Abstract
Transcription of mRNA consists of three critical steps - initiation, elongation, and termination - and is driven by RNA polymerase II (Pol II), whose activity is regulated by a unique C-terminal domain (CTD). The transcription-related kinases CDK7, CDK9, and CDK12 regulate transcription by differentially phosphorylating serine 2, serine 5, and serine 7 of the Pol II CTD, although their functional interactions are not yet fully understood. Since these CDKs are involved in cancer cell proliferation and survival, elucidating these interactions is useful for cancer treatment. We focused on CDK12, which plays an important role in the late phase of transcription and identified several novel autophosphorylation sites of CDK12. Among these, serine 423 on CDK12 was found to be a critical residue affecting the half-life of the CDK12 protein and its phosphorylation is mediated by both CDK12 and CDK7. Additionally, comprehensive phosphoproteomic analysis revealed that CDK7 and CDK9 affect the phosphorylation of CDK12 and the CDK12 interactome, suggesting crosstalk between these kinases. Inhibition of CDK7 disrupted the interaction between CDK12 and proteins phosphorylated by CDK12, including RNA processing factors, while inhibition of CDK7 and CDK9 enhanced the interaction between CDK12 and splicing factors. In conclusion, our results indicate that CDK7 and CDK9 functionally regulate CDK12 upstream, suggesting that transcriptional CDKs cooperatively regulate RNA transcription and subsequent transcriptional processes.
