the CTD in a whole-genomic fashion by leveraging the antibody masking effect with selectively removing the flanking interference. Using this method, we detected pT4pS5 double phosphosites occurring exclusively during the transcription of protein-coding genes. Furthermore, we showed that pT4pS5 marks recruit the Transcription and Export complex (TREX), which specifically facilitates mRNA processing and nucleocytoplasmic export of protein-coding mRNAs. The recruitment of TREX by pT4pS5 phosphosites is particularly important for the processing of lengthy neurogenesis-related genes. Our results provide experimental support for the notion that CTD coding system can function combinatorially and in a gene-specific manner, which encodes an exact information about the transcription of specific gene clusters. This method can be broadly applied to map all combinatorial PTM patterns on RNA polymerase II, paving the way for a deeper understanding of gene-specific transcription regulation at the molecular level.
2025-01-16 | GSE286362 | GEO
Project description:C-terminal domain (CTD) of RNA polymerase II is crucial for recruiting transcription regulators via specific post-translational modifications (PTM), especially phosphorylation. The hypothesis of combination of PTMs, or ‘CTD code’, that can allow precise and dynamic recruitment of transcription machinery is highly attractive, yet the experimental evidence to support this hypothesis has been scarce. Here, despite lacking specific antibodies for combinatorial CTD phosphorylation, we developed an innovative approach that detects double phosphorylation patterns on the CTD in a whole-genomic fashion by leveraging the antibody masking effect with selectively removing the flanking interference. Using this method, we detected pT4pS5 double phosphosites occurring exclusively during the transcription of protein-coding genes. Furthermore, we showed that pT4pS5 marks recruit the Transcription and Export complex (TREX), which specifically facilitates mRNA processing and nucleocytoplasmic export of protein-coding mRNAs. The recruitment of TREX by pT4pS5 phosphosites is particularly important for the processing of lengthy neurogenesis-related genes. Our results provide experimental support for the notion that CTD coding system can function combinatorially and in a gene-specific manner, which encodes an exact information about the transcription of specific gene clusters. This method can be broadly applied to map all combinatorial PTM patterns on RNA polymerase II, paving the way for a deeper understanding of gene-specific transcription regulation at the molecular level.
2025-01-16 | GSE286258 | GEO
Project description:C-terminal domain (CTD) of RNA polymerase II is crucial for recruiting transcription regulators via specific post-translational modifications (PTM), especially phosphorylation. The hypothesis of combination of PTMs, or CTD code, that can allow precise and dynamic recruitment of transcription machinery is highly attractive, yet the experimental evidence to support this hypothesis has been scarce. Here, despite lacking specific antibodies for combinatorial CTD phosphorylation, we developed an innovative approach that detects double phosphorylation patterns on the CTD in a whole-genomic fashion by leveraging the antibody masking effect with selectively removing the flanking interference. Using this method, we detected pT4pS5 double phosphosites occurring exclusively during the transcription of protein-coding genes. Furthermore, we showed that pT4pS5 marks recruit the Transcription and Export complex (TREX), which specifically facilitates mRNA processing and nucleocytoplasmic export of protein-coding mRNAs. The recruitment of TREX by pT4pS5 phosphosites is particularly important for the processing of lengthy neurogenesis-related genes. Our results provide experimental support for the notion that CTD coding system can function combinatorically and in a gene-specific manner, which encodes an exact information about the transcription of specific gene clusters. This method can be broadly applied to map all combinatorial PTM patterns on RNA polymerase II, paving the way for a deeper understanding of gene-specific transcription regulation at the molecular level
2025-01-11 | MSV000096830 | MassIVE
Project description:Combinatorial phosphorylation on CTD of RNA polymerase II selectively controls transcription and export of protein-coding mRNAs
| PRJNA1208493 | ENA
Project description:The C-terminal domain (CTD) of RNA polymerase II orchestrates transcription via specific post-translational modifications (PTMs), notably phosphorylation. The combinatorial phosphorylation of CTD can potentially encode the precise recruitment of transcription machinery but lacks robust evidence. Addressing the challenge of detecting combinatorial CTD phosphorylation in cells without specific antibodies, we developed a novel genome-wide approach exploiting antibody masking and selective interference removal to identify dual phosphorylation patterns. Using this method, we uncovered pT4pS5 phosphosites occurring exclusively during protein-coding gene transcription but excluded in non-coding genes. Using multiple sequencing methods, the role of T4 phosphorylation in elongation is highlighted. Particularly, pT4pS5 marks recruit the Transcription and Export complex to facilitate mRNA export. These findings provide robust evidence that CTD operates as a combinatorial, gene-specific coding system, encoding precise transcriptional instructions. Our approach offers a versatile framework for mapping all combinatorial PTMs on RNA polymerase II, advancing molecular insights into gene-specific transcriptional regulation.
