Browse
Submit Data
Databases
API
Help

Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

4sUDRB-seq: measuring transcription elongation and initiation genomewide

ABSTRACT: A new method to measure elongation and intitiation rates Reversal inhibition of transcription with DRB and tagging newly transcribed RNA with 4-thiouridine (4sU)

ORGANISM(S): Homo sapiens

SUBMITTER: Gilad Fuchs

PROVIDER: E-GEOD-57116 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Json Xml

Publications

4sUDRB-seq: measuring genomewide transcriptional elongation rates and initiation frequencies within cells.

Fuchs Gilad G Voichek Yoav Y Benjamin Sima S Gilad Shlomit S Amit Ido I Oren Moshe M

Genome biology 20140509 5

Although transcriptional elongation by RNA polymerase II is coupled with many RNA-related processes, genomewide elongation rates remain unknown. We describe a method, called 4sUDRB-seq, based on reversible inhibition of transcription elongation coupled with tagging newly transcribed RNA with 4-thiouridine and high throughput sequencing to measure simultaneously with high confidence genome-wide transcription elongation rates in cells. We find that most genes are transcribed at about 3.5 Kb/min, w ...[more]

PMID: 24887486

Similar Datasets

Rate of elongation by RNA polymerase II is influenced by specific gene features and histone modifications

Project description:The rate of transcription elongation plays important roles in the timing of expression of full-length transcripts as well as for the regulation of alternative splicing. In this study we coupled Bru-Seq technology with 5,6-dichlorobenzimidazole 1-M-NM-2-D-ribofuranoside (DRB) to estimate the elongation rates of over 2,000 individual genes in human cells. This technique, BruDRB-Seq, revealed gene-specific differences in elongation rates with a median rate of around 1.5 kb/min. We found that genes with fast elongation rates showed higher densities of H3K79m2 and H4K20me1 marks compared to slower elongating genes. Furthermore, fast elongation rates had a positive correlation with gene length, low complexity DNA sequence and distance from nearest active transcription unit. Features that negatively correlated with elongation rate included exon density and the number of LINE sequences in the gene. The BruDRB-Seq technique offers new opportunities to interrogate mechanisms of regulation of transcription elongation. Measurement of RNA Pol II elogation rate. Normal fibroblasts (HF1 and TM), Cockayne syndrome group B fibroblasts, K562 and MCF-7 cells were exposed to DRB for 60 minutes, after which a washout was performed. Nascent RNA was labeled using bromouridine for 10 minutes immediately after the washout. The genomic region extending from actice Trancription Start Sites was used to determine the gene's elongation rate. Please note that the nf_0h_3* samples are duplicated sample records of GSM1062445 and GSM1062446, for the convenient retrieval of the complete raw data from SRA.

2014-04-01 | E-GEOD-55534 | biostudies-arrayexpress

A function for the hnRNP A1/A2 proteins in transcription elongation

Project description:The hnRNP A1 and A2 proteins regulate processes such as alternative pre-mRNA splicing and mRNA stability. Here, we report that a reduction in the levels of hnRNP A1 and A2 by RNA interference or their cytoplasmic retention by osmotic stress drastically increases the transcription of a reporter gene. Based on previous work, we propose that this effect may be linked to a decrease in the activity of the transcription elongation factor P-TEFb. Consistent with this hypothesis, the transcription of the reporter gene was stimulated when the catalytic component of P-TEFb, CDK9, was inhibited with DRB. While low levels of A1/A2 stimulated the association of RNA polymerase II with the reporter gene, they also increased the association of CDK9 with the repressor 7SK RNA, and compromised the recovery of promoter-distal transcription on the Kitlg gene after the release of pausing. Transcriptome analysis revealed that more than 50% of the genes whose expression was affected by the siRNA-mediated depletion of A1/A2 were also affected by DRB. RNA polymerase II-chromatin immunoprecipitation assays on DRB-treated and A1/A2-depleted cells identified a common set of repressed genes displaying increased occupancy of polymerases at promoter-proximal locations, consistent with pausing. Overall, our results suggest that lowering the levels of hnRNP A1/A2 elicits defective transcription elongation on a fraction of P-TEFb-dependent genes, hence favoring the transcription of P-TEFb-independent genes. two treatements and one control

2015-04-07 | E-GEOD-67617 | biostudies-arrayexpress

Mapping of nascent RNA upon release of DRB in WT and KD of RECQL5

Project description:Global Run-On has been performed on WT or KD for RECQL5 cells after release from DRB. When RECQL5 is knocked-down the transcriptional wave front is more advanced, suggesting that transcription is faster. Constitutive knock-down cell lines expressing or not endogenous levels of shRNA resistant RECQL5 under a Doxycycline inducible promoter were treated with high doses of DRB to block transcription. Upon release into fresh medium we were able to follow how much and how fast the RNA Pol II progresses through genes by mapping nascent RNA by Run-On. The experiment was performed in two cell line clones.

2014-06-09 | E-GEOD-49133 | biostudies-arrayexpress

Rho transcription inhibitor CCG-1423 effect on PC-3 cells

Project description:Analysis of serum starved PC-3 cells treated with CCG-1423, Latrunculin B, or the transcription elongation inhibitor DRB for 2 or 24 hours. Results provide insights to potential therapeutic approaches to cancer metastasis. Twenty one samples in triplicate were analyzed and compared to the DMSO-treated control. The primary condition tested was the effect of the Rho-transcription pathway inhibitor, CCG-1423 As a biologically related control, the actin polymerization inhibitor, Latrunculin B, that also blocks Rho-stimulated gene transcription was tested. As a control for non-specific transcription inhibition, DRB was used. All samples, 2-hr and 24-hr treated samples were compared to the 24-hr DMSO sample.

