N-terminal tagging of RNA Polymerase II shapes transcriptomes more than C-terminal alterations
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE217243 | GEO | 2024/05/12
REPOSITORIES: GEO
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Project description:For overexpression of exogenous RPB1, 10 ug of purified HA-tagged RPB1 plasmids (wildtype or CTD-truncated, both α-amanitin resistant) were transfected into HeLa S3 cells per 10 cm dish using TurboFect transfection reagent (Thermo Fisher Scientific). Culture medium was changed to include 2 ug/ml of Doxycycline 12 hrs post-transfection for inducing exogenous gene expression and then treated with 2.5 ug/ml α-amanitin to block the function of endogenous RPB1. Cells were further cultured for 42 hrs before harvesting for subsequent analysis.
2024-05-27 | GSE175663 | GEO
Project description:RPB1, the largest subunit of RNA polymerase II, contains a highly modifiable C-terminal domain (CTD) that consists of variations of a consensus heptad repeat sequence (Y1S2P3T4S5P6S7). The consensus CTD repeat motif and tandem organization represent the ancestral state of eukaryotic RPB1, but across eukaryotes CTDs show considerable diversity in repeat organization and sequence content. These differences may reflect lineage-specific CTD functions mediated by protein interactions. Mammalian CTDs contain eight non-consensus repeats with a lysine in the seventh position (K7). Posttranslational acetylation of these sites was recently shown to be required for proper polymerase pausing and regulation of two growth factor-regulated genes. To investigate the origins and function of RPB1 CTD acetylation (acRPB1), we computationally reconstructed the evolution of the CTD repeat sequence across eukaryotes and analyzed the evolution and function of genes dysregulated when acRPB1 is disrupted. Modeling the evolutionary dynamics of CTD repeat count and sequence content across diverse eukaryotes revealed an expansion of the CTD in the ancestors of Metazoa. The new CTD repeats introduced the potential for acRPB1 due to the appearance of distal repeats with lysine at position seven. This was followed by a further increase in the number of lysine-containing repeats in developmentally complex clades like Deuterostomia. Mouse genes enriched for acRPB1 occupancy at their promoters and genes with significant expression changes when acRPB1 is disrupted are enriched for several functions, such as growth factor response, gene regulation, cellular adhesion, and vascular development. Genes occupied and regulated by acRPB1 show significant enrichment for evolutionary origins in the early history of eukaryotes through early vertebrates. Our combined functional and evolutionary analyses show that RPB1 CTD acetylation was possible in the early history of animals, and that the K7 content of the CTD expanded in specific developmentally complex metazoan lineages. The functional analysis of genes regulated by acRPB1 highlight functions involved in the origin of and diversification of complex Metazoa. This suggests that acRPB1 may have played a role in the success of animals. We used a HA-tagged mouse RPB1 construct in which all K7 residues were substituted with arginines (8KR). This mutation resembles unacetylated lysines by conserving the positive charge at these positions, but preventing acetylation. To examine the potential functions of K7 acetylation in regulating gene expression, we stably expressed wildtype or 8KR HA-RPB1 in murine NIH/3T3 fibroblasts and cultured these cells in media containing α-amanitin. Both were expressed at equivalent levels, but acetylation was present only in wildtype, and not mutant HA-RPB1. We then performed gene expression profiling using the Affymetrix Mouse Gene 1.0 ST microarray with three biological replicates.
2015-02-19 | E-GEOD-66088 | biostudies-arrayexpress
Project description:The p53 family is known as a family of transcription factors with functions in tumor suppression and development. Whereas the central DNA binding domain is highly conserved among the three family members p53, p63 and p73, the C-terminal domains (CTDs) are diverse and subject to alternative splicing and post-translational modification. Here we demonstrate that the CTDs strongly influence DNA binding and transcriptional activity. While p53 and the p73 isoform p73gamma have basic CTDs and form weak sequence-specific protein-DNA complexes, the major p73 isoforms alpha, beta and delta have neutral CTDs and bind DNA strongly. A basic CTD has been previously shown to enable sliding along the DNA backbone and to facilitate the search for binding sites in the complex genome. Our experiments, however, reveal that a basic CTD also reduces protein-DNA complex stability, intranuclear mobility, promoter occupancy in vivo, transgene activation and induction of cell cycle arrest or apoptosis. A basic CTD in p53 and p73gamma therefore provides both positive and negative regulatory functions presumably to enable rapid switching of protein activity in response to stress. In contrast, most p73 isoforms exhibit constitutive DNA binding activity consistent with a predominant role in developmental control. Keywords: Adenoviros, p73 / p53
2008-08-01 | GSE8660 | GEO
Project description:Xenopus laevis embryos were injected with alpha-amanitin to inhibit RNA polymerase II activity. Embryos were allowed to develop up to stage 10.5 (early gastrula, control and alpha-amanitin injected embryos) and subsequently collected for RNA isolation. The transcriptome profiles of alpha-amanitin injected and control embryos were compared.
