CDK12 regulates co-transcriptional splicing and RNA turnover in human cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA790622 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR17272530_1.fastq.gz | Fastqsanger.gz | |||
| SRR17272530_2.fastq.gz | Fastqsanger.gz | |||
| SRR17272531_1.fastq.gz | Fastqsanger.gz | |||
| SRR17272531_2.fastq.gz | Fastqsanger.gz | |||
| SRR17272532_1.fastq.gz | Fastqsanger.gz |
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Project description:The cyclin-dependent kinase CDK12 promotes transcription completion of long genes by suppressing the utilization of intronic polyadenylation sites that cause premature transcriptional termination. One class of genes that are particularly affected by loss of CDK12 activity are DNA damage response genes and therefore inhibitors of CDK12 have garnered interest as cancer therapeutic amplifiers. In this study, we used the CDK12/13 inhibitor THZ531 to treat HF1 (normal human fibroblasts), K562 and HeLa cells and the adenine analog 1-NM-PP1 to treat analog-sensitive HeLa cells and to assess the acute effects on transcription and RNA processing using Bru-seq and BruChase-seq. While acute transcriptional changes were small and limited to the 3’-ends of genes, RNA turnover was dramatically affected by CDK12 inhibition. Importantly, the downregulation of DNA damage response genes was predominantly due to increased mRNA turnover. Co-transcriptional splicing was suppressed by CDK12 inhibition. Finally, many introns that showed premature transcriptional termination were found to be resistant to degradation following CDK12 inhibition despite showing efficient splicing. We speculate that premature termination and addition of poly(A)s protect these spliced intronic fragments from degradation. These studies reveal previously unknown roles of CDK12 in regulating RNA turnover and co-transcriptional splicing.
2022-09-17 | GSE191222 | GEO
Project description:The cyclin-dependent kinase CDK12 has garnered interest as a cancer therapeutic target as DNA damage response genes are particularly suppressed by loss of CDK12 activity. In this study, we assessed the acute effects of CDK12 inhibition on transcription and RNA processing using nascent RNA Bru-seq and BruChase-seq. Acute transcriptional changes were overall small after CDK12 inhibition but over 600 genes showed intragenic premature termination, including DNA repair and cell cycle genes. Furthermore, many genes showed reduced transcriptional readthrough past the end of genes in the absence of CDK12 activity. RNA turnover was dramatically affected by CDK12 inhibition and importantly, caused increased degradation of many transcripts from DNA damage response genes. We also show that co-transcriptional splicing was suppressed by CDK12 inhibition. Taken together, these studies reveal the roles of CDK12 in regulating transcription elongation, transcription termination, co-transcriptional splicing, and RNA turnover.
| S-EPMC9468413 | biostudies-literature
Project description:We describe the epigenetic profiling of the H3K9me2 and HP1a in Drosophila third instar larvae before and after CDK12 depletion by RNA interference (RNAi). Here we show that CDK12 regulates heterochromatin dynamics in Drosophila chromosomes. Depletion of CDK12 induces the increased HP1a and H3K9me2 binding profile on the coding region of euchromatic genes, with the X chromosome being the most affected. These results are consistent with the polytene chromosome immunostaining pattern of HP1a and H3K9me2 after CDK12 knockdown in our initial cytological observations, which show that CDK12 depletion induce heterochromatin spreading on euchromatic arms, especially on the X chromosome. This study describes a novel role of the CDK12 complex in controlling the epigenetic transition between euchromatin and heterochromatin. Examination of the genome-wide H3K9me2 and HP1a binding profile in wildtype larvae (WT) and CDK12-depleted larvae (CDK12-KD). Examination of the genome-wide CDK12 binding profile in wildtype larvae (WT). Twelve independent immunoprecipitations were conducted for each antibody. Two biological replicates were performed.
2015-11-02 | E-GEOD-63011 | biostudies-arrayexpress
Project description:We performed mRNA sequencing on 184-hTert and SK-BR-3 and MDA-MB-231 cell lines treated with siRNA directed to CDK12 (Cyclin dependent kinase 12) or a scrambled siRNA control. For each cell line, we generated 6 RNA-seq libraries (3 treatment replicates and 3 control replicates) to study the differential expression and the differential splicing patterns regulated by CDK12.
