PAF1C-dependent activation of CDK12 is required for RNA polymerase II phosphorylation and transcript elongation

The RNA polymerase II (RNAPII) transcription cycle is regulated by dynamic changes in RNAPII C-terminal domain (CTD) phosphorylation, and such modification is crucial for gene expression. However, the mechanisms regulating the transcription-specific cyclin-dependent kinases (CDKs) during the transcription cycle remain poorly understood. Here, we show that human CDK12 co-phosphorylates CTD Serine2 and Serine5. This di-phosphorylated Serine2-Serine5 CTD mark can then act as a precursor for Serine2 mono-phosphorylated CTD through Serine5 de-phosphorylation. Notably, CDK12 is specifically regulated by association with the elongation-specific factor PAF1C, in which the CDC73 subunit contains a metazoan-specific peptide motif, capable of allosteric CDK12/Cyclin K activation. This motif is essential for cell proliferation and required for normal cellular levels of CTD phosphorylation. It is also crucial for transcript elongation, particularly across long human genes. Together, these findings provide new insight into the mechanisms governing RNAPII phospho-CTD dynamics that ensure progression through the human transcription cycle. Overall design: To investigate the role of CDC73-KIM for global RNA production, we first established a “switchover” system where endogenous CDC73 was knocked down by siRNA in HEK293 cells, and different recombinant, siRNA resistant, and Flag-tagged versions of CDC73 were inducibly expressed by doxycycline (Dox) addition. This was followed by metabolic labelling of nascent RNA using a short pulse of the uridine analog 4-thiouridine (4sU). Transient Transcriptome chem-sequencing (TTchem-seq) was then used to affinity purify, fragment and sequence 4sU-labelled RNA for high-resolution profiles of nascent transcription.