2016-03-07 | E-MTAB-3614 | biostudies-arrayexpress
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 RNA binding ability of the glucocorticoid receptor (GR) remains an understudied area of GR regulation. Through in vitro binding assays, we identified hairpin RNAs as GR's preferred binding motif. To study how GR-bound RNAs change with dexamethasone treatment, we first generated stable U2OS cells expressing wild-type GR-HaloTag. We then treated the cells with dexamethsone every hour over three hours, UV crosslinked, harvested, and pulled down GR using a specific and covalently-bound HaloTag ligand. GR-bound RNAs were then isolated, library prepped, and sequenced.
2022-12-17 | GSE217888 | GEO
Project description:Background and Aims: To verify the protective effect of Ezetimibe, an sodium taurocholate co-transporting polypeptide (NTCP) inhibitor, on α-amanitin poisoning in vitro and in vivo by inhibiting NTCP to prevent α-amanitin into hepatocytes. Approach and Results: In animal experiments, the survival rate was significantly improved in the treatment group. The pathomorphological characteristics of liver and kidney in the treatment group were significantly improved. In cell experiments,The cell viability of the treatment group was significantly improved, and the expression of NTCP in the treatment group was significantly decreased by immunofluorescence. In molecular docking simulations, we demonstrated the potential of NTCP to bind Ezetimibe and α-amanitin, respectively. Transcriptomics in high-throughput sequencing was used to detect the differential metabolic genes between α-amanitin poisoning group and the treatment group, and signal pathway enrichment was used to analyze the significantly different signal pathways. Conclusions: Ezetimibe, as an inhibitor of NTCP, can reduce the entry of α-amanitin into hepatocytes to play a protective role and improve the cell viability and survival rate of mice.
2023-06-30 | GSE235740 | GEO
Project description:Transcriptional profiling of HeLa cells treated with α-amanitin for 9 h. Relative abundance to untreated control cells was used to evaluate the biogenesis of lncRNAs by RNA polymerase II. Two-condition experiment, alpha-amanitin-treated vs. untretated HeLa cells. Biological replicates: 4 with dye-swap
2015-06-30 | E-GEOD-62989 | biostudies-arrayexpress
Project description:HEK293T cells were transfected with the Rbp1-amr or slow (R729H-amr) α-amanitin resistant subunit of RNA Pol II and selected with α-amanitin 24 hours after transfection for additional 24 hours. Total RNA was extracted and global changes in gene expression were determined using microarray chips. MiRNAs are transcribed by RNA pol II but the transcriptional features influencing their synthesis are poorly defined. Here we report that a TATA-box in miRNA and a subset of protein-coding genes is associated with increased sensitivity to a slow rate of transcription elongation. We also show that promoters driven by TATA-box or NF-κB elicit high transcription re-initiation rate, but paradoxically lower levels of miRNA. Interestingly, miRNA synthesis was converted to a more productive mode by decreasing initiation rate, but less productive when the re-initiation rate increased. This phenomenon was found to be associated with a delay in miR-146a induction by NF-κB. We also demonstrate that miRNAs are remarkably strong pause sites. Our findings suggest that lower efficiency of miRNA synthesis directed by the TATA-box or NF-κB is a consequence of frequent transcription initiation that lead to Pol II crowding at pause sites, thereby increasing the chance of collision and premature termination. These findings highlight the importance of the transcription initiation mechanism for miRNA synthesis, and have implications for TATA-box promoters in general. HEK293T cells were transfected with plasmids directing the expression of α-amanitin-resistant variants of Pol II (Rpb1-amr and R749H-amr). α-amanitin was added and RNA was prepared 24 and 48 h later, respectively. The data provided is from 3 Rpb1-amr vs 3 R749H-amr (6 samples).
2013-07-01 | E-GEOD-48163 | biostudies-arrayexpress
Project description:To identify the proteins synthesized from long-lived mRNAs at early phase of germination, proteome of embryos at 0 DAI (dry embryos), 1 DAI with α-amanitin and 1 DAI without α-amanitin were analyzed by using LC-MS/MS.
2019-08-13 | PXD013628 | Pride