RNA-seq analysis in WT and rpb1-CTD-S2A strains
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ABSTRACT:
PROVIDER: PRJNA719083 | ENA |
REPOSITORIES: ENA
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Project description:We report calibrated transcriptome of rpb1-CTD-S2A and WT of S. pombe cells. Phosphorylation of the RNA polymerase II (Pol II) C-terminal domain on heptad Y1S2P3T4S5P6S7 coordinates key events during transcription and when its deregulation leads to defects in transcription and RNA processing. Here we report that alanine substitution of all Ser2 in CTD result in increased antisense transcription.
2021-08-26 | GSE171307 | GEO
Project description:Transctriptome profiling of CTD-14 repeats, 2A, 5A mutants responding to 0.7N NaCl for 30mins. The study shows that phosphorylation at Ser5 sites plays a role in normal induction and repression of genes upon NaCl stress. The CTD14 strains harbors a plasmid expressing RPB1 with 14 wild-type CTD repeats. 5A strains carries a plasmid expressing a chimeric RPB1 in which the CTD was composed of 5 repeats of CTD-serine 5 substituted with Ala followed by 7 wild-type-sequenced repeats. The 2A strains carrys 8 repeats of CTD-serine 2 substituted with alanine followed by 7 wild-type-sequenced repeats.
2014-12-03 | GSE60700 | GEO
Project description:Transctriptome profiling of CTD-14 repeats, 2A, 5A mutants responding to 0.7N NaCl for 30mins. The study shows that phosphorylation at Ser5 sites plays a role in normal induction and repression of genes upon NaCl stress. The CTD14 strains harbors a plasmid expressing RPB1 with 14 wild-type CTD repeats. 5A strains carries a plasmid expressing a chimeric RPB1 in which the CTD was composed of 5 repeats of CTD-serine 5 substituted with Ala followed by 7 wild-type-sequenced repeats. The 2A strains carrys 8 repeats of CTD-serine 2 substituted with alanine followed by 7 wild-type-sequenced repeats. Two-color fluorescence arrays reporting on mRNA abunance in strains before and after 30 min with 0.7M NaCl treatment
2014-12-03 | E-GEOD-60700 | biostudies-arrayexpress
Project description:In fission yeast, the nuclear-localized Lsk1p-Lsc1p-Lsg1p cyclin dependent kinase complex is required for the reliable execution of cytokinesis and is also required for Ser-2 phosphorylation RNA pol II carboxy terminal domain. To address whether alterations in CTD phosphorylation might selectively alter expression of cytokinesis genes, expression profiling of site-directed CTD mutants was performed. Strains bearing the rpb1-12XCTD and rpb1-12XS2ACTD mutations were grown to mid-log phase in YES media and treated with 0.5uM LatA (or the solvent control, DMSO) for three hours at 30C. Three biological replicates were performed.
2011-08-31 | E-GEOD-31369 | biostudies-arrayexpress
Project description:The carboxy-terminal domain of RPB1 subunit of RNA Polymerase II (CTD) plays an essential function in the regulation of gene expression and the coordination of co-transcriptional processes. CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast and its length is crucial for spatial organization of transcriptional machinery in the nucleus. We found that the proximal half of the CTD is sufficient to support RNA metabolism and co-transcriptional processing in steady state conditions in human cells. Signal induced transcription, however, is severely impaired upon CTD shortening. Our data suggest that CTD length increased in evolution to allow for spatio-temporal control of gene expression patterns at least in part by facilitating enhancer function.
2021-01-04 | GSE159092 | GEO
Project description:The carboxy-terminal domain (CTD) of Rpb1, the largest component of the 12-subunit RNA polymerase II, consists of repeating Y1S2P3T4S5P6S7 heptapeptides (26 repeats in budding yeast). Each stage of transcription relies on the ordered recruitment and exchange of specific protein complexes that act on RNA polymerase II, its nascent transcripts, and the underlying chromatin. This dynamic process is orchestrated via patterned post-translational modifications of the CTD. To characterize the role of phosphorylation on Thr4, we examined the effect of Rpb1 alleles in which Thr4 was substituted with an alanine (T4A) or the phospho-mimic glutamate (T4E). Substitutions were made across all heptad repeats of the CTD.
