Combinatorial phosphorylation on CTD of RNA polymerase II selectively controls transcription and export of protein-coding mRNAs [pThr4]
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PROVIDER: PRJNA1208980 | ENA |
REPOSITORIES: ENA
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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 | 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 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 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: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:Oxidative phosphorylation selectively orchestrates tissue macrophage homeostasis
| PRJNA880299 | ENA
Project description:Nuclear export of mRNA is essential for eukaryotic cells to establish the flow of genetic information in the nucleus to protein synthesis in the cytoplasm. This transport process is highly regulated to ensure efficient and accurate gene expression. Viruses are well known for their ability to manipulate host gene expression. Here, we report that ORF10 of Kaposi’s sarcoma associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. This export inhibitory effect of ORF10 requires the interaction with an RNA export factor, Rae1. Genome-wide analysis by RNA sequencing revealed the subset of cellular mRNAs whose nuclear export is blocked by ORF10. The 3’ untranslated regions (3’ UTRs) of ORF10-targeted transcripts confer their sensitivity to nuclear export inhibition by ORF10. In the context of KSHV replication, the interaction of ORF10 with Rae1 is important for the virus to express viral genes and produce infectious virions. Our results suggest that a nuclear replicating DNA virus can selectively interfere with RNA export through Rae1 to restrict host gene expression for optimal viral replication.
2016-10-18 | GSE82241 | GEO
Project description:The switch from cellular proliferation to differentiation occurs to a large extend through specific programs of gene expression. In fission yeast, the high-mobility-group transcription factor Ste11 is the master regulator of sexual differentiation. ste11 is induced by environmental conditions, mostly nitrogen starvation, leading to mating and meiosis 1. We have used ChIP-chip and gene expression profiling to show that CDK-dependent phosphorylation of serine 2 in the C-terminal domain (CTD) of the largest subunit of the RNA polymerase II (PolII) holoenzyme plays a critical role in the induction of ste11 transcription during sexual differentiation while it has a minor impact on gene expression during vegetative growth. Moreover, we demonstrate that both the recruitment of the CTD serine 2 kinase and the phosphorylation event initiate at the promoter region of ste11 in contrast to the classical case where serine 2 phosphorylation occurs across the coding region 2. In the absence of CTD serine 2 phosphorylation, both PolII occupancy at the ste11 locus, and ste11 expression are impaired. This results in sterility that is rescued when ste11 is expressed from the canonical adh promoter. We conclude that a modification of the RNA polymerase II holoenzyme plays a specific and pivotal role in the sexual differentiation. For the ChIP-chip experiments, 3-4 biological replicates were performed for each tagged protein of interest. For the expression experiments, two biological samples were hybridized for each mutant strain (replicates 1 and 2), with two dye-swap technical replicates per sample (replicates 3 and 4).
2010-01-01 | E-GEOD-16498 | biostudies-arrayexpress
Project description:The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of RNA polymerase II’s C-terminal domain (CTD) and is essential for the transition from transcription initiation to elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and hinders understanding the transition from transcription initiation to elongation. Through in vitro reconstruction of CTD phosphorylation, mass spectrometry analysis, and chromatin immunoprecipitation-seq analysis we uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 phosphorylation causes the reduction of Ser2 and accumulation of RNA polymerase II in the promoter region as detected by ChIP-seq. We demonstrate the ability of Tyr1 phosphorylation to generate a heterogeneous CTD modification landscape that expands the CTD’s coding potential. These findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events by influencing the behavior of downstream enzymes.
2019-11-01 | GSE131838 | GEO