Characterization of human cyclin-dependent kinase 12 (CDK12) and CDK13 complexes in C-terminal domain phosphorylation, gene transcription, and RNA processing

Abstract

Cyclin-dependent kinase 9 (CDK9) and CDK12 have each been demonstrated to phosphorylate the RNA polymerase II C-terminal domain (CTD) at serine 2 of the heptad repeat, both in vitro and in vivo. CDK9, as part of P-TEFb and the super elongation complex (SEC), is by far the best characterized of CDK9, CDK12, and CDK13. We employed both in vitro and in vivo assays to further investigate the molecular properties of CDK12 and its paralog CDK13. We isolated Flag-tagged CDK12 and CDK13 and found that they associate with numerous RNA processing factors. Although knockdown of CDK12, CDK13, or their cyclin partner CCNK did not affect the bulk CTD phosphorylation levels in HCT116 cells, transcriptome sequencing (RNA-seq) analysis revealed that CDK12 and CDK13 losses in HCT116 cells preferentially affect expression of DNA damage response and snoRNA genes, respectively. CDK12 and CDK13 depletion also leads to a loss of expression of RNA processing factors and to defects in RNA processing. These findings suggest that in addition to implementing CTD phosphorylation, CDK12 and CDK13 may affect RNA processing through direct physical interactions with RNA processing factors and by regulating their expression.

Fig 1

Purification of CDK12 and CDK13 complexes. (A and B) Flag-purified CDK12 and CDK13 complexes were analyzed by silver staining (A) and Western blotting with the M2 Flag monoclonal antibody (B). Arrows indicate the positions of Flag-CDK12, Flag-CDK13, and cyclin K. (C) MudPIT analysis of CDK12 and CDK13 complexes. Flag-CDK12 and Flag-CDK13 purifications identified numerous RNA processing factors. (D) Gene ontology analysis showed that the CDK12- and CDK13-associated proteins are significantly enriched for factors involved in RNA processing, splicing, the spliceosome, and nuclear splicing speckles (nuclear speck). (E and F) Superose 6 size-exclusion chromatography of Flag-CDK12 and Flag-CDK13 purifications. Silver staining and anti-Flag Western blotting demonstrated that both CDK12 and CDK13 complexes peaked in fractions 13 and 14 (∼1 to 2 MDa). (G) MudPIT analysis of fractions 12 to 15 (red boxes in panels E and F) was performed, and the top interacting proteins are shown.

FIG 2

Characterization of CDK12 and CDK13 activities on Pol II CTD phosphorylation. (A) Pol II CTD kinase activities of CDK12, CDK13, and CDK9 complexes. In vitro CTD kinase assays were performed with purified CDK complexes, recombinant His-tagged CTD, and [γ-³²P]ATP. The reaction products were subjected to SDS-PAGE and autoradiography to assess phosphorylated Pol II levels. Triangles indicate increasing amounts of the indicated enzyme used in the assay mixtures. Arrows indicates the positions of CDK9, CDK12, CDK13, and the His-tagged CTD. (B) Silver staining of CDK9, CDK12, and CDK13 complexes used in the CTD kinase assays. (C) Western blotting of phosphorylated recombinant CTD by use of phosphorylation-specific antibodies. A reaction time of 1 h gave no detectable signal for the CDK12- or CDK13-phosphorylated CTD with the 3E10 (Ser2P), 3E8 (Ser5P), and 4E12 (Ser7P) monoclonal antibodies, while the CDK9-phosphorylated CTD was recognized by all of these antibodies. The 8WG16 monoclonal antibody preferentially recognizes unphosphorylated CTD repeats. After extension of the reaction time to 10 h, modest Ser2P and Ser5P signals could be detected with CDK12 and CDK13 complexes. (D) CDK12 and CDK13 complexes are not as sensitive as CDK9 to flavopiridol inhibition. Flavopiridol (100 nM, 1 μM, and 10 μM) was incubated with the indicated CDK, recombinant Pol II CTD, and [γ-³²P]ATP. Phosphorylated CTD levels were measured by SDS-PAGE and autoradiography. (E and F) CDK12, CDK13, and CCNK depletion by shRNA knockdown in HCT116 cells did not affect bulk Pol II CTD phosphorylation levels as assayed with the indicated antibodies. The nontargeting shRNA (GFPi) was used as a negative control. Cell lysates were made at 3 days posttransduction and were analyzed by Western blotting. N20 is a polyclonal antibody recognizing total Pol II levels. Tubulin served as a loading control.

FIG 3

CDK9, CDK12, and CDK13 have positive effects on gene expression in vivo. (A) RT-qPCR results showing the efficiencies of CDK9, CDK12, and CDK13 knockdowns by two individual shRNAs in HCT116 cells. (B) Knockdown efficiency was further confirmed by Western blotting with the indicated antibodies. (C) Venn diagram of the differentially expressed transcripts after RNAi of CDK9, CDK12, and CDK13. The majority of transcripts (74%) affected by CDK13 knockdown were affected by CDK12 knockdown. (D to F) Log₂ ratio (M) versus mean average (A) plots showing that most differentially regulated transcripts (indicated in color) were downregulated by CDK9 (D), CDK12 (E), or CDK13 (F) depletion. The y axis of each plot shows the log₂ fold change in transcript expression of the knockdown over the wild type. The x axis of each plot shows the log₂ average expression level.

Fig 4

CDK12 and CDK13 losses affect expression of different sets of genes. (A) Gene ontology analysis of CDK-regulated genes. CDK12 loss preferentially affected genes involved in RNA processing and DNA damage, while CDK13 loss preferentially affected genes involved in translation. (B and C) Genome browser track examples for DNA damage repair genes BRCA1 (B) and APEX1 (C), whose expression was affected by CDK12 but not CDK13 knockdown. (D) Depletion of CCNK also resulted in reduced expression of BRCA1 and APEX1 in HCT116 cells. (E) CDK13 loss affected snRNA and snoRNA gene expression more than CDK9 or CDK12 loss did. The Venn diagram shows the numbers of downregulated snRNA and snoRNA genes after knockdown of CDK9, CDK12, and CDK13. (F) Levels of snoRNA38 were downregulated by depletion of CDK13 but not CDK12.

FIG 5

Altered SRSF1 alternative splicing by CDK12 knockdown. (A) SRSF1 has two different isoforms in HCT116 cells. Compared to isoform 1, the isoform 2 transcripts lack an intron in the 3′ UTR. Arrows indicate the primers for RT-qPCR analysis of constitutive and isoform-specific SRSF1 transcripts. (B to D) Relative expression levels of SRSF1 and its specific isoforms after CDK12 and CDK13 depletion for 3 days. The CDK12 knockdown led to a significant decrease in expression of isoform 2, which lacks the 3′-UTR intron. (E) Relative expression levels of SRSF1 isoform 2 after CDK9 knockdown for 3 days. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.