We affinity purified HA-tagged Rpb1 from Saccharomyces cerevisiae strains bearing WT, T4A, and T4E CTDs. A control strain (Z26) lacking the HA-tagged Rpb1 was subjected to an identical affinity enrichment procedure. Three biological replicates were acquired for each type of affinity purification and analyzed independently. After TCA-precipitation, proteins were urea-denatured, reduced, alkylated, then digested with endoproteinase LysC followed by trypsin. The resulting peptide mixtures were analyzed by Multidimensional Protein Identification Technology (MudPIT). Label-free quantitative proteomics was used to identify and quantify the relative abundance of affinity-enriched complexes.
2017-02-24 | MSV000080573 | MassIVE
Project description:The modification of Ser 5 is important for the relocalization of RNAP II upon NaCl stress. The CTD14 strains harbors a plasmid expressing RPB1 with 14 wild-type CTD repeats. The 5A strain carries a plasmid expressing a chimeric RPB1 in which the CTD was composed of 5 repeats of CTD-serine 5 substituted with alanine followed by 7 wild-type-sequenced repeats.
2014-12-03 | GSE60707 | GEO
Project description:The modification of Ser 5 is important for the relocalization of RNAP II upon NaCl stress. The CTD14 strains harbors a plasmid expressing RPB1 with 14 wild-type CTD repeats. The 5A strain carries a plasmid expressing a chimeric RPB1 in which the CTD was composed of 5 repeats of CTD-serine 5 substituted with alanine followed by 7 wild-type-sequenced repeats. Two-color fluorescence arrays reporting on Rpb3 localization abundance in strains (input vs. IP) before and at 20 min after a shock with 0.7M NaCl
2014-12-03 | E-GEOD-60707 | biostudies-arrayexpress
Project description:The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) orchestrates dynamic recruitment of specific cellular machines during different stages of transcription. Signature phosphorylation patterns of Y1S2P3T4S5P6S7 heptapeptide repeats of the CTD engage specific “readers.” While phospho-Ser5 and phospho-Ser2 marks are ubiquitous, phospho-Thr4 is reported to only impact specific genes. Here, we investigate the RNA expression profile in WT and CTD-mutant strains of S. cerevisiae.
2017-05-02 | GSE95417 | GEO
Project description:Transcription by RNA polymerase II (RNAPII) is coupled to mRNA processing and chromatin modifications via the C-terminal domain (CTD) of its largest subunit, consisting of multiple repeats of the heptapeptide YSPTSPS. Pioneering studies showed that CTD serines are differentially phosphorylated along genes in a prescribed pattern during the transcription cycle. Genome-wide analyses challenged this idea, suggesting that this cycle is non-uniform among different genes. Moreover, the respective role of enzymes responsible for CTD modifications remains controversial. Here, we systematically profiled the location of the RNAPII phospho-isoforms in wild type cells and mutants for most CTD modifying enzymes. Together with results of in vitro assays, these data reveal a complex interplay between the modifying enzymes, and provide evidence that the CTD cycle is uniform across genes. We also identify Ssu72 as the Ser7 phosphatase and show that proline isomerization is a key regulator of CTD dephosphorylation at the end of genes. We took a systematic approach to examine the genome-wide distribution of the various CTD modifications using a panel of RNAPII CTD phospho-specific antibodies; both in wild type cells and in mutants for most of the CTD kinases, phosphatases and the isomerase. Immunoprecipitation of CTD phospho-isoforms were done using the following antibodies: H14 and 3E8 for Ser5, H5 and 3E10 for Ser2, 4E12 for Ser7, 8WG16 (anti-Rpb1-CTD) and W0012 (anti-Rpb3) for RNAPII (global localization). A list of the mutant strains and their genotypes can be found in the supplemental files of the related publication. Most ChIPs (in Cy5) were hybridyzed against a non-immunoprecipitated (whole cell extract, WCE) in Cy3. Ssu72, Pti1 and Rpb1 were immunoprecipitated using tagged proteins (3myc-Ssu72, Pti1-3myc, Rpb1-9myc) and the ChIP DNA hybridized in competition with a control ChIP DNA prepared from an isogenic untagged strain (NoTag). ChIP from wild type strains yFR116 (W303) and yFR117 (S288C) were used to obtains wild type profiles that can be compared to mutants strains of the same background. All ChIP-chip experiments were done at least in duplicates. Each microarray was normalized using the Lima Loess and replicates were combined using a weighted average method as previously described (Pokholok et al., 2005).
2012-02-01 | E-GEOD-29403 | biostudies-arrayexpress