The C-terminal domain (CTD) of the large subunit of RNA polymerase (pol) II comprises conserved heptad repeats, and post-translational modification of the CTD regulates transcription and cotranscriptional RNA processing. Recently, the spatial patterns of modification of the CTD repeats have been investigated, and new functions of CTD modification have been revealed. In addition, there are new insights into the roles of the enzymes that decorate the CTD. We review these new findings and reassess the role of the pol II CTD in the regulation of gene expression.

Transcription through early-elongation checkpoints requires phosphorylation of negative transcription elongation factors (NTEFs) by the cyclin-dependent kinase (CDK)9. Using CDK9 inhibitors and global run-on sequencing (GRO-seq), we have mapped CDK9 inhibitor-sensitive checkpoints genome-wide in human (Homo sapiens) cells. Our data indicate that early-elongation checkpoints are a general feature of RNA polymerase (pol) II-transcribed human genes and occur independently of polymerase stalling. Pol II that has negotiated the early-elongation checkpoint can elongate in the presence of inhibitors but, remarkably, terminates transcription prematurely close to the terminal polyadenylation (poly(A)) site. Our analysis has revealed a hitherto-unsuspected poly(A)-associated elongation checkpoint, which has major implications for the regulation of gene expression. Interestingly, the pattern of modification of the carboxyl-terminal domain (CTD) of pol II terminated at this novel checkpoint largely mirrors the pattern normally found downstream of the poly(A) site, suggesting common mechanisms of termination.

SummaryThe carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II) comprises multiple heptapeptide repeats of the consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Reversible phosphorylation of Ser2, Ser5, and Ser7 during the transcription cycle mediates the sequential recruitment of transcription/RNA processing factors. Phosphorylation of Ser7 is required for recruitment of the gene type-specific Integrator complex to the Pol II-transcribed small nuclear (sn)RNA genes. Here, we show that RNA Pol II-associated protein 2 (RPAP2) specifically recognizes the phospho-Ser7 mark on the Pol II CTD and also interacts with Integrator subunits. siRNA-mediated knockdown of RPAP2 and mutation of Ser7 to alanine cause similar defects in snRNA gene expression. In addition, we show that RPAP2 is a CTD Ser5 phosphatase. Taken together, our results indicate that during transcription of snRNA genes, Ser7 phosphorylation facilitates recruitment of RPAP2, which in turn both recruits Integrator and dephosphorylates Ser5.

Cyclin-dependent kinase 12 (CDK12) phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) but its roles in transcription beyond the expression of DNA damage response genes remain unclear. Here, we have used TT-seq and mNET-seq to monitor the direct effects of rapid CDK12 inhibition on transcription activity and CTD phosphorylation in human cells. CDK12 inhibition causes a genome-wide defect in transcription elongation and a global reduction of CTD Ser2 and Ser5 phosphorylation. The elongation defect is explained by the loss of the elongation factors LEO1 and CDC73, part of PAF1 complex, and SPT6 from the newly-elongating pol II. Our results indicate that CDK12 is a general activator of pol II transcription elongation and indicate that it targets both Ser2 and Ser5 residues of the pol II CTD.

The Herpes Simplex Virus 1 (HSV-1)-encoded ICP22 protein plays an important role in viral infection and affects expression of host cell genes. ICP22 is known to reduce the global level of serine (Ser)2 phosphorylation of the Tyr1Ser2Pro3Thr4Ser5Pro6Ser7 heptapeptide repeats comprising the carboxy-terminal domain (CTD) of the large subunit of RNA polymerase (pol) II. Accordingly, ICP22 is thought to associate with and inhibit the activity of the positive-transcription elongation factor b (P-TEFb) pol II CTD Ser2 kinase. We show here that ICP22 causes loss of CTD Ser2 phosphorylation from pol II engaged in transcription of protein-coding genes following ectopic expression in HeLa cells and that recombinant ICP22 interacts with the CDK9 subunit of recombinant P-TEFb. ICP22 also interacts with pol II in vitro. Residues 193 to 256 of ICP22 are sufficient for interaction with CDK9 and inhibition of pol II CTD Ser2 phosphorylation but do not interact with pol II. These results indicate that discrete regions of ICP22 interact with either CDK9 or pol II and that ICP22 interacts directly with CDK9 to inhibit expression of host cell genes.