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-PO4 mark (as in S7A) results in constitutive derepression of Pho1 expression in phosphate-replete medium. In contrast, indelible installation of a Ser7-PO4 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-PO4 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.

Keywords: acid phosphatase; phosphate transporter; phosphate-responsive gene expression.

FIGURE 1.

Effect of eliminating CTD phosphorylation sites on phosphate homeostasis. Isogenic heterothallic S. pombe rpb1-CTD strains in which 14 heptad repeats (either wild-type YSPTSPS or mutated heptads as specified) are appended to amino acid 1577 of Rpb1 (11,12) were grown in liquid culture in YES medium. Cells were then harvested, washed with water, and incubated for 3 h in PMG medium containing 15.5 mM phosphate (+ phosphate) or lacking exogenous phosphate (− phosphate). Pho1 phosphatase was assayed by conversion of p-nitrophenylphosphate to p-nitrophenol. The y-axis specifies the phosphatase activity (A₄₁₀) normalized to input cells (A₆₀₀).

FIGURE 2.

Contribution of CTD Ser5 to regulated Pho1 expression. Cells with Rpb1-CTDs composed of the indicated mixtures of S5 heptads and S5A heptads (A), or CTD-MCE fusions in which the 14 heptads were all wild-type, S5A, or P6A (B), were assayed for Pho1 activity after incubation for 3 h in PMG medium containing 15.5 mM phosphate (+ phosphate) or lacking exogenous phosphate (− phosphate).

FIGURE 3.

5′-end mapping of RNAs transcribed from the fission yeast phosphate regulon. (A) Schematic illustration of the adjacent pho84⁺ (SPBC8E4.01c) and pho1⁺ (SPBP4G3.02) genes on fission yeast chromosome II, with the open reading frames denoted by arrows in the direction of mRNA synthesis. The experimentally mapped 5′ ends of the pho84 and pho1 mRNAs and the intervening noncoding prt RNA are indicated by their distance (nt) from the translations start sites of the pho84 and pho1 mRNAs. (B) To map the 5′ ends of the pho84 and prt RNAs, we analyzed the reverse transcriptase primer extension product (lanes *) in parallel with a series of DNA-directed primer extension reactions that contained mixtures of standard and chain-terminating nucleotides (the chain terminator is specified above the lanes). The primer extension products were analyzed by electrophoresis through a 42-cm denaturing 8% polyacrylamide gel and visualized by autoradiography of the dried gel. The 5′ RNA ends are denoted by ◂ to the right of the gel. The coding strand DNA sequences flanking the pho84 and prt transcription start sites are shown at right, with the start sites denoted by ↱. (C) ³²P-labeled oligonucleotide primers complementary to pho1 and act1 mRNAs (left panel), pho84 mRNA RNA (top right panel), or prt RNA (bottom right panel) were annealed to total RNA from pho7⁺ (WT) or pho7Δ strains and extended with reverse transcriptase. The reaction products were analyzed by denaturing PAGE and visualized by autoradiography.

FIGURE 4.

Effects of CTD mutations on expression of phosphate-responsive mRNAs. (A) ³²P-labeled primers complementary to pho1, pho84, and act1 mRNAs (top panel) or prt RNA (bottom panel) were annealed to total RNA isolated from the indicated fission yeast strains that had been incubated for 3 h in PMG medium containing 15.5 mM phosphate (+ phosphate) or lacking exogenous phosphate (− phosphate). After primer extension with reverse transcriptase, the reaction products were analyzed by denaturing PAGE and visualized by autoradiography. (B) The pho1 primer extension product in panel A was quantified and normalized to that of act1 measured for the same RNA sample. The bar graph shows the fold-change in pho1 relative to the wild-type + phosphate control (defined as 1.0). (C) RT-qPCR analysis was performed as described in Materials and Methods. The level of pho1 transcript was normalized to that of act1 measured for the same RNA sample. The bar graph shows the fold-change in pho1 RNA relative to the wild-type + phosphate control (defined as 1.0). Each datum in the bar graph is the average of values from RT-qPCR analyses of RNAs from three independent yeast cultures. The error bars denote SEM.

FIGURE 5.

rrp6Δ delays the induction of both pho1 and pho84. (A) Fission yeast rpb1⁺ rrp6⁺ (WT) and rpb1⁺ rrp6Δ strains were grown in YES medium to A₆₀₀ of 0.5 to 0.7. The cells were harvested, washed in water, and after withdrawing an aliquot to measure Pho1 activity (time 0), the cells were transferred to PMG medium lacking exogenous phosphate. Pho1 activity was assayed after incubation for the times specified. (B,C) Total RNA isolated from WT and rrp6Δ cells at time 0 and at 3 and 6 h post-transfer to phosphate-free medium was used to assay by RT-qPCR the levels of pho1 and pho84 mRNAs (B) and prt RNA (C). The transcript levels were normalized to those of act1 measured for the same RNA samples. The bar graph shows the fold-change in RNA relative to the WT time 0 value (defined as 1.0). Each datum in the bar graphs is the average of values from RT-qPCR analyses of RNAs from three independent yeast cultures. The error bars denote SEM.

FIGURE 6.

Effects of manipulating Csk1 and Cdk9 kinases and Ssu72 phosphatase on Pho1 regulation. rpb1⁺ cells, either wild-type or bearing additional mutations as specified, were assayed for Pho1 activity after incubation for 3 h in PMG medium containing 15.5 mM phosphate (+ phosphate) or lacking exogenous phosphate (− phosphate).

FIGURE 7.

Genetic and functional interaction of Ssu72 with the Pol2 CTD. (A) Exponentially growing cultures of S. pombe strains with the indicated chromosomal rpb1-CTD and ssu72 alleles were adjusted to A₆₀₀ of 0.1, and aliquots of serial fivefold dilutions were spotted to YES agar and incubated at the indicated temperatures. (B) Fission yeast cells with genotypes as specified were assayed for Pho1 activity after incubation for 3 h in PMG medium containing 15.5 mM phosphate (+ phosphate) or lacking exogenous phosphate (− phosphate).