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Rpb9 plays a critical role in global co-transcriptional splicing (Fraction RNA-seq)

ABSTRACT: Rpb9 plays a critical role in global co-transcriptional splicing (Fraction RNA-seq)

PROVIDER: PRJNA681178 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR13156632_1.fastq.gz	Fastqsanger.gz
	SRR13156632_2.fastq.gz	Fastqsanger.gz
	SRR13156633_1.fastq.gz	Fastqsanger.gz
	SRR13156633_2.fastq.gz	Fastqsanger.gz
	SRR13156634_1.fastq.gz	Fastqsanger.gz

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U2AF1 plays a critical role in global co-transcriptional splicing (Fraction chrRNA-seq)

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Rpb9 plays a critical role in global co-transcriptional splicing in mouse ESC (Fraction chrRNA-seq)

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U2AF1 plays a critical role in global co-transcriptional splicing (Fraction chrRNA-seq)

Project description:In order to measure the impact of U2AF1 on co-transcriptional splicing, we treated HeLa cells with control siRNA or U2AF1 siRNA. To measure co-transcriptional splicing, we fractionated HeLa cells into cytosolic (Cyt), nucleoplasm (NP), and chromatin/particle (Chr) fractions. We purified total RNA from Chr compartment, spiked in with in vitro transcribed EGFP and FireFly luciferase RNA for data normalization, depleted ribosomal RNAs, and constructed two independent libraries for RNA-seq.

2023-05-27 | GSE175671 | GEO

Rpb9 plays a critical role in global co-transcriptional splicing (Fraction RNA-seq)

Project description:In order to measure the impact of Rpb9 on co-transcriptional splicing, we treated HeLa cells with control siRNA or siRpb9, and for comparative analysis, we also depleted Rpb1 and confirmed by Western blotting the knockdown efficiency in all cases. To measure co-transcriptional splicing, we fractionated HeLa cells into cytosolic (Cyt), nucleoplasm (NP), and chromatin/particle (Chr) fractions. We purified total RNA from each compartment, spiked in with in vitro transcribed EGFP and FireFly luciferase RNA for data normalization, depleted ribosomal RNAs, and constructed two independent libraries for RNA-seq, which showed excellent reproducibility .

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

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Rpb9 plays a critical role in global co-transcriptional splicing in mouse ESC (Fraction chrRNA-seq)

Project description:To define the function of Rpb9 on co-transcriptional splicing, we used auxin-inducible Rpb9 degron system in mouse embryonic stem cells (mESCs) with auxin treatment for 3 hours to sufficiently deplete Rpb9. To measure co-transcriptional splicing, we fractionated mESCs cells (spiked in with Drosophila S2 cells for data normalization) into cytosolic (Cyt), nucleoplasm (NP), and chromatin/particle (Chr) fractions. We purified total RNA from Chr compartment, depleted ribosomal RNAs, and constructed two independent libraries for RNA-seq, which showed excellent reproducibility.

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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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The conserved ubiquitin-like protein Hub1 plays a critical role in splicing in human cells

Project description:Different from canonical ubiquitin-like proteins, Hub1 does not form covalent conjugates with substrates but binds proteins non-covalently. In Saccharomyces cerevisiae, Hub1 associates with spliceosomes and mediates alternative splicing of SRC1, without affecting pre-mRNA splicing generally. Human Hub1 is highly similar to its yeast homolog, but its cellular function remains largely unexplored. Here, we show that human Hub1 binds to the spliceosomal protein Snu66 as in yeast, however, unlike its S. cerevisiae homolog, human Hub1 is essential for viability. Prolonged in vivo depletion of human Hub1 leads to various cellular defects, including splicing speckle abnormalities, partial nuclear retention of mRNAs, mitotic catastrophe and consequently cell death by apoptosis. Early consequences of Hub1 depletion are severe splicing defects, however, only for specific splice sites leading to exon skipping and intron retention. Thus, the ubiquitin-like protein Hub1 is not a canonical spliceosomal factor needed generally for splicing, but rather a modulator of spliceosome performance and facilitator of alternative splicing. Human U2OS cells were transfected with siRNAs to specifically knockdown the ubiquitn-like protein Hub1. Cells treated with non-targeting oligos (GL2) served as negative control. Total RNAs of three biological replicates of each knockdown experiment were isolated and changes of splicing patterns were subsequently analyzed by Affymetrix GeneChip Human Exon 1.0 ST arrays

2014-05-30 | E-GEOD-56878 | biostudies-arrayexpress

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