Poly(A) site choice and Pol2 CTD Serine-5 status govern lncRNA control of phosphate-responsive tgp1 gene expression in fission yeast

Abstract

Expression of fission yeast glycerophosphate transporter Tgp1 is repressed in phosphate-rich medium and induced during phosphate starvation. Repression is enforced by transcription of the nc-tgp1 locus upstream of tgp1 to produce a long noncoding (lnc) RNA. Here we identify two essential elements of the nc-tgp1 promoter: a TATA box ^-30TATATATA^-23 and a HomolD box ^-64CAGTCACA^-57, mutations of which inactivate the nc-tgp1 promoter and de-repress the downstream tgp1 promoter under phosphate-replete conditions. The nc-tgp1 lncRNA poly(A) site maps to nucleotide +1636 of the transcription unit, which coincides with the binding site for Pho7 (¹⁶³²TCGGACATTCAA¹⁶⁴³), the transcription factor that drives tgp1 expression. Overlap between the lncRNA template and the tgp1 promoter points to transcriptional interference as the simplest basis for lncRNA repression. We identify a shorter RNA derived from the nc-tgp1 locus, polyadenylated at position +508, well upstream of the tgp1 promoter. Mutating the nc-tgp1-short RNA polyadenylation signal abolishes de-repression of the downstream tgp1 promoter elicited by Pol2 CTD Ser5Ala phospho-site mutation. Ser5 mutation favors utilization of the short RNA poly(A) site, thereby diminishing transcription of the lncRNA that interferes with the tgp1 promoter. Mutating the nc-tgp1-short RNA polyadenylation signal attenuates induction of the tgp1 promoter during phosphate starvation. Polyadenylation site choice governed by CTD Ser5 status adds a new level of lncRNA control of gene expression and reveals a new feature of the fission yeast CTD code.

Keywords: CTD code; phosphate homeostasis; polyadenylation.

FIGURE 1.

Transcripts derived from the nc-tgp1 and tgp1 loci and plasmid reporter for dissection of nc-tgp1 control of tgp1 expression. (A) The tandem nc-tgp1–tgp1 locus is shown with the nc-tgp1 transcription start site indicated by the blue arrow and the tgp1 ORF depicted as a green bar with arrowhead indicating the direction of transcription. The distance between the nc-tgp1 transcription initiation site and the ATG translation start site of the tgp1 ORF is indicated by the bracket. The two nc-tgp1 transcripts detected by northern analysis (in panel B) are depicted as wavy lines below the DNA. (B) Northern blotting. Total RNA from wild-type (WT), rrp6Δ, and mmi1Δ cells was resolved by agarose gel electrophoresis, and ethidium bromide-stained ribosomal RNAs (EtBr, right panel) were visualized prior to transfer of the gel contents to membrane, which was hybridized to ³²P-labeled nc-tgp1 (left panel) and tgp1 (middle panel) DNA probes. The positions and sizes (kb) of RNA size markers are indicated on the left panel. (C) tgp1 mRNA. The nc-tgp1–tgp1 locus is shown with the distance between the nc-tgp1 and tgp1 transcription initiation sites indicated by the bracket. The DNA sequence surrounding the tgp1 transcription start site (mapped by primer extension and indicated by the black arrow) is shown on the top line; the tgp1 ATG translation start codon is underlined. The cDNA sequence including and preceding the poly(A) tail of the tgp1 mRNA is shown on the bottom line; the consensus poly(A) signal is highlighted in white on black background. (D) The plasmid-borne nc-tgp1–tgp1•pho1 reporter cassette is shown with the mapped transcription start sites of the nc-tgp1 lncRNA and tgp1 mRNA indicated by arrows. The pho1 ORF is depicted as a gold bar with arrowhead. (E) The nc-tgp1–tgp1•pho1 plasmid was introduced into pho1Δ yeast cells that were either pho7⁺ (WT) or pho7Δ. Cells grown for 3 h in synthetic medium containing 15.5 mM phosphate (+PO₄) or in medium lacking exogenous phosphate (−PO₄) to elicit the starvation response were assayed for acid phosphatase activity by conversion of p-nitrophenylphosphate to p-nitrophenol. The y-axis specifies the phosphatase activity (A₄₁₀) normalized to input cells (A₆₀₀). The error bars denote SEM. (F) CTD mutations de-repress regulated tgp1 expression during phosphate-replete growth. The nc-tgp1–tgp1•pho1 reporter plasmid was introduced into pho1Δ cells in which the rpb1 chromosomal locus was replaced by alleles rpb1-CTD-WT, CTD-T4A, CTD-S7A, or CTD-S5₃S5A₁₁. Cells grown in phosphate-replete medium were assayed for acid phosphatase activity. (G) Time course of the phosphate starvation response. The indicated CTD strains bearing the nc-tgp1–tgp1•pho1 reporter plasmid were assayed for acid phosphatase activity immediately prior to and at hourly intervals after transfer to medium lacking exogenous phosphate.

