Functions of CDK12 and CDK13 in pre-mRNA Processing

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This application will define the roles of the cyclin dependent kinases (CDKs) CDK12 and CDK13 in the coordinate regulation of transcription and RNA processing. While there is a deep understanding of the key proteins and biochemical mechanisms required for RNA splicing by the spliceosome, little is known about the processes that govern coordinate transcription elongation, polyadenylation, and spliceosome activity. CDKs typically respond to signal transduction cascades and regulate central cellular processes (e.g. cell cycle, transcription) through phosphorylation of target proteins. The paralogous proteins CDK12 and CDK13 phosphorylate RNA Polymerase 2 to maintain processive transcription elongation. These kinases also contain domains typically found in splicing factors and signaling proteins, and we have shown that they interact with a broad range of splicing and RNA processing factors. Recently, we demonstrated that CDK12 also regulates a specific form of alternative 3' end mRNA processing. This regulation is highly cell type- and gene-specific, suggesting additional layers of regulation that have yet to be defined. The basic biological functions of both CDK12 and CDK13 remain poorly understood. Thus, understanding their functional mechanisms is essential for defining key or novel aspects of the coordinated regulation of transcription and splicing. We will study CDK12 and CDK13 using these approaches: Aim 1. Determine mechanisms of CDK12 and CDK13 complexes in regulating RNA processing. Proximity labeling will be used to identify proteins and RNA sequences associated with CDK12 and CDK13. This will determine the temporal association of CDK12 and CDK13 with RNA, RNA binding and splicing proteins to determine how these kinases regulate RNA processing. Aim 2. Map phospho-regulation by CDK12 and CDK13 complexes in RNA processing. Using global phospho-proteomics and a potent CDK12/CDK13 covalent inhibitor we will independently identify the dynamics of signaling pathways downstream of CDK12 and CDK13. Aim 3. Define cellular roles of CDK12 and CDK13 by functional genomics. Using RNA-seq and proteomics, we will ascertain the genes and pathways regulated by CDK12 and CDK13. We will employ transient transcriptome (TT)-seq to examine temporal changes in RNA regulated by CDK12 and CDK13. These experiments will define the cellular roles of CDK12 and CDK13. While CDK12 and CDK13 have specific features to suggest that they perform the coordinated regulation of transcription and RNA processing, the molecular mechanism of these processes have not been thoroughly investigated. Our program will define the specific functions of CDK12 and CDK13 complexes in RNA processing, transcription, polyadenylation, and spliceosome regulation. The general significance of our program will be an increased understanding of the critical cellular regulation processes that control coordinated transcription and RNA processing affect the global gene expression state.