CDK12 and CDK13 cooperatively regulate RNA polymerase II elongation and alternative polyadenylation of mRNA

© 2020 Zheng FanTranscription driven by RNA Polymerase II (POLII) is a multi-step process that is strictly regulated by Cyclin dependent kinases (CDKs) at multiple checkpoints. Compared to the regulation of transcription initiation and pause release, the roles of CDKs in regulating transcription elongation remain poorly defined. To investigate the individual and shared roles of CDK12 and CDK13 in transcription regulation, a set of cell lines containing analog-sensitive variants of CDK12 and CDK13 were constructed using CRISPR-Cas9 homology-directed repair (HDR) technology. Multiple Next-Generation Sequencing (NGS) based assays including RNA-Seq, ChIP-Seq, PRO-Seq, TT-Seq were utilised to provide a comprehensive characterisation of the consequences of acute inhibition of CDK12, CDK13 or both kinases using these analog sensitive CDK12 and CDK13 cell lines. Selective inhibition of CDK12 or CDK13 led to various molecular responses including selective changes in gene expression, alternations in polyadenylation site usage as well as mild reduction in POLII elongation rate and processivity. In contrast, dual inhibition of CDK12 and CDK13 caused dramatic changes in transcription genome-wide, including the induction of widespread alternative polyadenylation events, reduction in POLII elongation rates and processivity, substantial changes in gene expression, as well as the near complete loss of POLII Ser2 phosphorylation. These observations illustrated that both CDK12 and CDK13 are regulators of POLII transcription elongation. Furthermore, the substantial differences between selective and simultaneous inhibition of CDK12 and CDK13 revealed the redundant and individual roles of CDK12 and CDK13 in maintaining global transcription elongation. To identify substrates of CDK12 and CDK13 that might be responsible for the phenotypes caused by CDK12 and CDK13 inhibition, phospho-proteomic analysis was performed to identify putative CDK12 and CDK13 substrates. The analysis revealed that CDK12 and CDK13 shared multiple substrates and functional redundancy between CDK12 and CDK13 in phosphorylating these substrates was identified. In order to identify the putative substrates that were responsible for the transcriptional changes upon CDK12 and CDK13 inhibition, a novel siRNA screen method “mini-bulk” CEL-Seq2 siRNA screen was developed and utilised. The screen revealed that SF3B1 and SRRM2 could be the potential substrates of CDK12 and CDK13 that were partially responsible for the transcriptional phenotype caused by the dual inhibition of CDK12 and CDK13, as depletion of SF3B1 and SRRM2 led to similar differential gene expression and alternative polyadenylation profiles as CDK12 and CDK13 inhibition. Finally, as the phospho-proteomic analysis also revealed that CDK12 and CDK13 might regulate phosphorylation of multiple translation regulators, the effect of CDK12 and CDK13 inhibition on protein translation was also investigated. Both nascent protein labelling as well as polysome profiling revealed that CDK12 and CDK13 function was required to maintain global translation. In conclusion, this thesis explored the role of CDK12 and CDK13 in POLII driven transcription and protein translation. CDK12 and CDK13 were shown to cooperatively regulate POLII transcription elongation processivity and alternative polyadenylation, potentially through regulating POLII Ser2 phosphorylation and the phosphorylation of other CDK12 and CDK13 substrates