FIGURE 2.

HomolD and TATA boxes are essential for nc-tgp1 promoter activity and regulation of tgp1. (A) Plasmid reporter of nc-tgp1 promoter activity. The pho1 ORF (beginning at the underlined ATG translation initiation codon in blue font) was fused downstream from a fragment of genomic DNA containing the nc-tgp1 transcription start site (indicated by the black arrow above the DNA sequence) and 301 nt of 5′ flanking nc-tgp1 DNA (presumed to include the nc-tgp1 promoter). Serial truncations of the upstream margin of the 5′ flanking nc-tgp1 DNA were made at positions −211, −134, −98, −71, and −55 (indicated by red arrows below the DNA sequence) relative to the nc-tgp1 transcription start site. A putative TATA element is outlined by a blue box. Three putative HomolD elements are shaded in gold (forward orientation) or orange (reverse orientation). (B) Acid phosphatase activity of pho1Δ cells bearing the indicated nc-tgp1 promoter-driven pho1 reporter plasmids. (C) Acid phosphatase activity of pho1Δ pho7⁺ (WT) and pho1Δ pho7Δ cells bearing the −301 nc-tgp1 promoter-driven pho1 plasmid. (D) Acid phosphatase activity of pho1Δ rpb1-CTD strains bearing the −301 nc-tgp1–pho1 plasmid. (E,F) The indicated mutated versions of individual HomolD sites (hd-1, hd-2, and hd-3) and the TATA box of the nc-tgp1 promoter (shown at right in panel F) were introduced into the −301 nc-tgp1•pho1 reporter and phosphate-replete pho1Δ cells bearing the indicated nc-tgp1•pho1 reporter plasmids were assayed for acid phosphatase activity (panel E). (F) The HomolD and the TATA box mutations of the nc-tgp1 promoter were introduced into the nc-tgp1–tgp1•pho1 reporter plasmid (shown at the top of the panel). Phosphate-replete pho1Δ cells bearing the indicated reporter plasmids were assayed for acid phosphatase activity.

FIGURE 3.

Effect of rpb1-CTD mutations on tgp1 promoter activity. A schematic of the -871 tgp1 promoter-driven pho1 reporter (Schwer et al. 2017) is shown at the top, with the Pho7 binding site in the tgp1 promoter highlighted. Acid phosphatase activity of pho1Δ rpb1-CTD-WT, rpb1-CTD-T4A, rpb1-CTD-S7A, and rpb1-CTD-S5₃S5A₁₁ cells bearing the tgp1•pho1 reporter plasmid is shown.

FIGURE 4.