2013-05-01 | E-GEOD-30188 | biostudies-arrayexpress

Identification of CDK9 targets using DRB and quantitative phosphoproteomics

Project description:CDK9 is a critical kinase required for the productive transcription of protein-coding genes by RNA polymerase II (pol II) in higher eukaryotes. Phosphorylation of targets including the elongation factor SPT5 and the carboxyl-terminal domain (CTD) of RNA pol II allow the polymerase to pass an early elongation checkpoint (EEC), which is encountered soon after initiation. In addition to halting RNA polymerase II at the EEC, CDK9 inhibition also causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, suggesting that CDK9 and PP2A, working together, regulate the coupling of elongation and transcription termination to RNA maturation. Our phosphoproteomic analyses, using either DRB or an analog-sensitive CDK9 cell line confirm the splicing factor SF3B1 as an additional key target of this kinase. CDK9 inhibition causes loss of interaction of splicing and export factors with SF3B1, suggesting that CDK9 also helps to co-ordinates coupling of splicing and export to transcription.

2022-08-22 | PXD026720 | Pride

MYCN overexpression in SH-SY5Y/6TR(EU)/pTrex-Dest-30/MYCN cell line

Project description:MYCN overexpression in the doxycline MYCN inducible cell line SH-SY5Y/6TR(EU)/pTrex-Dest-30/MYCN (SY5Y-MYCN) using the dynamic transcriptome analysis (DTA) protocol. Samples at different time-points after MYCN over-expression and uninduced controls (0h) were sequenced in duplicates. The experimental setup consists of [A] total mRNA at 0h, 1h, 4h, 24h, which corresponds to the standard mRNA-seq protocol, [B] 4-thioUridine (4sU) labelled mRNA 30 min before extraction at time-points 0h, 1h, 4h, which is the freshly transcribed mRNA within the last 30 min before the time-point and [C] the counter-part, 4sU unlabelled mRNA 30 min before extraction which corresponds to the mRNA from before 30 min of extraction. The samples were sequenced on an Illumina GA IIx using the Illumina protocols.

2014-12-30 | E-MTAB-2691 | biostudies-arrayexpress

Genome-wide miRNA turnover profile in various cell types after transcription inhibition

Project description:We measured miRNA turnover rates in 8 mammalian cell types post transcription inhibitors ActinomycinD (ActD) or 5,6-Dichlorobenzimidazole 1-M-NM-2-D-ribofuranoside (DRB) treatment with miRNA expression profiling, generating 218 expression profiles on 528 endogenous miRNAs. Cells were plated in six-well plates at around 200,000 cells/well. A media change was performed one day after plating. ActD and DRB treatment were started after confluency (around two days after plating) for 0, 2, 4, 6, 12, and 24 hours post treatment with biological duplicate (ActD treatment) or biological triplicate (DRB treatment). The concentration of ActD and DRB for different cell lines were titrated to achieve rapid c-myc down-regulation after 2 hours of ActD and DRB treatment. After treatment at indicated time points, cells were washed with PBS once and lysed in TriZol reagent immediately (Invitrogen). RNA extraction was performed following the manufacturerM-bM-^@M-^Ys protocol. miRNA profiling was performed using a Luminex bead-based method.

2015-02-05 | E-GEOD-46877 | biostudies-arrayexpress

Pre-mRNA Splicing is Facilitated by an Optimal RNA Polymerase II Elongation Rate

Project description:Transcription and pre-mRNA alternative splicing was analyzed by in isogenic human HEK293 cell lines that inducibly express a-amanitin resistant mutants of the RNA polymerase II large subunit with slow and fast elongation rates. Alternative splicing was analyzed by, RNA-seq and RASL-seq of polyA+ RNA from a-amanitin treated cells; transcription elongation rate was analyzed by BrUTP-labelled GRO-seq of a-amanitin treated cells at time points after release from a DRB (5,6-dichloro-1-bold beta-D-ribofuranosylbenzimidazole) block.

2014-11-25 | E-GEOD-63375 | biostudies-arrayexpress

Wide-spread disruption of transcription termination in HSV-1 infection: Next generation sequencing of total and newly transcribed (4sU-RNA) RNA

Project description:Primary human foreskin fibroblasts (HFF) were infected with wild-type simplex virus 1 (HSV-1) strain 17 at a multiplicity of infection (MOI) of 10. Newly transcribed RNA was labelled by adding 500µM 4-thiouridine (4sU) to the cell culture media for 1h. Total cellular RNA was isolated using Trizol. Newly transcribed RNA was purified following the protocol described in Raedle et al. JoVE 2013. Newly transcribed RNA was labelled in one hour intervals during the first eight hours of HSV-1 infection. All nine 4sU-RNA samples as well as total cellular RNA of every second hour of infection were sent for sequencing. Two independent biological replicates were analysed.

2015-05-26 | E-GEOD-59717 | biostudies-arrayexpress

Global Run-On sequencing (GRO-seq) of HeLa cells under CDK9 inhibitor tratment to study elongation checkpoints at both ends of RNA polymerase II-transcribed genes.

Project description:Global Run-On sequencing (GRO-seq) was carried out on nuclei isolated from HeLa cells after 30 minutes of treatment with CDK9 inhibitors KM05382 and DRB and from two untreated controls. Libraries of nascent RNAs were generated and sequenced using Illumina Hi-seq technology. GRO-seq reads were processed, deduplicated, mapped to human genome and normalised to reads in the 5’ ETS of the 45S rRNA gene.

2015-02-26 | E-MTAB-3360 | biostudies-arrayexpress

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data