Splicing Factor 3b Subunit 1 (SF3B1), a core component of the spliceosome, undergoes dynamic phosphorylation and dephosphorylation during the splicing cycle to regulate pre-mRNA splicing. Twenty-eight threonine/proline repeats are phosphorylated by CDK11 during spliceosome activation and remain phosphorylated in the catalytically active spliceosomes. The function of phosphorylated SF3B1 (P-SF3B1), and the identity of spliceosomes stalled by CDK11 inhibition remain unclear. Using quantitative proteomics of chromatin-associated spliceosomes, we identify a previously uncharacterized intermediate complex B-OTS964, arrested by CDK11 inhibitor OTS964, that incorporates the nineteen-related (NTR) but not nineteen (NTC) complex. iCLIP-seq revealed that P-SF3B1 engages with the U6 snRNA internal stem-loop (ISL), suggesting a potential role in stabilizing the RNA catalytic core. We further demonstrate that P-SF3B1 is recognized by forkhead-associated (FHA) domain of SNIP1, which promotes recruitment of retention and splicing (RES) complex during spliceosome activation. Acute SNIP1 depletion disrupts RES incorporation, causes widespread splicing defects, and promotes hyperphosphorylation of SF3B1 by CDK11. Mutations in SNIP1 FHA domain, including the neurodevelopmental disorder-associated E366G variant, impair P-SF3B1 binding, pre-mRNA splicing, and cell viability. Together, these findings uncover the phosphorylation-dependent CDK11/P-SF3B1/SNIP1 signaling axis that is critical for pre-mRNA splicing and cellular proliferation and provide a mechanistic insight into its dysregulation in disease.

Cyclin-dependent kinase 11 (CDK11) is essential for the regulation of pre-mRNA splicing via phosphorylation of the core spliceosome component SF3B1. This phosphorylation is a marker of the catalytically active spliceosomes; thus, it is important to identify the mechanisms that regulate CDK11 itself. Here, we report that a small subset of CDK11 is phosphorylated on the activation T-loop threonine 595 (Thr595) and is associated with the activated spliceosome on chromatin in gene bodies. Mutational analyses revealed that Thr595 is essential for the formation of the active CDK11 complex with cyclin L and SAP30BP. CDK11 transiently associates with CDK7, a transcriptional kinase that also promotes the activation of other CDKs. Inhibition of CDK7 initially decreases transcription, but longer durations of inhibition lead to production of unspliced pre-mRNAs. The onset of the CDK7-mediated splicing defect correlates with the sequential dephosphorylation of CDK11 Thr595 and SF3B1. SILAC-based phosphoproteomics upon brief CDK11 inhibition identified SF3B1, CDC5L, and ESS2 as CDK11 substrates, which overlap with the previously identified CDK7 substrates in the spliceosome. In summary, our study suggests that CDK7 likely acts via CDK11 Thr595 phosphorylation to regulate pre-mRNA splicing in cells. The identification of additional CDK11 substrates points to its broader role in spliceosome regulation.

The ability of a cell to properly express its genes depends on optimal transcription and splicing. RNA polymerase II (RNAPII) transcribes protein-coding genes and produces pre-mRNAs, which undergo, largely co-transcriptionally, intron excision by the spliceosome complex. Spliceosome activation is a major control step, leading to a catalytically active complex. Recent work has showed that cyclin-dependent kinase (CDK)11 regulates spliceosome activation via the phosphorylation of SF3B1, a core spliceosome component. Thus, CDK11 arises as a major coordinator of gene expression in metazoans due to its role in the rate-limiting step of pre-mRNA splicing. This review outlines the evolution of CDK11 and SF3B1 and their emerging roles in splicing regulation. It also discusses how CDK11 and its inhibition affect transcription and cell cycle progression.

