Dissecting cyclin-dependent kinase functions in mRNA quality control in fission yeast

RNA Polymerase (Pol) II transcribes protein-coding genes in eukaryotic cells. Transcription by Pol II is coordinated with processes through the carboxyl-terminal domain (CTD) of its largest subunit, Rpb1. Cyclin-dependent kinases (CDKs) phosphorylate CTD heptad repeats to influence their binding properties and thereby govern co-transcriptional mRNA processing. Transcription factor (TF) IIH-associated Cdk7 phosphorylates Ser5 residues to recruit capping enzymes, while Cdk9 promotes elongation by phosphorylating the CTD and the processivity factor Spt5. I examined the functions of these essential kinases in fission yeast in a putative mRNA quality control mechanism that could ensure that only fully capped transcripts are elongated. I found that Cdk9 forms a constitutive and stable complex with the cyclin Pch1 and the 5'-cap methyltransferase Pcm1 in vivo. Using chemical genetics, we showed that Mcs6 activity is required to recruit the Cdk9-Pch1-Pcm1 complex to a subset of CDK-dependent genes. In vitro, I showed that phosphorylation of the CTD by Mcs6 stimulates subsequent phosphorylation by Cdk9. Together, these observations support a model in which Mcs6 and Cdk9 act sequentially to coordinate elongation and capping of select pre-mRNAs.;To test that model, I undertook a functional dissection of the fission yeast Cdk9-Pch1-Pcm1 complex. I show that a Cdk9 carboxyl-terminal extension, distinct from the catalytic domain, mediates binding to Pcm1 and the Pol II CTD. Removal of this segment leads to growth defects and transcriptional deficiencies. These phenotypes are suppressed by Pcm1 overproduction, suggesting that normal transcript elongation and gene expression depend on physical linkage between Cdk9 and Pcm1. The extension is dispensable, however, for recognition by Cdk9 of CTD substrates "primed" by Mcs6. Cdk9 prefers CTD peptides phosphorylated at Ser7 over unmodified repeats. In vivo, Ser7 phosphorylation depends on Mcs6 activity, suggesting a mechanism to order transcriptional CDK functions, independent of chromatin recruitment. Therefore, fission yeast Cdk9 comprises a catalytic domain sufficient for primed substrate recognition, and a multivalent recruitment module that couples transcription with capping. Together, these results support a proposed model whereby CTD phosphorylation by Mcs6 stimulates subsequent Cdk9 activity and the Cdk9/Pcm1 complex serves to coordinate capping with transcript elongation, thereby protecting mRNA quality.