CDK12/13 promote splicing of proximal introns by enhancing the interaction between RNA polymerase II and the splicing factor SF3B1

Abstract

Transcription-associated cyclin-dependent kinases (CDKs) regulate the transcription cycle through sequential phosphorylation of RNA polymerase II (RNAPII). Herein, we report that dual inhibition of the highly homologous CDK12 and CDK13 impairs splicing of a subset of promoter-proximal introns characterized by weak 3' splice sites located at larger distance from the branchpoint. Nascent transcript analysis indicated that these introns are selectively retained upon pharmacological inhibition of CDK12/13 with respect to downstream introns of the same pre-mRNAs. Retention of these introns was also triggered by pladienolide B (PdB), an inhibitor of the U2 small nucelar ribonucleoprotein (snRNP) factor SF3B1 that recognizes the branchpoint. CDK12/13 activity promotes the interaction of SF3B1 with RNAPII phosphorylated on Ser2, and disruption of this interaction by treatment with the CDK12/13 inhibitor THZ531 impairs the association of SF3B1 with chromatin and its recruitment to the 3' splice site of these introns. Furthermore, by using suboptimal doses of THZ531 and PdB, we describe a synergic effect of these inhibitors on intron retention, cell cycle progression and cancer cell survival. These findings uncover a mechanism by which CDK12/13 couple RNA transcription and processing, and suggest that combined inhibition of these kinases and the spliceosome represents an exploitable anticancer approach.

Graphical Abstract

CDK12/13 promote splicing of proximal introns by enhancing the interaction between RNA polymerase II and the splicing factor SF3B1.

Figure 1.

Inhibition of transcriptional CDKs causes widespread intron retention. (A) Venn diagram showing the overlap between genes regulated at the gene expression (GE) and alternative splicing (AS) level by THZ1 (6 h, 100 nM). Statistical analyses were performed by Fisher's exact test. (B) Pie chart showing percentages of the indicated splicing patterns affected by THZ1. (C) Bar graph showing percentages of events annotated in FAST-DB (white columns) and of those regulated by THZ1 treatment (grey columns) within each AS pattern. Statistical analyses of comparisons between THZ1-regulated events and their expected representation in the reference database were performed by modified Fisher's test. (D) Pie charts showing percentages of intronic or exonic events in unannotated events. (E) Pie charts showing percentages of intronic or exonic events in total events. (F) Pie charts showing percentages of up- and down-regulated introns in THZ1-treated cells. (G and H) Gene Ontology of up- and down-regulated IR events performed by the g-profiler tool.

Figure 2.

THZ1 treatment leads to widespread retention of proximal introns. (A) Metagene representation of IR location within the gene body. (B) Bar graph showing GE fold change between the first and second introns and all other introns. Statistical analysis was performed by Student's test. (C) Bar graph showing percentages of IR genes that were regulated at the GE level or not between the first and second up-regulated introns and all other introns (left panel) and between the first and second up-regulated and down-regulated introns (right panel). Statistical analyses were performed by Fisher's exact test. (D) Graphic representation of POLH and SOGA1 genes showing the IR event in the first intron and the reduction in read coverage in downstream portions of the transcript after THZ1 treatment. (E–H) Visualization of the RNA-seq reads profile of the intron-retaining region and PCR primer strategy used in POLH (E) and SOGA1 (G) genes. Bar graphs showing the results of qPCR analyses for the expression of the retained first intron (ex1-int1) and the reduction in downstream portions of the transcript (ex5-ex6) in POLH (F) and SOGA1 (H) genes relative to FKBP9. Data represent the mean of at least three independent experiments with relative standard devation (SD). Statistical analyses were performed by Student's test *P <0.05; **P <0.01; ***P <0.001.

Figure 3.