Human cyclin‐dependent kinases (CDKs) direct the progression of the cell cycle and transcription. They are deregulated in tumours, and despite their involvement in the regulation of basic cellular processes, many CDKs are promising targets for cancer therapy. CDK11 is an essential gene for the growth of many malignancies; however, its primary cellular function has been obscure, and the mode‐of‐action of OTS964, the first CDK11 inhibitor and antiproliferative compound, has been unknown. A recent study has shown that OTS964 prevents spliceosome activation, revealing a key role of CDK11 in the regulation of pre‐mRNA splicing. In light of these findings, we discuss the therapeutic potential of CDK11 in cancer.

RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA-protein complex that assembles de novo on each pre-mRNA intron through an ordered assembly of intermediate complexes1,2. Spliceosome activation is a major control step that requires substantial protein and RNA rearrangements leading to a catalytically active complex1-5. Splicing factor 3B subunit 1 (SF3B1) protein-a subunit of the U2 small nuclear ribonucleoprotein6-is phosphorylated during spliceosome activation7-10, but the kinase that is responsible has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N terminus during spliceosome activation. The phosphorylation is important for the association between SF3B1 and U5 and U6 snRNAs in the activated spliceosome, termed the Bact complex, and the phosphorylation can be blocked by OTS964, a potent and selective inhibitor of CDK11. Inhibition of CDK11 prevents spliceosomal transition from the precatalytic complex B to the activated complex Bact and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation and characterize OTS964 as a highly selective CDK11 inhibitor that suppresses spliceosome activation and splicing.

Cyclin-dependent kinase 12 (CDK12) phosphorylates the C-terminal domain of RNA polymerase II and is needed for the optimal transcription elongation and translation of a subset of human protein-coding genes. The kinase has a pleiotropic effect on the maintenance of genome stability, and its inactivation in prostate and ovarian tumours results in focal tandem duplications, a CDK12-unique genome instability phenotype. CDK12 aberrations were found in many other malignancies and have the potential to be used as biomarkers for therapeutic intervention. Moreover, the inhibition of CDK12 emerges as a promising strategy for treatment in several types of cancers. In this review, we summarize mechanisms that CDK12 utilizes for the regulation of gene expression and discuss how the perturbation of CDK12-sensitive genes contributes to the disruption of cell cycle progression and the onset of genome instability. Furthermore, we describe tumour-suppressive and oncogenic functions of CDK12 and its potential as a biomarker and inhibition target in anti-tumour treatments.

Replication-dependent histones (RDH) are required for packaging of newly synthetized DNA into nucleosomes during the S phase when their expression is highly upregulated. However, the mechanisms of this upregulation in metazoan cells remain poorly understood. Using iCLIP and ChIP–seq, we found that human cyclin-dependent kinase 11 (CDK11) associates with RNA and chromatin of RDH genes primarily in the S phase. Moreover, its amino-terminal region binds FLASH, an RDH-specific 3′-end processing factor, which keeps the kinase on the chromatin. CDK11 phosphorylates serine 2 (Ser2) of the carboxy-terminal domain of RNA polymerase II (RNAPII), which is initiated when RNAPII reaches the middle of RDH genes and is required for further RNAPII elongation and 3′-end processing. CDK11 depletion leads to decreased number of cells in S phase, likely owing to the function of CDK11 in RDH gene expression. Thus, the reliance of RDH expression on CDK11 could explain why CDK11 is essential for the growth of many cancers. CDK11 functions with histone pre-mRNA-processing factor FLASH to upregulate expression of replication-dependent histone genes in S phase and to couple transcription and processing of nascent histone transcripts in mammalian cells.

CDK12 is a kinase associated with elongating RNA polymerase II (RNAPII) and is frequently mutated in cancer. CDK12 depletion reduces the expression of homologous recombination (HR) DNA repair genes, but comprehensive insight into its target genes and cellular processes is lacking. We use a chemical genetic approach to inhibit analog-sensitive CDK12, and find that CDK12 kinase activity is required for transcription of core DNA replication genes and thus for G1/S progression. RNA-seq and ChIP-seq reveal that CDK12 inhibition triggers an RNAPII processivity defect characterized by a loss of mapped reads from 3'ends of predominantly long, poly(A)-signal-rich genes. CDK12 inhibition does not globally reduce levels of RNAPII-Ser2 phosphorylation. However, individual CDK12-dependent genes show a shift of P-Ser2 peaks into the gene body approximately to the positions where RNAPII occupancy and transcription were lost. Thus, CDK12 catalytic activity represents a novel link between regulation of transcription and cell cycle progression. We propose that DNA replication and HR DNA repair defects as a consequence of CDK12 inactivation underlie the genome instability phenotype observed in many cancers.

