Single-cell RNA-seq methods can be used to delineate cell types and states at unprecedented resolution but do little to explain why certain genes are expressed. Single-cell ATAC-seq and multiome (ATAC + RNA) have emerged to give a complementary view of the cell state. It is however unclear what additional information can be extracted from ATAC-seq data besides transcription factor binding sites. Here, we show that ATAC-seq telomere-like reads counter-inituively cannot be used to infer telomere length, as they mostly originate from the subtelomere, but can be used as a biomarker for chromatin condensation. Using long-read sequencing, we further show that modern hyperactive Tn5 does not duplicate 9 bp of its target sequence, contrary to common belief. We provide a new tool, Telomemore, which can quantify nonaligning subtelomeric reads. By analyzing several public datasets and generating new multiome fibroblast and B-cell atlases, we show how this new readout can aid single-cell data interpretation. We show how drivers of condensation processes can be inferred, and how it complements common RNA-seq-based cell cycle inference, which fails for monocytes. Telomemore-based analysis of the condensation state is thus a valuable complement to the single-cell analysis toolbox.

• MeSH
• Single-Cell Analysis * methods   MeSH
• B-Lymphocytes metabolism   MeSH
• Cell Cycle * genetics   MeSH
• Chromatin Immunoprecipitation Sequencing methods   MeSH
• Chromatin * genetics   metabolism   MeSH
• Fibroblasts metabolism   MeSH
• Humans MeSH
• RNA-Seq methods   MeSH
• Telomere genetics   MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chromatin * MeSH

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.

• MeSH
• Enzyme Activation drug effects   MeSH
• Quinolones pharmacology   MeSH
• Chromatin metabolism   MeSH
• Cyclin-Dependent Kinases * antagonists & inhibitors   metabolism   MeSH
• Phosphoproteins * chemistry   metabolism   MeSH
• Phosphorylation MeSH
• Ribonucleoprotein, U2 Small Nuclear * chemistry   metabolism   MeSH
• RNA Precursors * genetics   metabolism   MeSH
• RNA Splicing * drug effects   MeSH
• Spliceosomes * drug effects   metabolism   MeSH
• Threonine metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Quinolones MeSH
• Chromatin MeSH
• Cyclin-Dependent Kinases * MeSH
• Phosphoproteins * MeSH
• Ribonucleoprotein, U2 Small Nuclear * MeSH
• OTS964 MeSH Browser
• RNA Precursors * MeSH
• Threonine MeSH

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.

• Publication type
• Journal Article MeSH

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.

• Keywords
• CDK12, CTD Ser2 phosphorylation, G1/S, premature termination and polyadenylation, tandem duplications,
• MeSH
• Cyclin-Dependent Kinases genetics   metabolism   MeSH
• Phosphorylation MeSH
• HCT116 Cells MeSH
• G1 Phase Cell Cycle Checkpoints genetics   physiology   MeSH
• Humans MeSH
• DNA Repair genetics   physiology   MeSH
• DNA Replication genetics   physiology   MeSH
• RNA Polymerase II genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CDK12 protein, human MeSH Browser
• Cyclin-Dependent Kinases MeSH
• RNA Polymerase II MeSH

Congenital heart defects, dysmorphic facial features and intellectual developmental disorders (CHDFIDD) syndrome in humans was recently associated with mutation in CDK13 gene. In order to assess the loss of function of Cdk13 during mouse development, we employed gene trap knock-out (KO) allele in Cdk13 gene. Embryonic lethality of Cdk13-deficient animals was observed by the embryonic day (E) 16.5, while live embryos were observed on E15.5. At this stage, improper development of multiple organs has been documented, partly resembling defects observed in patients with mutated CDK13. In particular, overall developmental delay, incomplete secondary palate formation with variability in severity among Cdk13-deficient animals or complete midline deficiency, kidney failure accompanied by congenital heart defects were detected. Based on further analyses, the lethality at this stage is a result of heart failure most likely due to multiple heart defects followed by insufficient blood circulation resulting in multiple organs dysfunctions. Thus, Cdk13 KO mice might be a very useful model for further studies focused on delineating signaling circuits and molecular mechanisms underlying CHDFIDD caused by mutation in CDK13 gene.

• Keywords
• cyclin, cyclin K, cyclin-dependent kinase (CDK), cyclin-dependent kinase 13, development, mouse, transcription regulation,
• Publication type
• Journal Article MeSH

• Publication type
• Journal Article MeSH
• Comment MeSH