CDK12/13 kinase activity is required for optimal splicing of THZ1-regulated proximal introns. (A) Schematic representation of the substrates inhibited by THZ1 (CDK7, CDK12 and CDK13), SY-1365 (CDK7) and THZ531 (CDK12/13). (B) Proliferation assays of MiaPaCa-2 cells after 72 h of THZ1, THZ531 and SY-1365 treatment at the indicated doses. Data represent the mean of at least three independent experiments with relative SD. (C and D) Bar graphs showing the results of qPCR analyses for the expression of the retained first intron (ex1-int1) in POLH and SOGA1 genes relative to FKBP9 in cells treated for 6 h with THZ531 (100 nM) or SY-1365 (33 nM) (C) or knocked down for CDK12/13 (shCDK12/13) or CDK7 (shCDK7) or transfected with empty vector (shCTRL) (D). Data represent the mean of at least three independent experiments with relative SD. Statistical analyses were performed by one-way analysis of variance (ANOVA), *P <0.05; **P <0.01, ns = not significant. (E and F) Correlation of the expression of CDK12 (E) or CDK13 (F) with that of POLH and SOGA1 in patients from the pancreatic adenocarcinoma project (TCGA, Firehose Legacy). Statistical analyses were performed by Pearson's correlation coefficient (E and F).

Figure 4.

CDK12/13 inhibition causes a splicing defect of proximal introns in target genes. (A) Schematic representation for the isolation of nascent RNAs labelled with 4sU. Transcription of cells was blocked by treatment with 100 μM DRB for 6 h. In the last hour, 200 nM THZ531 or DMSO was added; then DRB was removed by washing the cells with PBS. In the last 30 min, nascent RNAs were labelled with 4sU in the presence or not of THZ531. (B and C) Bar graphs showing the results of qRT–PCR analysis of POLH (B) and SOGA1 (C) to evaluate the level of IR in nascent RNA (4sU labelled) using primers spanning the 5′ (ex1-i1 5′) and 3′ (i1/2 3′-ex2/3) splice site regions of the regulated intron with respect to the spliced mRNA (ex1-ex2/3) and primers spanning the 5′ splice site region of a non-regulated intron (ex5-i5 5′) with respect to the spliced mRNA (ex5-ex6). A graphical representation of the genomic regions analysed and of the PCR primer strategy used is illustrated. Data represent the mean of at least three independent experiments with relative SD. Statistical analyses were performed by Student's test *P <0.05; **P <0.01, ns = not significant.

Figure 5.

Proximal introns regulated by CDK12/13 activity are characterized by weak 3′ splice sites. (A) Box plots representing comparison of the 5′ and 3′ splice site strength (MaxEnt score) between proximal introns (first and second) up- and down-regulated by THZ1 treatment, reference IR events not affected by THZ1 treatment (Ref. IR), constitutively spliced introns (Ref. const.) and introns from genes regulated by THZ1 only at the GE level (Ref. GE-reg). (B and C) Boxplots showing comparison between the groups described in (A) for distance (B) and percentage of pyrimidine (C) between the branchpoint (BP) and the 3′ splice site. Statistical analyses were performed by Student's test *P <0.05; **P <0.01; ***P <0.001, ns = not significant.

Figure 6.

