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

Abstract

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.

Keywords: Biological sciences; cell biology; molecular biology.

Graphical abstract

Figure 1

Effects of THZ531 on nascent transcription (A) HF1, K562, and HeLa cells were treated with 400 nM THZ531 for 1 h at 37°C with the addition of bromouridine for the last 30 min and Bru-labeled RNA was then prepared for Bru-seq. Nascent RNA signal from Bru-seq libraries is represented as reads per kilobase per million (RPKM) and is shown for the control (blue) and THZ531 (yellow) for each cell line. The RPKM sign indicates the transcribed strand (positive values for plus strand signal; negative values for minus strand signal). Gene models are above the signal tracks and transcript isoform models are below (green for plus strand; red for minus strand). The nascent RNA reads were mapped to Hg38. Examples of genes showing upregulated transcription following THZ531 treatment in all three cell lines are RALY and SERTAD2. (B) Gene set enrichment analysis (GSEA) of Bru-seq data showing gene sets upregulated across all three cell lines following a 1-h THZ531 treatment. Gene sets in the Hallmark and KEGG pathways are shown and the data is expressed as normalized enrichment scores (NES). (C) Nascent transcription of genes, represented as in (A), showing reduced signal toward their 3′-ends following THZ531 treatment. (D) Aggregated profile of gene transcription end sites (TESs) showing that THZ531 reduces transcription toward the 3′-ends and past the TES. For the aggregate graphs, 2637 genes >20kb in length and expressed at >0.5 RPKM in the HF1 control sample were used.

Figure 2

Effects of the adenine analog 1-NM-PP1 on nascent transcription in HeLa-AS cells (A) HeLa-AS cells were treated in two biological experiments with DMSO or 1-NM-PP1 for 30 min in the presence of bromouridine and Bru-labeled RNA was then prepared for Bru-seq. Inhibition of CDK12 resulted in the rapid reduction of transcription reads at the 3′-ends of the indicated genes (represented as in Figure 1A). No transcriptional effects of 1-NM-PP1 treatment were observed in parental cells. (B) Aggregate view of gene transcription end sites (TESs) from Bru-seq data showing that the inhibition of CDK12 results in reduced transcription toward the 3′-end of genes and past the TES. For the aggregate graphs, 2619 genes >20kb in length and expressed at >0.5 RPKM in the HeLa + DMSO sample are represented. (C) Contiguous regions of transcription at different expression levels (shown as rectangular overlays; one overlay per replicate) for the DOCK5 gene. A single, long segment overlays the gene body in the control (top), but is partitioned into two segments (high and low expression) after CDK12 inhibition, indicating the region containing a presumptive intergenic termination site. (D) Genes with intragenic termination events identified by transcription segment partitioning. 688 genes were found to be common to both biological replicates. (E–H) DAVID analyses using the 688 common gene list show enrichment for gene sets. E) KEGG pathways, F) tissues and cluster enrichment, G) DNA damage responses, H) cell cycle and J) kinetochore and chromosomes. Enrichment is expressed as -log₁₀ p value.

Figure 3

Effects of THZ351 treatment on the turnover of RNAs (A) Treatment with THZ531 stabilized several transcripts such as MAT2A, CCNL1, and PDP1 while de-stabilized transcripts such as SRSF1. HF1 cells were either mock-treated or treated for 1h with 400 nM THZ531 with labeling with bromouridine for the last 30 min. Cells were then chased in 20 mM uridine in the presence or absence of THZ531 for 6 h and prepared for BruChase-seq. Bru-seq and BruChase-seq signal is presented in Figure 1A. (B) Enriched gene sets from BruChase-seq differential expression in THZ531-treated HF1, K562, and HeLa cells and presented as normalized expression scores (NES) from GSEA.

Figure 4

Effect of 1-NM-PP1 treatment of HeLa-AS cells on RNA turnover (A) HeLa-AS cells were mock-treated with DMSO or 1-NM-PP1 for 30 min in the presence of bromouridine. The cells were then chased in uridine in the presence of either DMSO or 1-NM-PP1 for 2 h before preparing the Bru-RNA for BruChase-seq (n = 3). The top panels are displaying Bru-seq reads and the bottom panels are displaying BruChase-seq reads (2-h chase). For a transcript to be considered stabilized by 1-NM-PP1 treatment, the ratio of the treated sample (yellow) to control (blue) is higher in the BruChase-seq data than in Bru-seq data (e.g. MAT2A)and lower for de-stabilized transcripts (e.g. CBX5, MY O 19 and SRSF1). (B) Volcano plot showing transcripts stabilized (red) and transcripts de-stabilized (blue) after CDK12 inhibition (adjusted p value < 0.01; log₂ fold-change > 1 or < −1). (C) Enrichment of Hallmark and KEGG pathway gene sets using BruChase-seq stability following CDK12 inhibition shown as normalized enrichment scores (NES) from GSEA.

Figure 5

Effect of CDK12 inhibition on co-transcriptional splicing (A) THZ531 suppresses global co-transcriptional splicing in HF1, K562, and HeLa cells. Splicing index (SI) values were calculated from Bru-seq data using intron junctions with sufficient reads to make the cut-off as previously described (Bedi et al., 2021). The number of introns qualifying for the analyses is shown below the violin plots. The median SI values are shown in the white rectangles. (B) Inhibition of CDK12 with 1NMPP1 in HeLa-AS cells shows slight but significantly reduced co-transcriptional splicing. (C) SI-values for HF1, K562, and HeLa cells from BruChase-seq data show no significant difference between THZ531-treated and control cells for any of the cell lines. (D) SI-values for 1NMPP1-treated HeLa-AS cells show a slight, but significant difference following a 2-h chase. ∗∗∗ = p value<10⁻¹⁵, ∗∗ = p value<0.0007.

Figure 6

Model of the roles of CDK12 in transcription and post-transcriptional processes Our results implement CDK12 playing roles in transcription both in suppressing premature termination at cryptic polyadenylation sites (PAS) as well as promoting transcriptional readthrough past transcription end sites (TES). CDK12 also was found to promote more efficient general splicing of transcripts. Finally, our study shows that CDK12 is involved in the regulation of transcript stability where transcripts encoding DNA repair enzymes are stabilized by CDK12 while transcripts encoding proteins in the p53, G2/M checkpoint, hypoxia pathways as well as the spliceosome are de-stabilized.