Transcriptional profiling of fission yeast RNA polymerase II CTD mutants
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ABSTRACT:
PROVIDER: PRJNA674640 | ENA |
REPOSITORIES: ENA
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Project description:This RNA-Seq analysis compares gene expression of the RNA polymerase II CTD-mutants contrasting to the WT RNA polymerase II CTD (with isogenic 3' selection marker) in the fission yeast S. pombe
2021-02-25 | GSE160852 | GEO
Project description:The RNA polymerase II carboxy-terminal domain (CTD) consists of conserved repeats of the consensus sequence Y1-S2-P3-T4-S5-P6-S7, which can be phosphorylated to influence distinct stages of the transcription cycle, including RNA processing. Although affinity purification coupled with mass spectrometry has defined CTD-associated proteins, phospho-dependent CTD interactions have remained largely elusive. Proximity-dependent biotinylation (PDB) provides an alternative approach to identify protein-protein associations in the native cellular environment. Here we present a PDB-based map of the fission yeast RNAPII CTD in live cells. The proteomic screen identified known CTD-associated proteins, but also captured new and unexpected CTD proximal proteins. We also used PDB to identify phospho-dependent CTD interactions by using a mutant in which Ser2 was replaced by alanine in every repeat of the fission yeast CTD. Surprisingly, CTD-mediated biotinylation of most 3’ end processing factors was not affected in the S2A mutant, consistent with RNA-seq and ChIP-seq analysis indicating that CTD Ser2 phosphorylation is not required for 3’ end processing and transcription termination. Conversely, we found that CTD Ser2 phosphorylation is critical for the association between RNAPII and the histone methyltransferase Set2 during transcription elongation. We show that loss of CTD Ser2 phosphorylation disables the Set2-Clr6(II) axis, resulting in a global increase in cryptic antisense transcription that correlates with elevated levels of histone acetylation in gene bodies. Our findings reveal that the fundamental role of CTD Ser2 phosphorylation is to establish a chromatin-based repressive state that prevents cryptic intragenic transcription initiation.
2024-05-06 | PXD047023 | Pride
Project description:The primary structure and phosphorylation pattern of the tandem YSPTSPS repeats of the RNA polymerase II CTD comprise an informational code that coordinates transcription, chromatin modification, and RNA processing. To gauge the contributions of individual CTD coding “letters” to gene expression, we analyzed the poly(A)+ transcriptomes of fission yeast mutants that lack each of the four inessential CTD phosphoacceptors: Tyr1, Ser2, Thr4, and Ser7. There was a hierarchy of CTD mutational effects with respect to the number of dysregulated protein-coding RNAs, with S2A (n=227) >> Y1F (n=71) > S7A (n=58) >> T4A (n=7). The majority of the protein-coding RNAs affected in Y1F cells were coordinately affected by S2A, suggesting that Tyr1-Ser2 constitutes a two-letter code “word”. Y1F and S2A elicited increased expression of genes encoding proteins involved in iron uptake (Frp1, Fip1, Fio1, Str3, Str1, Sib1), without affecting the expression of the genes that repress the iron regulon, implying that Tyr1-Ser2 transduces a repressive signal. Y1F and S2A cells had increased levels of ferric reductase activity and were hypersensitive to phleomycin, indicative of elevated intracellular iron. The T4A and S7A mutations had opposing effects on the phosphate response pathway. T4A reduced the expression of two genes encoding proteins involved in phosphate acquisition (the Pho1 acid phosphatase and the phosphate transporter SPBC8E4.01c), without affecting the expression of known genes that regulate the phosphate response pathway, while S7A increased pho1+ expression. Meiotic genes were enriched among those up-regulated in S7A cells. These results highlight specific cellular gene expression programs that are responsive to distinct CTD cues.