2017-03-16 | GSE80409 | GEO
Project description:Mutations that attenuate DNA repair by homologous recombination (HR) promote tumorigenesis and sensitize cells to chemotherapeutic agents that cause replication fork collapse, a phenotype known as “BRCAness.” BRCAness tumors arise from loss-of-function mutations in 22 genes. Of these genes, all but one (Cdk12) directly function in the HR repair pathway. Cdk12 phosphorylates Serine 2 of the RNA Polymerase II (RNAPII) C-terminal domain (CTD) heptapeptide repeat, a modification that regulates transcription elongation, splicing, and cleavage/polyadenylation. Genome-wide expression studies suggest that Cdk12 depletion abrogates the expression of several HR genes relatively specifically, blunting HR repair. This observation suggests that Cdk12 mutational status may predict tumor sensitivity to targeted treatments against BRCAness, such as Parp 1 inhibitors, and that small-molecule inhibitors of Cdk12 may induce sensitization of otherwise HR-competent tumors to these treatments. Despite this growing clinical interest, the mechanism behind the apparent specificity of Cdk12 in regulating HR genes remains unknown. Here we find that Cdk12 globally suppresses intronic polyadenylation events, enabling the production of full-length gene products. Many HR genes harbor significantly more intronic polyadenylation sites compared to all expressed genes, and the cumulative effect of these sites accounts for the increased sensitivity of HR gene expression to Cdk12 loss. Finally, we find evidence that Cdk12 loss-of-function mutations cause increased intronic polyadenylation within HR genes in human tumors, suggesting that this mechanism is conserved. This work clarifies the biological function of CDK12 and underscores its potential both as a chemotherapeutic target and as a tumor biomarker.
2018-11-28 | GSE116017 | GEO
Project description:The majority of genic transcription is intronic. Introns are removed by splicing as branched lariat RNAs which require rapid recycling. The branch site is recognized during splicing catalysis and later debranched by Dbr1 in the rate-limiting step of lariat turnover. Through generation of the first viable DBR1 knockout cell line, we find the predominantly nuclear Dbr1 enzyme to encode the sole debranching activity in human cells. Dbr1 preferentially debranches substrates that contain canonical U2 binding motifs, suggesting that branchsites discovered through sequencing do not necessarily represent those favored by the spliceosome. We find that Dbr1 also exhibits specificity for particular 5' splice site sequences. We identify Dbr1 interactors through co-immunoprecipitation mass spectroscopy. We present a mechanistic model for Dbr1 recruitment to the branchpoint through the intron-binding protein AQR. In addition to a 20-fold increase in lariats, Dbr1 depletion increases exon skipping. Using ADAR fusions to timestamp lariats, we demonstrate a defect in spliceosome recycling. In the absence of Dbr1, spliceosomal components remain associated with the lariat for a longer period of time. As splicing is co-transcriptional, slower recycling increases the likelihood that downstream exons will be available for exon skipping.
2023-06-26 | MSV000092263 | MassIVE
Project description:Co-transcriptional splicing regulates 3' end cleavage during mammalian erythropoiesis
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Project description:Inactivation of CDK12 characterizes an aggressive sub-group of castration-resistant prostate cancer (CPRC), and CRPC is currently a lethal disease. Hyper-activation of MYC transcription factor is sufficient to confer the CRPC-phenotype. Here, we show that the loss of CDK12 promotes MYC activity, which renders the cells addicted on the splicing regulatory kinase SRPK1. High MYC expression is associated with increased levels of SRPK1 in patient samples, and over-expression of MYC sensitizes prostate cancer cells to SRPK1 inhibition using pharmacological and genetic strategies. We show that Endovion (SCO-101), a compound currently in clinical trials against pancreatic cancer, phenocopies the effects of the well-characterized SRPK1 inhibitor (SRPIN340) on the nascent transcription, splicing and the overall transcriptional program. Inhibition of SRPK1 with either of the compounds promotes transcription elongation, causes defects in splicing and transcriptionally activates the unfolded protein response. In brief, here we show that CDK12 inactivation promotes MYC-signaling in an SRPK1-dependent manner and identify the clinical grade compound Endovion, which selectively targets the cells with CDK12 inactivation.
2024-10-16 | GSE236089 | GEO
Project description:We performed Total RNA-seq on MV4;11 cells after 6 hours treatment of THZ531 to determine whether CDK12 and CDK13 inhibtion to cause splicing defects.
2020-04-29 | GSE141372 | GEO
Project description:In a forward genetic screen, we have previously identified a null mutant of Cdk12 that results in alterations in actin dynamics, the axon initial segment and electrophysiology in Drosophila melanogaster. To decipher how Cdk12 may be having these effects, we extracted RNA from pooled Drosophila heads and compared Cdk12-null mutants to controls at the transcriptome level.
2023-01-01 | E-MTAB-12090 | biostudies-arrayexpress