Abstract The Cdk12/CycK complex is a novel transcription elongation-associated kinase that promotes expression of a subset of RNA polymerase II (Pol II) genes, including those of the DNA damage response (DDR) network. CDK12 is mutated in many cancers, particularly in high-grade serous ovarian carcinoma (HGS-OvCa), where it is one of only nine genes with statistically recurrent somatic mutations. However, the influence of these mutations on the kinase and their link to cancerogenesis remain ill-defined. Here, we show that most CDK12 mutations interfere with formation of the complex, rendering the kinase inactive. By examining the mutations within the Cdk12/CycK structure, we find that they likely provoke structural rearrangements detrimental to Cdk12 activation. Our mRNA expression analysis of the patient samples containing the CDK12 mutations identifies coordinated down-regulation of key DDR genes, particularly those of the homologous recombination (HR) repair pathway. Moreover, we note that production of nascent HR gene transcripts is compromised significantly upon CDK12 and CYCK knockdown. Indeed, Cdk12 occupies these genes and promotes phosphorylation of the Pol II's C-terminal domain (CTD) at Serine 2 (Ser2-P), the hallmark of effective transcription elongation, indicating that the down-regulated genes are directly controlled by Cdk12/CycK. Functionally, we demonstrate that the mutant Cdk12 proteins fail to stimulate the faithful repair of DNA double strand breaks via HR. In summary, we provide molecular basis of how mutated CDK12 ceases to function in ovarian carcinoma. We propose that CDK12 is a tumor suppressor of which the loss-of-function mutations could elicit defects in HR-mediated and possibly other DNA repair pathways, leading to genomic instability underlying the genesis of HGS-OvCa. Citation Format: Kingsley M. Ekumi, Hana Paculova, Tina Lenasi, Vendula Pospichalova, Christian A. Bösken, Jana Rybarikova, Vitezslav Bryja, Matthias Geyer, Dalibor Blazek and Matjaz Barboric. OVARIAN CARCINOMA CDK12 MUTATIONS IMPAIR GENE-SPECIFIC POL II ELONGATION AND FAITHFUL REPAIR OF DNA DOUBLE STRAND BREAKS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-043.

new study reports THZ531 as a covalent CDK12/CDK13 inhibitor affecting transcription. Application of the compound in cells decreases transcription elongation of DNA damage response genes and key super-enhancer-associated transcription factor genes, with important implications for targeted cancer therapy.

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A CD8+ cell non-cytotoxic antiviral response (CNAR), mediated by a CD8+ cell antiviral factor (CAF), is associated with a long-term healthy state in human immunodeficiency virus (HIV) infection. CNAR/CAF reduces viral transcription without a known effect on specific viral sequences in the HIV genome. In studies to define the mechanism involved in the block in viral transcription, we now report that transcription from the HIV-LTR reporter is reduced in infected CD4+ cells upon treatment with CAF. In agreement with this observation, the amount of RNA polymerase II (RNAPII) on the HIV promoter and other viral regions was strongly diminished in HIV-infected CD4+ cells co-cultivated with CNAR-expressing CD8+ cells. These results demonstrate further that CNAR/CAF has a specific role in regulating HIV transcription and a step during the preinitiation complex assembly appears to be sensitive to CNAR/CAF.

