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Human telomerase RNA processing and quality control

ABSTRACT: RNA sequencing of HeLa cells treated with siRNA against the RNA exosome components hRRP40, hRRP6, hDIS3, and hRRP6/hDIS3 or the splicing inhibitors Isoginkgetin and spliceostatin A, respectively. Stranded, ribo-depleted RNA seq profiles of HeLa cells treated with exosome targeting siRNAs or splicing inhibitors using Illumina HiSeq. All experiments were carried out in triplicate starting with independent cell cultures

ORGANISM(S): Homo sapiens

SUBMITTER: Peter Baumann

PROVIDER: E-GEOD-72055 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Human telomerase RNA processing and quality control

Project description:RNA sequencing of HeLa cells treated with siRNA against the RNA exosome components hRRP40, hRRP6, hDIS3, and hRRP6/hDIS3 or the splicing inhibitors Isoginkgetin and spliceostatin A, respectively.

2015-11-25 | GSE72055 | GEO

An atlas of active enhancers across human cell types and tissues

Project description:Sequencing of 5' ends of RNA molecules from control and exosome-depleted HeLa-S3 cells. CAGE library construction from RNA extracted from control and exosome-depleted cells.

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Project description:Sequencing of 5' ends of RNA molecules from control and exosome-depleted HeLa-S3 cells. CAGE library construction from RNA extracted from control and exosome-depleted cells.

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Effect of splicing on histone H3 K36 methylation

Project description:Histone H3 K36me3 and RNA pol II were localized by ChIP before and after inhibition of splicing by spliceostatin for 12 hr. Total matched reads for the control and SSA treated Hela samples were 11.6 and 8.2 million for anti-H3K36me3 and 6.6 and 5.0 million for anti-pol II.

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Spliceostatin treatment on HeLa cells

Project description:HeLa cell line treated with Spliceostatin or methanol

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Effect of splicing on histone H3 K36 methylation

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Transcriptome changes in spliceostatin A treated cells

Project description:Pre-mRNA in eukaryotes is subjected to post-transcriptional modifications, such as capping, polyadenylation and splicing. It is well known that transcription and post-transcriptional modifications are coupled and that this coupling stimulates post-transcriptional modifications, however, the effects of post-transcriptional modifications on transcription are not fully understood. Here, we report that inhibition of U2 snRNP by a splicing inhibitor, spliceostatin A (SSA), and antisense morpholino oligonucleotide to U2 snRNA caused 3’ end down regulation in a gene-specific manner. We also show that U2 snRNP inhibition resulted in the dephosphorylation of second serine residues (Ser2) within heptad repeats of the C-terminal domain (CTD) of Pol II, which is important for transcription elongation, and this dephosphorylation was correlated with splicing inactivation. Interestingly, removal of SSA from the culture media caused restoration of the Ser2 phosphorylation and expression of the 3’ end, suggesting that transcription elongation resumed. These findings suggest that a novel checkpoint mechanism prevents pre-mRNA accumulation and the production of aberrant proteins translated from pre-mRNA upon splicing deficiencies through the dephosphorylation of CTD Ser2. Treated HeLa cells with methanol or spliceosotain A for 3 hours followed by labelling of nascent transcripts with EU for 1 hour. The EU labelled nascent RNA was purified and analyzed on human exon 1.0ST array. Three biological replicates were used for each treatment.

2013-03-28 | E-GEOD-45379 | biostudies-arrayexpress

Human telomerase RNA processing and quality control

Project description:Primary telomerase RNA transcripts are processed into shorter mature forms that assemble into a complex with the catalytic subunit and provide the template for telomerase activity. In diverse fungi telomerase RNA 3â end processing involves a single cleavage reaction by the spliceosome akin to the first step of splicing. Longer forms of human telomerase RNA (hTR) have been reported, but how the mature form of precisely 451 nucleotides is generated is still unknown. We now show that the splicing inhibitor isoginkgetin causes accumulation of long hTR transcripts, but find no evidence for a direct role for splicing in hTR processing. Instead, isoginkgetin mimics the effects of inhibiting the RNA exosome. Depletion of exosome components and accessory factors reveals functions for the cap binding complex (CBC) and the nuclear exosome targeting (NEXT) complex in hTR turnover. Whereas longer transcripts are predominantly degraded, shorter precursor RNAs are oligo-adenylated by TRF4-2 and either processed by poly (A) specific ribonuclease (PARN) or degraded by the exosome. Our results reveal that hTR biogenesis involves a kinetic competition between RNA processing and quality control pathways and suggest new treatment options for dyskeratosis congenita caused by mutations in RNA processing factors. We cloned and sequenced 3â ends by RLM-RACE coupled with high-throughput sequencing to gain further insights into hTR processing.

2015-11-25 | E-GEOD-73776 | biostudies-arrayexpress

Homo sapiens

Project description:Spliceostatin treatment on HeLa cells

| PRJNA128345 | ENA

Quantifying the transcriptional effects of lncRNA depletion in HeLa cells with RNA-seq

Project description:Long noncoding RNAs (lncRNAs) are a major transcriptional output of the mammalian genome, yet their functions are poorly characterized. In this experiment, we used RNA interference (RNAi) and locked nucleic acid antisense oligonucleotides (LNAs) to deplete the lncRNAs LINC00899 and C1QTNF1-AS1 (C1) in HeLa cells. Both of these lncRNAs are of interest as their depletion results in mitotic delay, suggesting a role in mitotic progression. After depletion of each lncRNA with RNAi or LNAs, we performed high-throughput RNA sequencing and tested for differential expression compared to negative control samples (using control siRNAs or negative control LNAs) to identify downstream regulatory targets of each lncRNA. We generated 3-4 replicates for each condition, spread across multiple batches and experiments. Samples with the same batch number were treated at roughly the same time (within the same month), while the experiment number is nested within batch and refers to cells treated on the same day. Samples were pooled and sequenced across multiple lanes, yielding 8-9 technical replicates per sample.

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