2026-03-19 | GSE324962 | GEO
Project description:The C-terminal domain (CTD) of RNA polymerase II orchestrates transcription via specific post-translational modifications (PTMs), notably phosphorylation. The combinatorial phosphorylation of CTD can potentially encode the precise recruitment of transcription machinery, but lacks robust evidence. Addressing the challenge of detecting combinatorial CTD phosphorylation in cells without specific antibodies, we developed a novel genome-wide approach exploiting antibody masking and selective interference removal to identify dual phosphorylation patterns. Using this method, we uncovered pT4pS5 phosphosites occurring exclusively during protein-coding gene transcription but excluded in non-coding genes. Using multiple sequencing methods, the role of T4 phosphorylation in elongation is highlighted. Particularly, pT4pS5 marks recruit the Transcription and Export complex to facilitate mRNA export. These findings provide robust evidence that CTD operates as a combinatorial, gene-specific coding system, encoding precise transcriptional instructions. Our approach offers a versatile framework for mapping all combinatorial PTMs on RNA polymerase II, advancing molecular insights into gene-specific transcriptional regulation.
2026-03-18 | GSE324921 | GEO
Project description:Decoding a combinatorial RNA polymerase II CTD grammar that specifies protein-coding gene transcription and mRNA export
| PRJNA1442667 | ENA
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.
2012-06-29 | E-MTAB-1060 | biostudies-arrayexpress
Project description:The carboxy-terminal domain (CTD) of RNA polymerase II (Pol II) consists of heptad repeats with the consensus motif Y1-S2-P3-T4-S5-P6-S7. Dynamic phosphorylation of the CTD coordinates Pol II progression through the transcription cycle. Monoclonal antibodies have been used to study in vivo the potentially phosphorylated CTD amino acids (Y1, S2, T4, S5 and S7). However, the epitopes detected by antibodies can be masked by proteins or modifications at neighbouring sites. Therefore, the effectiveness of antibodies in western blot or ChIP analysis reflects the number of accessible CTD phosphorylation marks, but not the total number of phosphorylations. Most importantly, CTD phospho-specific antibodies do not provide any heptad - (location) specific information of CTD phosphorylation. Due to these limitations, the principles and patterns of CTD phosphorylation remained elusive. Here, we use genetic and mass spectrometric approaches to directly detect and map phosphosites along the entire CTD. We confirm phosphorylation of CTD residues Y1, S2, T4, S5 and S7 in mammalian and yeast cells. Although specific phosphorylation signatures dominate, adjacent CTD repeats can be differently phosphorylated, leading to a high variation of coexisting phosphosites in mono- and di-heptad CTD repeats. Inhibition of CDK9 kinase specifically reduces S2 phosphorylation levels within the CTD.
2016-01-25 | PXD003159 | Pride
Project description:Decoding a combinatorial RNA polymerase II CTD grammar that specifies protein-coding gene transcription and mRNA export [TT-seq]
| PRJNA1438170 | ENA