Abstract The Cdk12/CycK complex promotes gene expression by phosphorylating the C-terminal domain of RNA polymerase II. In previous work we found that CDK12/CycK complex maintains genomic stability via regulation of expression of several key DNA damage response genes such as BRCA1, ATR, FANCD2 or FANCI. CDK12 is among only nine genes with recurrent somatic mutations in high-grade serous ovarian carcinoma. However, the influence of these mutations on the Cdk12/CycK complex and their link to cancerogenesis remain ill-defined. Here, we show that most mutations interfere with the Cdk12/CycK complex formation, rendering the kinase inactive. By examining the mutations within the Cdk12/CycK structure, we find that they likely provoke structural rearrangements detrimental to Cdk12 activation. Our mRNA expression analysis of the patient samples containing the CDK12 mutations identifies coordinated down-regulation of key DNA damage response genes. Accordingly, we observed that the mutant Cdk12 proteins fail to promote the repair of DNA double strand breaks via homologous recombination. Together, we provide the molecular basis of how mutated CDK12 ceases to function in ovarian carcinoma. We propose that CDK12 is a tumor suppressor of which the loss-of-function mutations may elicit defects in multiple DNA repair pathways, leading to genomic instability that underlies the genesis of the cancer.. Citation Format: Kingsley M. Ekumi, Hana Paculova,Vendula Pospichalova, Christian A. Bosken,Vitezslav Bryja, Matthias Geyer, Dalibor Blazek, Matjaz Barboric. Ovarian carcinoma CDK12 mutations misregulate DNA repair genes via deficient formation and function of the Cdk12/CycK complex [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS21.

The Cdk12/CycK complex promotes expression of a subset of RNA polymerase II genes, including those of the DNA damage response. CDK12 is among only nine genes with recurrent somatic mutations in high-grade serous ovarian carcinoma. However, the influence of these mutations on the Cdk12/CycK complex and their link to cancerogenesis remain ill-defined. Here, we show that most mutations prevent formation of the Cdk12/CycK complex, rendering the kinase inactive. By examining the mutations within the Cdk12/CycK structure, we find that they likely provoke structural rearrangements detrimental to Cdk12 activation. Our mRNA expression analysis of the patient samples containing the CDK12 mutations reveals coordinated downregulation of genes critical to the homologous recombination DNA repair pathway. Moreover, we establish that the Cdk12/CycK complex occupies these genes and promotes phosphorylation of RNA polymerase II at Ser2. Accordingly, we demonstrate that the mutant Cdk12 proteins fail to stimulate the faithful DNA double strand break repair via homologous recombination. Together, we provide the molecular basis of how mutated CDK12 ceases to function in ovarian carcinoma. We propose that CDK12 is a tumor suppressor of which the loss-of-function mutations may elicit defects in multiple DNA repair pathways, leading to genomic instability underlying the genesis of the cancer.

Cyclin-dependent kinases regulate the cell cycle and transcription in higher eukaryotes. We have determined the crystal structure of the transcription kinase Cdk13 and its Cyclin K subunit at 2.0 Å resolution. Cdk13 contains a C-terminal extension helix composed of a polybasic cluster and a DCHEL motif that interacts with the bound ATP. Cdk13/CycK phosphorylates both Ser5 and Ser2 of the RNA polymerase II C-terminal domain (CTD) with a preference for Ser7 pre-phosphorylations at a C-terminal position. The peptidyl-prolyl isomerase Pin1 does not change the phosphorylation specificities of Cdk9, Cdk12, and Cdk13 but interacts with the phosphorylated CTD through its WW domain. Using recombinant proteins, we find that flavopiridol inhibits Cdk7 more potently than it does Cdk13. Gene expression changes after knockdown of Cdk13 or Cdk12 are markedly different, with enrichment of growth signaling pathways for Cdk13-dependent genes. Together, our results provide insights into the structure, function, and activity of human Cdk13/CycK.

