PXD009533-1

PXD009533 is an original dataset announced via ProteomeXchange.

Title	CDK12 regulates DNA damage response genes through suppression of premature cleavage and polyadenylation
Description	Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle or transcription, and are emerging as promising therapeutic targets in cancer cells. The transcriptional kinase, CDK12, is especially intriguing, as it modulates transcription elongation by phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase (Pol II) with subsequent effects on expression of DNA damage response (DDR) genes. Yet the exact mechanism by which CDK12 regulates this vital process remains unclear. Using the selective inhibitor of CDK12/13, THZ5317 and transient transcriptome sequencing (TT-seq), we sought to capture the dynamic transcriptional changes linked to CDK12 by analyzing the immediate, early effects on nascent RNA production after CDK12 depletion in neuroblastoma (NB) cells. CDK12-induced inhibition resulted in gene length-dependent defects in transcription elongation, leading to a disproportionate loss of 3' reads in long genes. Short transcripts, by contrast, including those of replication-dependent histone genes, underwent 3' UTR extension. The effect on long genes was accompanied by premature cleavage and polyadenylation (PCPA), followed by early termination and loss of gene expression. DDR gene transcripts were the most prominent among those terminated by PCPA. Phosphoproteomic analysis indicated that pre-mRNA processing factors, including those involved in CPA , are direct phospho-targets of CDK12 and that CDK depletion phenocopied their effects on transcription. Importantly, sensitivity to CDK12 inhibition was predicted by a lower proportion of U1 snRNP binding sites compared to polyadenylation sites (PAS) and hence a higher propensity to intronic poly(A) site selection - a characteristic observed in most DDR genes. These results support a model in which CDK12 regulates expression of DDR genes not only by regulating transcription elongation through its effects on Pol II CTD phosphorylation, but also through direct phosphorylation of pre-mRNA processing factors. These mechanistic insights may enable the development of new anti-cancer strategies targeting multiple vulnerable steps in CDK12-dependent regulation of DNA damage repair.
HostingRepository	MassIVE
AnnounceDate	2018-04-18
AnnouncementXML	Submission_2018-04-18_10:31:56.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Mark Adamo
SpeciesList	scientific name: Homo sapiens; common name: human; NCBI TaxID: 9606;
ModificationList	Label:13C(6)15N(2); Label:13C(6)15N(4); Oxidation; Phospho; Carbamidomethyl
Instrument	Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2018-04-18 09:34:36	ID requested
⏵ 1	2018-04-18 10:31:57	announced

no publication

submitter keyword: Cyclin-dependent kinase, CDK, CDK12, CDK13, DNA damage, premature cleavage, polyadenylation, THZ5317, transcription, phosphorylation, SILAC, phosphoproteomics

Scott Gerber
contact affiliation	The Geisel School of Medicine at Dartmouth
contact email	scott.a.gerber@dartmouth.edu
lab head
Mark Adamo
contact affiliation	Dartmouth College
contact email	mark.e.adamo@dartmouth.edu
dataset submitter

MassIVE dataset URI

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