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

Abstract

At the 3′ end of protein-coding genes, RNA polymerase (Pol) II is dephosphorylated at tyrosine (Tyr1) residues of its C-terminal domain (CTD). In addition, the associated cleavage and polyadenylation (pA) factor (CPF) cleaves the transcript and adds a polyA tail. Whether these events are coordinated and how they lead to transcription termination remains poorly understood. Here we show that CPF from Saccharomyces cerevisiae is a Pol II CTD phosphatase and that the CPF subunit Glc7 dephosphorylates Tyr1 in vitro. In vivo, the activity of Glc7 is required for normal Tyr1 dephosphorylation at the pA site, for recruitment of termination factors Pcf11 and Rtt103, and for normal Pol II termination. These results show that transcription termination involves Tyr1 dephosphorylation of the CTD and indicate that pre-mRNA processing by CPF and transcription termination are coupled via Glc7-dependent Pol II Tyr1 dephosphorylation.

Dephosphorylating eukaryotic RNA polymerase II

“…Interestingly, yeast mutants with decreased Ssu72 activity accumulated Ser5, but not Ser2 CTD phosphorylation [143]. This result is consistent with in vitro phosphatase assays against phosphorylated CTD peptide substrate, showing that Ssu72 is specific for Ser5 and not Ser2 or Tyr1 [110, 152]. …”

“…Purified yeast cleavage and poly-adenylation factor (CPF) complex can dephosphorylate the CTD at Tyr1, Ser2, and Ser5 [152]. Ssu72 and Glc7 are the only two known phosphatases in this complex [160].…”

“…Ssu72 and Glc7 are the only two known phosphatases in this complex [160]. Ssu72 has no significant Tyr1 phosphatase activity in vitro or in vivo , even though it is structurally similar to established tyrosine phosphatases [110, 152]. This leaves Glc7, a di-metal ion phosphatase that is essential for the termination of snoRNA [172] and mRNA export [173].…”

“…Since EDTA and microcystin do not affect the activity of PTP phosphatases like Ssu72, it was reasoned that Glc7 is responsible for the Tyr1 and Ser2 phosphatase activity of CPF in vitro . Depletion of Glc7 in vivo led to dephosphorylation defects specific to Tyr1 and resulted in increased occupancy of RNA Pol II downstream of genes, suggesting defects in termination [152]. In vitro purified Glc7 isolated from CPF has not been tested for phosphatase activity against CTD, owing in part to the low specificity expected of di-metal dependent phosphatases in the absence of auxiliary domains or binding partners [97, 152].…”

Modifications of RNA polymerase II CTD: Connections to the histone code and cellular function

“…An FCP1-like phosphatase, SCP1, preferentially catalyzes the dephosphorylation of CTD S5-P, a modification that is important for transitioning from transcription initiation to elongation (Yeo et al, 2003;Zhang et al, 2010b). Finally, yeast Glc7, a cleavage/poly(A) factor, acts as a Y1 CTD phosphatase at the 3′-ends of protein-coding genes both in vitro and in vivo (Schreieck et al, 2014).…”