Expression of fission yeast Pho1 acid phosphatase is repressed during growth in phosphate-rich medium. Repression is mediated by transcription of the prt locus upstream of pho1 to produce a long noncoding (lnc) prt RNA. Repression is also governed by RNA polymerase II CTD phosphorylation status, whereby inability to place a Ser7-PO₄ mark (as in S7A) derepresses Pho1 expression, and inability to place a Thr4-PO₄ mark (as in T4A) hyper-represses Pho1 in phosphate replete cells. Here we find that basal pho1 expression from the prt–pho1 locus is inversely correlated with the activity of the prt promoter, which resides in a 110-nucleotide DNA segment preceding the prt transcription start site. CTD mutations S7A and T4A had no effect on the activity of the prt promoter or the pho1 promoter, suggesting that S7A and T4A affect post-initiation events in prt lncRNA synthesis that make it less and more repressive of pho1, respectively. prt lncRNA contains clusters of DSR (determinant of selective removal) sequences recognized by the YTH-domain-containing protein Mmi1. Altering the nucleobase sequence of two DSR clusters in the prt lncRNA caused hyper-repression of pho1 in phosphate replete cells, concomitant with increased levels of the prt transcript. The isolated Mmi1 YTH domain binds to RNAs with single or tandem DSR elements, to the latter in a noncooperative fashion. We report the 1.75 Å crystal structure of the Mmi1 YTH domain and provide evidence that Mmi1 recognizes DSR RNA via a binding mode distinct from that of structurally homologous YTH proteins that recognize m⁶A-modified RNA.

Fission yeast phosphate acquisition genes pho1, pho84, and tgp1 are repressed in phosphate-rich medium by transcription of upstream lncRNAs. Here, we show that phosphate homeostasis is subject to metabolite control by inositol pyrophosphates (IPPs), exerted through the 3′-processing/termination machinery and the Pol2 CTD code. Increasing IP8 (via Asp1 IPP pyrophosphatase mutation) de-represses the PHO regulon and leads to precocious termination of prt lncRNA synthesis. pho1 de-repression by IP8 depends on cleavage-polyadenylation factor (CPF) subunits, termination factor Rhn1, and the Thr4 letter of the CTD code. pho1 de-repression by mutation of the Ser7 CTD letter depends on IP8. Simultaneous inactivation of the Asp1 and Aps1 IPP pyrophosphatases is lethal, but this lethality is suppressed by mutations of CPF subunits Ppn1, Swd22, Ssu72, and Ctf1 and CTD mutation T4A. Failure to synthesize IP8 (via Asp1 IPP kinase mutation) results in pho1 hyper-repression. Synthetic lethality of asp1Δ with Ppn1, Swd22, and Ssu72 mutations argues that IP8 plays an important role in essential 3′-processing/termination events, albeit in a manner genetically redundant to CPF. Transcriptional profiling delineates an IPP-responsive regulon composed of genes overexpressed when IP8 levels are increased. Our results establish a novel role for IPPs in cell physiology.

Significance: The carboxyl-terminal domain (CTD) code encrypted within the YSPTSPS heptad repeats of RNA polymerase II is deeply rooted in eukaryal biology. This paper exploits fission yeast CTD mutants in which single coding cues (or combinations of cues) are subtracted from all consensus heptads to gauge the roles of the Tyr1, Ser2, Thr4, and Ser7 phosphoacceptors in gene expression. Transcriptome profiling revealed distinctive signatures for the CTD phosphoacceptors that were verified by cell-based activity assays. The results highlight ( i ) the role of Tyr1 and Ser2 in repressing the iron uptake regulon and ( ii ) the distinctive roles of Thr4 and Ser7 in the phosphate starvation response, whereby Thr4 facilitates and Ser7 represses the induction of Pho1 acid phosphatase.

