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

Effect of mutating the DSR clusters in the prt lncRNA on pho1 expression. (A) Cartoon of the prt and pho1 genes, highlighting the presence of two clusters of DSR elements (boxes 1 and 2) within the 5′ segment of the prt transcription unit. The nucleotide sequence of the 5′ segment of prt lncRNA is shown below the cartoon, with the transcription start site indicated by the arrow. DSR consensus hexamers UUAAAC and variant hexamers UUAAAU are shaded gray. Clustered DSR triple-repeats DSRx3-1 (box 1) and DSRx3-2 (box 2) are underlined. Mutant 1 and mutant 2 versions of the prt–pho1 reporter cassette had DNA mutations that resulted in the indicated changes in the sequence of the prt lncRNA. (B, left panel) Acid phosphatase activity of pho1Δ cells bearing the indicated prt–pho1 reporter plasmids (kanMX). (Right panel) RT-qPCR analysis of the prt transcript was performed with total RNA isolated from pho1Δ cells bearing the DSR WT or DSR mut1 + mut2 reporter plasmids. The prt transcript levels are normalized to that of the wild-type DSR reporter (defined as 1.0). (C) Effect of DSR mutations on the phosphate starvation response. Fission yeast pho1Δ cells bearing the indicated prt–pho1 reporter plasmids were grown in YES medium to A₆₀₀ of 0.5–0.7. The cells were harvested, washed in water and, after withdrawing an aliquot to measure phosphatase activity (time 0), were transferred to PMG medium (+ kanamycin) lacking exogenous phosphate. Pho1 activity was assayed after incubation for the times specified. Error bars indicate SEM.