The Cdk12/CycK complex promotes expression of a subset of RNA polymerase II genes, including those of the DNA damage response. CDK12 is among only nine genes with recurrent somatic mutations in high-grade serous ovarian carcinoma. However, the influence of these mutations on the Cdk12/CycK complex and their link to cancerogenesis remain ill-defined. Here, we show that most mutations prevent formation of the Cdk12/CycK complex, rendering the kinase inactive. By examining the mutations within the Cdk12/CycK structure, we find that they likely provoke structural rearrangements detrimental to Cdk12 activation. Our mRNA expression analysis of the patient samples containing the CDK12 mutations reveals coordinated downregulation of genes critical to the homologous recombination DNA repair pathway. Moreover, we establish that the Cdk12/CycK complex occupies these genes and promotes phosphorylation of RNA polymerase II at Ser2. Accordingly, we demonstrate that the mutant Cdk12 proteins fail to stimulate the faithful DNA double strand break repair via homologous recombination. Together, we provide the molecular basis of how mutated CDK12 ceases to function in ovarian carcinoma. We propose that CDK12 is a tumor suppressor of which the loss-of-function mutations may elicit defects in multiple DNA repair pathways, leading to genomic instability underlying the genesis of the cancer.

Phosphorylation of the RNA polymerase II C-terminal domain (CTD) by cyclin-dependent kinases is important for productive transcription. Here we determine the crystal structure of Cdk12/CycK and analyse its requirements for substrate recognition. Active Cdk12/CycK is arranged in an open conformation similar to that of Cdk9/CycT but different from those of cell cycle kinases. Cdk12 contains a C-terminal extension that folds onto the N- and C-terminal lobes thereby contacting the ATP ribose. The interaction is mediated by an HE motif followed by a polybasic cluster that is conserved in transcriptional CDKs. Cdk12/CycK showed the highest activity on a CTD substrate prephosphorylated at position Ser7, whereas the common Lys7 substitution was not recognized. Flavopiridol is most potent towards Cdk12 but was still 10-fold more potent towards Cdk9. T-loop phosphorylation of Cdk12 required coexpression with a Cdk-activating kinase. These results suggest the regulation of Pol II elongation by a relay of transcriptionally active CTD kinases.

Cytochrome P450 1B1 (CYP1B1) is an enzyme that has a unique tumor-specific pattern of expression and is capable of bioactivating a wide range of carcinogenic compounds. We have reported previously that coordinated upregulation of CYP1B1 by inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and the aryl hydrocarbon receptor ligands, may increase bioactivation of promutagens, such as benzo[a]pyrene (BaP) in epithelial cells. Here, we extend those studies by describing a novel mechanism participating in the regulation of CYP1B1 expression, which involves activation of the p38 mitogen-activated protein kinase (p38) and mitogen- and stress-activated protein kinase 1 (MSK1). Using inhibitors of p38 and MSKs, as well as mouse embryonic cells derived from p38α-deficient and MSK1/2 double knockout mice, we show here that TNF-α potentiates CYP1B1 upregulation via the p38/MSK1 kinase cascade. Effects of this inflammatory cytokine on CYP1B1 expression further involve the positive transcription elongation factor b (P-TEFb). The inhibition of the P-TEFb subunit, cyclin-dependent kinase 9 (CDK9), which phosphorylates RNA polymerase II (RNAPII), prevented the enhanced CYP1B1 induction by a combination of BaP and inflammatory cytokine. Furthermore, using chromatin immunoprecipitation assays, we found that cotreatment of epithelial cells with TNF-α and BaP resulted in enhanced recruitment of both CDK9 and RNAPII to the Cyp1b1 gene promoter. Overall, these results have implications concerning the contribution of inflammatory factors to carcinogenesis, since enhanced CYP1B1 induction during inflammation may alter metabolism of exogenous carcinogens, as well as endogenous CYP1B1 substrates playing role in tumor development.

The cyclin-dependent kinases (Cdks) regulate many cellular processes, including the cell cycle, neuronal development, transcription, and posttranscriptional processing. To perform their functions, Cdks bind to specific cyclin subunits to form a functional and active cyclin/Cdk complex. This review is focused on Cyclin K, which was originally considered an alternative subunit of Cdk9, and on its newly identified partners, Cdk12 and Cdk13. We briefly summarize research devoted to each of these proteins. We also discuss the proteins' functions in the regulation of gene expression via the phosphorylation of serine 2 in the C-terminal domain of RNA polymerase II, contributions to the maintenance of genome stability, and roles in the onset of human disease and embryo development.

Comment on: Blazek D, et al. Genes Dev 2011; 25:2158–72