RNA polymerase II termination involves C-terminal-domain tyrosine dephosphorylation by CPF subunit Glc7

The synthesis of mRNA by Pol II involves cotranscriptional premRNA processing including 5′ capping, splicing and 3′-end processing. Pol II recruits pre-mRNA–processing factors via its CTD throughout transcription1–3, and this process is regulated by changes in CTD phosphorylation4. The CTD forms a flexible extension of Pol II consisting of 26 (yeast) or 52 (human) heptapeptide repeats of the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. When Pol II is recruited to promoters, the CTD is not phosphorylated4. As transcription proceeds, Ser5 phosphorylation recruits the mRNA 5′-capping machinery near the promoter5,6, whereas Ser2 phosphorylation recruits 3′-processing and termination factors near the polyadenylation (pA) site7. Tyr1 phosphorylation prevents premature recruitment of termination factors within gene bodies8. At the pA site, Tyr1 phosphorylation levels drop, whereas Ser2 phosphorylation remains, apparently enabling termination-factor recruitment8. Thus, CTD kinases and phosphatases have key roles in mRNA synthesis4. Addition of a 3′ poly(A) tail to mRNA facilitates nuclear export, regulates the stability of mRNAs and enhances translation9–11. 3′-end processing is performed by a large number of proteins including the ~1-MDa CPF complex (CPSF in mammals), which cleaves pre-mRNA with its endonuclease subunit (Ysh1 (CPSF73)) and then polyadenylates the new 3′ end with its poly(A) polymerase (Pap1 (PAP))12,13. Accessory proteins, such as CF IA (comprising Pcf11, Clp1, Rna14 and Rna15) and CF IB in yeast, contribute to the specificity and efficiency of 3′-end–processing reactions. The cleavage-and-polyadenylation machinery is recruited to nascent transcripts early in the transcription cycle. CPF associates with the Pol II CTD and TFIID at promoters and is presumed to travel with the transcription complex until it reaches the pA site12. Interestingly, the Pol II CTD itself is proposed to have a role in 3′-end processing because it is required for efficient pre-mRNA processing in vivo14,15 and stimulates the cleavage event in vitro16. Disruption of 3′-end processing can lead to defects in transcription termination, thus suggesting an intimate link between these two processes17. For example, temperaturesensitive mutations in CF IA subunits result in defective transcription termination18. Pcf11 contains a CTD-interacting domain (CID) that binds Ser2-phosphorylated CTD and has a role in transcription termination that is separable from its role in 3′-end processing15,19–21. The transition from transcription elongation to termination involves CTD Tyr1 dephosphorylation by an unknown phosphatase. We reasoned that the Tyr1 phosphatase might be part of the mRNA 3′-end– processing and termination machinery. In particular, CPF contains two candidate phosphatases, Ssu72 (refs. 22,23) and Glc7 (ref. 24). Ssu72 can dephosphorylate CTD residue Ser5 during transcription elongation22,25,26. Glc7 is implicated in transcription termination on genes encoding small nucleolar RNAs27, in mRNA export28 and in the polyadenylation activity of CPF29 but has no known CTD-related activity. We therefore set out to investigate the role of CPF in CTD phosphorylation. Here, we show that Glc7 is a Tyr1 phosphatase both in vitro and in vivo. We further show that Glc7 is required for normal recruitment of termination factors and for Pol II termination in vivo.