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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE49133 | GEO | 2014/06/09

SECONDARY ACCESSION(S): PRJNA213130

REPOSITORIES: GEO

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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

Rpb9 plays a critical role in global co-transcriptional splicing (Time-course GRO-seq)

Project description:To investigate the question that whether Rpb9 affects elongation velocity, thereby indirectly affecting splicing the regulation, we performed time-course GRO-seq after DRB release. In this assay, DRB was first used to block transcription elongation, which caused Pol II to accumulate at TSSs. Upon DRB wash-off, we performed GRO-seq at 0, 10, 20, and 30min to determine the movement of Pol II wave toward gene 3’ end.Based on the hidden Markov model established earlier, we detected a marked reduction of Pol II re-initiation upon DRB removal in response to Rpb9 knockdown (KD) in HeLa cells. This is consistent with the role of Rpb9 in transcription initiation. By determining the mean elongation rate between 10 and 20min and between 20 and 30m, we found accelerated Pol II elongation, as reported earlier, and interestingly, Rpb9 knockdown appeared to have slightly decreased, rather increased, the rate of transcription. We divided all impacted splicing events 4 equal bins and then asked if a higher degree of induced exon skipping tends to occur on genes with faster Pol II movement, finding that this is not the case. Conversely, we divided Rpb9 KD-altered Pol II rate into 4 equal binds and asked whether splicing shows any correlation to Pol II Rpb9 KD-altered Pol II elongation rate, and again, we detected no correlation. We conclude from these data that Rpb9 KD has some minor impact on the Pol II elongation rate in vivo, and importantly, whatever measurable changes in Rpb9 KD-induced Pol II elongation rate are not linked to changes in Rpb9 KD-induced co-transcriptional splicing.

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Project description:Confluent U2OS cells were released to serum-free medium, and continued to grow for 36 h (Per2-dLuc peak) or 48 h (Per2-dLuc trough), then added DRB (10uM) treatment for 0.5, 1.5, 3.5 h, then added BrU (250uM) for 0.5 h, harvested total RNA, captured with anti-BrU antibody, and conduced hi-seq.

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