Phosphorylation patterns of the C-terminal domain (CTD) of largest subunit of RNA polymerase II (called the CTD code) orchestrate the recruitment of RNA processing and transcription factors. Recent studies showed that not only serines and tyrosines but also threonines of the CTD can be phosphorylated with a number of functional consequences, including the interaction with yeast transcription termination factor, Rtt103p. Here, we report the solution structure of the Rtt103p CTD-interacting domain (CID) bound to Thr4 phosphorylated CTD, a poorly understood letter of the CTD code. The structure reveals a direct recognition of the phospho-Thr4 mark by Rtt103p CID and extensive interactions involving residues from three repeats of the CTD heptad. Intriguingly, Rtt103p's CID binds equally well Thr4 and Ser2 phosphorylated CTD A doubly phosphorylated CTD at Ser2 and Thr4 diminishes its binding affinity due to electrostatic repulsion. Our structural data suggest that the recruitment of a CID-containing CTD-binding factor may be coded by more than one letter of the CTD code.

• Klíčová slova
• NMR, RNA processing, RNAPII CTD code, structural biology,
• MeSH
• fosforylace MeSH
• genetická transkripce MeSH
• proteinkinasy metabolismus   MeSH
• proteolýza MeSH
• RNA-polymerasa II chemie   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• serin metabolismus   MeSH
• terciární struktura proteinů MeSH
• threonin chemie   metabolismus   MeSH
• transkripční faktory chemie   metabolismus   MeSH
• tyrosin metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteinkinasy MeSH
• RNA-polymerasa II MeSH
• Rtt103 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• serin MeSH
• threonin MeSH
• transkripční faktory MeSH
• tyrosin MeSH

CDK12 is a kinase associated with elongating RNA polymerase II (RNAPII) and is frequently mutated in cancer. CDK12 depletion reduces the expression of homologous recombination (HR) DNA repair genes, but comprehensive insight into its target genes and cellular processes is lacking. We use a chemical genetic approach to inhibit analog-sensitive CDK12, and find that CDK12 kinase activity is required for transcription of core DNA replication genes and thus for G1/S progression. RNA-seq and ChIP-seq reveal that CDK12 inhibition triggers an RNAPII processivity defect characterized by a loss of mapped reads from 3'ends of predominantly long, poly(A)-signal-rich genes. CDK12 inhibition does not globally reduce levels of RNAPII-Ser2 phosphorylation. However, individual CDK12-dependent genes show a shift of P-Ser2 peaks into the gene body approximately to the positions where RNAPII occupancy and transcription were lost. Thus, CDK12 catalytic activity represents a novel link between regulation of transcription and cell cycle progression. We propose that DNA replication and HR DNA repair defects as a consequence of CDK12 inactivation underlie the genome instability phenotype observed in many cancers.

• Klíčová slova
• CDK12, CTD Ser2 phosphorylation, G1/S, premature termination and polyadenylation, tandem duplications,
• MeSH
• cyklin-dependentní kinasy genetika   metabolismus   MeSH
• fosforylace MeSH
• HCT116 buňky MeSH
• kontrolní body fáze G1 buněčného cyklu genetika   fyziologie   MeSH
• lidé MeSH
• oprava DNA genetika   fyziologie   MeSH
• replikace DNA genetika   fyziologie   MeSH
• RNA-polymerasa II genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CDK12 protein, human MeSH Prohlížeč
• cyklin-dependentní kinasy MeSH
• RNA-polymerasa II MeSH

Cyclin-dependent kinases regulate the cell cycle and transcription in higher eukaryotes. We have determined the crystal structure of the transcription kinase Cdk13 and its Cyclin K subunit at 2.0 Å resolution. Cdk13 contains a C-terminal extension helix composed of a polybasic cluster and a DCHEL motif that interacts with the bound ATP. Cdk13/CycK phosphorylates both Ser5 and Ser2 of the RNA polymerase II C-terminal domain (CTD) with a preference for Ser7 pre-phosphorylations at a C-terminal position. The peptidyl-prolyl isomerase Pin1 does not change the phosphorylation specificities of Cdk9, Cdk12, and Cdk13 but interacts with the phosphorylated CTD through its WW domain. Using recombinant proteins, we find that flavopiridol inhibits Cdk7 more potently than it does Cdk13. Gene expression changes after knockdown of Cdk13 or Cdk12 are markedly different, with enrichment of growth signaling pathways for Cdk13-dependent genes. Together, our results provide insights into the structure, function, and activity of human Cdk13/CycK.

• Klíčová slova
• Cdk13, Cyclin K, Pin1, RNA polymerase II, flavopiridolIntroduction, transcription,
• MeSH
• cyklin-dependentní kinasy genetika   metabolismus   MeSH
• fosforylace MeSH
• lidé MeSH
• signální transdukce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cyklin-dependentní kinasy MeSH