Characterization of the nc-tgp1 lncRNA and a novel nc-tgp1 short RNA. (A) The nucleotide sequence of the first 1395 nt of the nc-tgp1 lncRNA is shown. A cluster of three putative DSR sequences in the region from nucleotides +821 to +850 is highlighted in gold. Other DSR hexamers are highlighted in green. The polyadenylation signal for the nc-tgp1-short RNA is shown in white font on black background. (B) Phosphate starvation response. The indicated mutated version of the +821 to +850 DSR cluster (mut) was introduced into the nc-tgp1–tgp1•pho1 reporter plasmid shown in panel A. Yeast pho1Δ cells bearing the DSR WT or mut nc-tgp1–tgp1•pho1 plasmids were assayed for acid phosphatase activity immediately prior to and at hourly intervals after transfer to medium lacking exogenous phosphate. (C) Northern blotting. Total RNA from pho1Δ cells bearing the DSR WT or mut nc-tgp1–tgp1•pho1 plasmids was resolved by agarose gel electrophoresis, transferred to membrane, and probed with ³²P-labeled nc-tgp1 (left panel) and pho1 (right panel) DNAs. The positions and sizes (kb) of RNA size markers are indicated on the right. (D–F) Mapping the 3′ ends of the polyadenylated nc-tgp1 lncRNA by 3′ RACE of RNA from mmi1Δ cells expressing nc-tgp1 from the native chromosomal locus (panel D; sequencing three independent cDNA clones), and pho1Δ cells expressing nc-tgp1 from the nc-tgp1(DSR-WT)–tgp1•pho1 and nc-tgp1(DSR-mut)–tgp1•pho1 reporter plasmids (panel E; sequencing two and four independent cDNA clones, respectively). (D–F) Mapping the 3′ ends of the polyadenylated nc-tgp1-short RNA by 3′ RACE of RNA from cells expressing nc-tgp1 from the native chromosomal locus (sequencing 10 independent cDNA clones), and pho1Δ cells expressing nc-tgp1 from the nc-tgp1(DSR-WT)–tgp1•pho1 and nc-tgp1(DSR-mut)–tgp1•pho1 reporter plasmids (sequencing 15 and 2 independent cDNA clones, respectively). (F) The lncRNA and short RNA sequences preceding the poly(A) tails are shown. Poly(A) nucleotides are depicted in lower case font. A canonical fission yeast polyadenylation signal is shown in white font on black background.

FIGURE 5.

Mutating the poly(A) signal of the nc-tgp1-short RNA abolishes de-repression of the tgp1 promoter by CTD S5A mutation. (A) The short RNA polyadenylation signal (PAS) in the nc-tgp1–tgp1•pho1 reporter cassette (highlighted in white font on black background) was replaced by the mutant sequence shown in red font below the wild-type PAS. (B) Acid phosphatase activity of pho1Δ rpb1-CTD-WT or rpb1-CTD-S5₃S5A₁₁ cells bearing nc-tgp1–tgp1•pho1 reporter plasmids with wild-type (WT) or mutant (mut) short RNA PAS elements. (C) RNase protection analysis of nc-tgp1-short PAS utilization in rpb1-CTD-WT cells (CTD WT; lanes 3,4,9) or rpb1-CTD-S5₃S5A₁₁ cells (CTD Mut; lanes 5,6,10) bearing nc-tgp1–tgp1•pho1 reporter plasmids with wild-type (PAS–WT; lanes 3–6) or mutant (PAS-Mut; lanes 9,10) short RNA PAS elements was performed as described in Materials and Methods. The RNase T1-digested samples (T1+) and untreated controls (T1−; lanes 1,7) were analyzed by denaturing PAGE. The positions and lengths (nt) of radiolabeled DNA size markers (lane 11) are indicated on the right.

FIGURE 6.

Mutating the poly(A) signal of the nc-tgp1-short RNA attenuates the induction of the tgp1 promoter during phosphate starvation. Cells bearing the nc-tgp1–tgp1•pho1 reporter in which the nc-tgp1-short RNA polyadenylation signal (PAS) was either wild-type or mutant were assayed for acid phosphatase activity immediately prior to and at hourly intervals after transfer to medium lacking exogenous phosphate.