RNA polymerase II CTD phospho-sites Ser5 and Ser7 govern phosphate homeostasis in fission yeast

Abstract

Phosphorylation of the tandem YSPTSPS repeats of the RNA polymerase II CTD inscribes an informational code that orchestrates eukaryal mRNA synthesis. Here we interrogate the role of the CTD in phosphate homeostasis in fission yeast. Expression of Pho1 acid phosphatase, which is repressed during growth in phosphate-rich medium and induced by phosphate starvation, is governed strongly by CTD phosphorylation status, but not by CTD repeat length. Inability to place a Ser7-PO 4 mark (as in S7A) results in constitutive derepression of Pho1 expression in phosphate-replete medium. In contrast, indelible installation of a Ser7-PO 4 mimetic (as in S7E) hyper-represses Pho1 in phosphate-replete cells and inhibits Pho1 induction during starvation. Pho1 phosphatase is derepressed by ablation of the CTD Ser5-PO 4 mark, achieved either by mutating Ser5 in all consensus heptads to alanine, or replacing all Pro6 residues with alanine. We find that Ser5 status is a tunable determinant of Pho1 regulation, i.e., serial decrements in the number of consensus Ser5 heptads from seven to two elicits a progressive increase in Pho1 expression in phosphate-replete medium. Pho1 is also derepressed by hypomorphic mutations of the CTD kinase Cdk9. Inactivation of the CTD phosphatase Ssu72 attenuates Pho1 induction in wild-type cells and blocks Pho1 derepression in S7A cells. These experiments implicate Ser5, Pro6, and Ser7 as component letters of a CTD coding "word" that transduces a repressive transcriptional signal via serine phosphorylation.

Transcription of lncRNA prt, clustered prt RNA sites for Mmi1 binding, and RNA polymerase II CTD phospho-sites govern the repression of pho1 gene expression under phosphate-replete conditions in fission yeast

“…The prt transcript, initiating 1198 nt upstream of the pho1 start codon, was also detected in wild-type rrp6 + cells by primer extension analysis . The level of the prt transcript, as gauged by RT-qPCR, was 50-fold higher in phosphate replete rrp6Δ cells versus rrp6 + cells , which accords with the hyper-repression of Pho1 acid phosphate activity in phosphate replete rrp6Δ cells (Shah et al 2014;Schwer et al 2015). Shah et al (2014) noted that the prt RNA contains a cluster of three DSR consensus sequences (5 ′ -UUAAAC) in the region from +399 to +438 (underlined in Fig.…”

“…Induction of the phosphate-regulated genes is strictly dependent on transcription factor Pho7 (Henry et al 2011;Carter-O'Connell et al 2012). Repression of pho1 under phosphate replete conditions is itself an active process involving protein kinases Csk1 and Cdk9 and the phosphorylation of the carboxyl-terminal domain (CTD) of the Rpb1 subunit of RNA polymerase II (Pol2) (Carter-O'Connell et al 2012;Schwer et al 2014Schwer et al , 2015.…”

“…Phosphorylation of the CTD Tyr1, Ser2, Thr4, Ser5, and Ser7 residues inscribes a dynamic "CTD code" that orchestrates transcription, cotranscriptional RNA processing, and chromatin modifications (Schwer and Shuman 2011;Schwer et al 2012;Corden 2013;Eick and Geyer 2013;Doamekpor et al 2014;Mbogning et al 2015). Deletion of Csk1, missense mutations of the Cdk9 T-loop threonine, and alanine mutations of Pol2 CTD phospho-sites Ser5, Pro6, or Ser7 cause derepression of pho1 in phosphate replete cells (Carter-O'Connell et al 2012;Schwer et al 2014Schwer et al , 2015. In contrast, CTD mutations T4A and S7E, and inactivation of CTD phosphatase Ssu72, hyper-repress pho1 in phosphate replete cells .…”

A long noncoding (lnc)RNA governs expression of the phosphate transporter Pho84 in fission yeast and has cascading effects on the flanking prt lncRNA and pho1 genes

“…The phosphate regulon in the fission yeast Schizosaccharomyces pombe comprises three genes that specify, respectively, a cell surface acid phosphatase Pho1, an inorganic phosphate transporter Pho84, and a glycerophosphate transporter Tgp1 (2). Expression of pho1 and tgp1 is actively repressed during growth in phosphate-rich medium by the transcription in cis of a long noncoding (lnc) RNA from the 5' flanking genes prt and nc-tgp1 (3)(4)(5)(6)(7)(8). The prt lncRNA initiates 1147 nucleotides (nt) upstream of the pho1 mRNA transcription start site (Fig.…”

“…For example, CTD mutations that prevent installation of the Ser7-PO 4 or Ser5-PO 4 marks de-repress pho1 in phosphate-replete cells. By contrast, prevention of the Thr4-PO 4 mark hyper-represses pho1 under phosphate-rich conditions (5). Because such CTD mutations do not affect the activity of the prt or pho1 promoters per se, it is proposed that CTD status affects Pol2 termination during prt lncRNA synthesis, and thus the propensity to displace Pho7 from the pho1 promoter (6).…”