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CDK12 regulates co-transcriptional splicing and RNA turnover in human cells

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE191222 | GEO | 2022/09/17

REPOSITORIES: GEO

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Project 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.

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CDK12 regulates co-transcriptional splicing and RNA turnover in human cells

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