2014-03-13 | GSE52370 | GEO
Project description:The primary structure and phosphorylation pattern of the tandem YSPTSPS repeats of the RNA polymerase II CTD comprise an informational code that coordinates transcription, chromatin modification, and RNA processing. To gauge the contributions of individual CTD coding M-bM-^@M-^\lettersM-bM-^@M-^] to gene expression, we analyzed the poly(A)+ transcriptomes of fission yeast mutants that lack each of the four inessential CTD phosphoacceptors: Tyr1, Ser2, Thr4, and Ser7. There was a hierarchy of CTD mutational effects with respect to the number of dysregulated protein-coding RNAs, with S2A (n=227) >> Y1F (n=71) > S7A (n=58) >> T4A (n=7). The majority of the protein-coding RNAs affected in Y1F cells were coordinately affected by S2A, suggesting that Tyr1-Ser2 constitutes a two-letter code M-bM-^@M-^\wordM-bM-^@M-^]. Y1F and S2A elicited increased expression of genes encoding proteins involved in iron uptake (Frp1, Fip1, Fio1, Str3, Str1, Sib1), without affecting the expression of the genes that repress the iron regulon, implying that Tyr1-Ser2 transduces a repressive signal. Y1F and S2A cells had increased levels of ferric reductase activity and were hypersensitive to phleomycin, indicative of elevated intracellular iron. The T4A and S7A mutations had opposing effects on the phosphate response pathway. T4A reduced the expression of two genes encoding proteins involved in phosphate acquisition (the Pho1 acid phosphatase and the phosphate transporter SPBC8E4.01c), without affecting the expression of known genes that regulate the phosphate response pathway, while S7A increased pho1+ expression. Meiotic genes were enriched among those up-regulated in S7A cells. These results highlight specific cellular gene expression programs that are responsive to distinct CTD cues. Interrogation of the S. pombe transcriptome using polyA+ strand specific RNA sequencing (Illumina HiSeq 2000) in cultures. A total of 16 samples were analysed: two biological repeates of each WT, Y1F, S2A, T4A, S7A,Y1F-S7A, S2A-S7A and T4A-S7A strains
2014-03-13 | E-GEOD-52370 | biostudies-arrayexpress
Project description:The switch from cellular proliferation to differentiation occurs to a large extent through specific programs of gene expression. In fission yeast, the master regulator of sexual differentiation, ste11, is induced by environmental conditions leading to mating and meiosis. We show that phosphorylation of serine 2 (S2P) in the C-terminal domain (CTD) of the largest subunit of the RNA polymerase II (PolII) enzyme by the Lsk1 cyclin dependent kinase has only a minor impact on global gene expression during vegetative growth but is critical for the induction of ste11 transcription during sexual differentiation. The recruitment of the Lsk1 kinase initiates in the vicinity of the transcription start site of ste11, resulting in a marked increase of S2P on the ste11 unit including an extended 5’-untranslated region (5’-UTR). This pattern contrasts with the classical gradient of S2P towards the 3’ region. In the absence of S2P, both PolII occupancy at the ste11 locus and ste11 expression are impaired. This results in sterility which is rescued by expression of the ste11 coding sequence from the adh1 promoter. Thus, the S2P polymerase plays a specific, regulatory role in cell differentiation through the induction of ste11.
2010-01-01 | GSE16498 | GEO
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:The aim of the project was to analyse the functional role of Tyrosine-1 of mammalian RNA polymerase II CTD in the regulation of transcription-coupled processes. We studied mammalian CTD mutants to analyze the function of tyrosine1 residues. Mutation of 3/4 of the tyrosine residues in a mutant YFFF resulted in a massive read-through transcription phenotype. Read-through was observed for 3’ processed and polyadenylated RNAs. The YFFF mutant shows a loss of interaction with the Mediator and Integrator complexes in MS analysis.
2018-01-09 | PXD008270 | Pride
Project description:Whole genome analysis of total RNA pol II, Ser2-, Ser5- and Ser7-phosphorylated RNA pol II, in WT and mutants of the C-terminal domain (CTD) kinases Ctk1 and Kin28, and localization of the termination factors Pcf11, Nrd1 and Rat1.
2010-09-15 | GSE23960 | GEO
Project description: Not available
| S-EPMC3142328 | biostudies-literature