Rpb4/7 facilitates RNA polymerase II CTD dephosphorylation

Abstract

The Rpb4 and Rpb7 subunits of eukaryotic RNA polymerase II (RNAPII) participate in a variety of processes from transcription, DNA repair, mRNA export and decay, to translation regulation and stress response. However, their mechanism(s) of action remains unclear. Here, we show that the Rpb4/7 heterodimer in Saccharomyces cerevisiae plays a key role in controlling phosphorylation of the carboxy terminal domain (CTD) of the Rpb1 subunit of RNAPII. Proper phosphorylation of the CTD is critical for the synthesis and processing of RNAPII transcripts. Deletion of RPB4, and mutations that disrupt the integrity of Rpb4/7 or its recruitment to the RNAPII complex, increased phosphorylation of Ser2, Ser5, Ser7 and Thr4 within the CTD. RPB4 interacted genetically with genes encoding CTD phosphatases (SSU72, FCP1), CTD kinases (KIN28, CTK1, SRB10) and a prolyl isomerase that targets the CTD (ESS1). We show that Rpb4 is important for Ssu72 and Fcp1 phosphatases association, recruitment and/or accessibility to the CTD, and that this correlates strongly with Ser5P and Ser2P levels, respectively. Our data also suggest that Fcp1 is the Thr4P phosphatase in yeast. Based on these and other results, we suggest a model in which Rpb4/7 helps recruit and potentially stimulate the activity of CTD-modifying enzymes, a role that is central to RNAPII function.

Genetic and structural analysis of the essential fission yeast RNA polymerase II CTD phosphatase Fcp1

“…Whereas it is likely that SpFcp1 is needed to erase Ser2-PO 4 and Ser5-PO 4 marks in fission yeast, based on its competence to do so in vitro, we do not rule out the prospect that SpFcp1 might dephosphorylate Ser7-PO 4 , Thr4-PO 4 or Tyr1-PO 4 on the fission yeast Pol2 CTD (Sakurai and Ishihama 2002) or that its essential activity extends to other phospho-protein substrates. Two recent studies have invoked a Pol2 CTD Thr4 phosphatase function for Fcp1 in S. cerevisiae and chicken DT40 cells (Allepuz-Fuster et al 2014;Hsin et al 2014).…”

Dephosphorylating eukaryotic RNA polymerase II

“…Depletion of Rpb4 results in decreased Fcp1 and accumulated CTD phosphorylation in immunoprecipitated RNA Pol II holoenzyme complexes [111]. A more recent report corroborates these earlier findings and demonstrate that Rpb4 mutation results in reduced Fcp1 occupancy at genes and accumulated Ser2 phosphorylation, although similar data is presented for another major CTD phosphatase Ssu72 and Ser5 phosphorylation suggesting Rpb4 is important to additional facets of CTD dephosphorylation [108]. The association of Fcp1 with TFIIH and Rpb4 likely contributes to its association with RNA Pol II throughout elongation, it's stimulation of elongation, and helps prime RNA Pol II for timely dephosphorylation by Fcp1 at the end of transcription where Fcp1 levels peak.…”

“…1) [8, 62]. Fcp1 has also been implicated as the phosphatase responsible for the newly identified Thr4 dephosphorylation in vivo [108, 109] and Fcp1 is active against phosphorylated Ser7 in vitro , but the physiological significance of this activity is unclear [62, 110]. These results together suggest that Fcp1 fulfills the specific and vital role of Ser2 CTD dephosphorylation, but maintains secondary activity against Thr4, Ser5, and Ser7 to ensure the complete dephosphorylation of the CTD at the end of transcription.…”

“…Ser5 and Ser7 are both phosphorylated by TFIIH [11, 185] and dephosphorylated by Ssu72 [91, 143]. Similarly, Ser2 and Thr4 phosphorylation is catalyzed by the same kinase in yeast, PTEFb [12, 186] and are dephosphorylated at least in part by Fcp1 [108, 109]. Higher order interactions between these phosphorylation marks and the transcriptional machinery also occur in cells.…”

“…Scps, Ssu72, Cdc14, and Glc7 all operate within protein complexes that position these CTD phosphatases at the correct point in the transcription cycle [120, 160, 170]. Fcp1 and RPAP2, the human homologue of Rtr1, rely on binding partners for association with the RNA polymerase II holoenzyme [108, 167]. However, gaps in our knowledge of the of binding partners for CTD phosphatases limit our biological interpretations and more transient protein-protein interactions may not even be apparent with current technologies.…”