CDK12/13 inhibition impairs the physical interaction of SF3B1 with RNAPII. (A) Representative western blot analysis of the co-immunoprecipitation between RNAPII and SF3B1 from MiaPaCa-2 nuclear extracts treated or not with RNase A. Rabbit non-immune IgGs were used as negative control. To assess degradation, RNA was isolated from both RNase A-untreated and -treated cell lysates and run on a 1% agarose gel to determine the integrity of the 28S:18S rRNA. The bar graph represents densitometric analysis of the co-immunoprecipitated SF3B1 with respect to the levels of immunoprecipitated RNAPII (mean of three independent experiments with relative SD). Statistical analyses were performed by Student's test, ns = not significant. (B) Representative western blot and densitometric analyses of RNAPII phosphorylated on Ser2, Ser5 or total RNAPII that co-immunoprecipitates with SF3B1 from MiaPaCa-2 nuclear extracts (mean of three independent experiments with relative SD). Statistical analyses were performed by one-way ANOVA, ***P <0.001. (C) Representative western blot and densitometric analyses of phospho-Ser2, phospho-Ser5 and total RNAPII in MiaPaCa2 cells treated or not with 200 nM THZ531 for 3 h. HSP90 was used as loading control (mean of three independent experiments with relative SD). Statistical analyses were performed by Student's test, **P <0.01, ns = not significant. (D) Representative western blot and densitometric analyses of SF3B1 and U1A co-immunoprecipitated with RNAPII in MiaPaCa-2 cells treated or not with 200 nM THZ531 for 3 h (mean of at least three independent experiments with relative SD). Statistical analyses were performed by Student's test, **P <0.01. (E) Representative western blot of SF3B1, RNAPII, PRP6 and PRP8 in cytosol, nucleoplasm and chromatin fractions of MiaPaCa-2 cells treated for 3 h with 200 nM THZ531. TUBULIN, U1-70K and H3 expression were evaluated as loading controls of the indicated fractions. For densitometric analyses, SF3B1 levels were normalized to U170K for the nucleoplasm fraction, and SF3B1 levels were normalized to H3 for the chromatin fraction. Data represent the mean of at least three independent experiments with relative SD. Statistical analyses were performed by Student's test, *P <0.05, ns = not significant. (F and G) qPCR analyses of RIP (F) and CLIP (G) assays of the binding of SF3B1 to the 3′ splice site of POLH intron 1 and SOGA1 intron 2 from cells treated for 3 h with DMSO or THZ531 (200 nM). Non-immune IgGs were used as control of the assays (mean of three independent experiments with relative SD). Statistical analyses were performed by one-way ANOVA, *P <0.05, **P <0.01, ***P <0.001.

Figure 7.

THZ531 synergizes with spliceosome inhibition in IR regulation and suppression of cell proliferation. (A) Results of qPCR analyses for the expression of the retained first intron in POLH and SOGA1 genes relative to FKBP9 in MiaPaCa-2 cells treated with suboptimal doses of THZ531 (50 nM) and PdB (3.3 nM) either alone or in combination for 6 h (mean of at least three independent experiments with relative SD). Statistical analyses were performed by one-way ANOVA, ***P <0.001. (B) Cell proliferation analysis of MiaPaCa-2 cells treated with suboptimal doses of THZ531 (25 nM) and PdB (1 nM) either alone or in combination (mean of at least three independent experiments with relative SD). Statistical analyses are reported in light blue when referring to 25 nM THZ531 treatment, in green when referring to 1 nM PdB treatment and in grey when referring to DMSO. Statistical analyses were performed by two-way ANOVA *P <0.05; **P <0.01; ***P <0.001. (C) Representative images and bar graph of MiaPaCa-2 cells labelled with cytotoxic green and NIR dyes for live cells after 96 h of treatment with suboptimal doses of THZ531 and PdB either alone or in combination analysed by the IncuCyte SX5 technology to evaluate the percentage of dead cells in each sample (mean of three independent experiments with relative SD). Statistical analyses were performed by one-way ANOVA, ***P <0.001. Scale bar 150 μm. (D) Representative western blot analysis of PARP1, γH2AX, H3 and HSP90 in MiaPaCa-2 cells treated for 48 h as indicated. (E–H) Fluorescence-activated cell sorting (FACS) analyses showing DNA content (7-AAD) to evaluate the cell cycle state of MiaPaCa2 cells after 24 h at optimal (E) or suboptimal doses (G) of the indicated drugs. Bar graphs (F and H) show the percentage of cells in G₁, S and G₂ phase. Data represent the mean of four independent experiments with relative SD. Statistical analyses were performed by one-way ANOVA, *P <0.05; **P <0.01; ***P <0.001.