Interactions between RNA guanylyltransferase (GTase) and the C-terminal domain (CTD) repeats of RNA polymerase II (Pol2) and elongation factor Spt5 are thought to orchestrate cotranscriptional capping of nascent mRNAs. The crystal structure of a fission yeast GTase•Pol2 CTD complex reveals a unique docking site on the nucleotidyl transferase domain for an 8-amino-acid Pol2 CTD segment, S_5PPSYSPTS_5P, bracketed by two Ser5-PO₄ marks. Analysis of GTase mutations that disrupt the Pol2 CTD interface shows that at least one of the two Ser5-PO₄-binding sites is required for cell viability and that each site is important for cell growth at 37°C. Fission yeast GTase binds the Spt5 CTD at a separate docking site in the OB-fold domain that captures the Trp4 residue of the Spt5 nonapeptide repeat T¹PAW⁴NSGSK. A disruptive mutation in the Spt5 CTD-binding site of GTase is synthetically lethal with mutations in the Pol2 CTD-binding site, signifying that the Spt5 and Pol2 CTDs cooperate to recruit capping enzyme in vivo. CTD phosphorylation has opposite effects on the interaction of GTase with Pol2 (Ser5-PO₄ is required for binding) versus Spt5 (Thr1-PO₄ inhibits binding). We propose that the state of Thr1 phosphorylation comprises a binary “Spt5 CTD code” that is read by capping enzyme independent of and parallel to its response to the state of the Pol2 CTD.

The primary structure and phosphorylation pattern of the tandem Y ¹ S ² P ³ T ⁴ S ⁵ P ⁶ S ⁷ repeats of the RNA polymerase II carboxyl-terminal domain (CTD) convey information about the transcription apparatus—a CTD code—to a large ensemble of CTD-binding receptor proteins. Four of the seven coding “letters” of the fission yeast CTD (Tyr1, Pro3, Ser5, Pro6) are essential in vivo, but the grammatical rules of the code are obscure. Here we show that the minimal fission yeast CTD coding unit is a decapeptide Y ¹ S ² P ³ T ⁴ S ⁵ P ⁶ S ⁷ Y ¹ S ² P ³ and the spacing between coding units is flexible; the coding unit must contain two Tyr1 residues and the spacing between consecutive tyrosines is important; Ser5-PO ₄ -Pro6 comprises an essential two-letter code “word” that is read by the mRNA capping apparatus; and a threshold number of Ser5-PO ₄ -Pro6 words are needed to comprise a readable “sentence” of CTD information. Bypassing the essentiality of the Ser5 and Pro6 letters by fusion of capping enzymes to the CTD helped reveal how CTD phosphorylation circuits are wired in vivo. We found that the Ser2-PO ₄ mark is independent of Ser5, Pro6, Ser7, and Thr4, whereas the Ser5-PO ₄ mark is independent of Ser2, Ser7, and Thr4. These results provide unique insights to the reading and writing of the CTD code.

The expression of the phosphate transporter Pho84 in fission yeast Schizosaccharomyces pombe is repressed in phosphate-rich medium and induced during phosphate starvation. Two other phosphate-responsive genes in S. pombe (pho1 and tgp1) had been shown to be repressed in cis by transcription of a long noncoding (lnc) RNA from the upstream flanking gene, but whether pho84 expression is regulated in this manner is unclear. Here we show that repression of pho84 is enforced by transcription of the SPBC8E4.02c locus upstream of pho84 to produce a lncRNA that we name prt2 (pho-repressive transcript 2) We identify two essential elements of the prt2 promoter: HomolD box and TATA box, mutations of which inactivate the prt2 promoter and derepress the downstream pho84 promoter under phosphate-replete conditions. We find that prt2 promoter inactivation also elicits a cascade effect on the adjacent downstream prt (lncRNA) and pho1 (acid phosphatase) genes, whereby increased pho84 transcription down-regulates prt lncRNA transcription and thereby de-represses pho1. Our results establish a unified model for the repressive arm of fission yeast phosphate homeostasis, in which transcription of prt2, prt, and nc-tgp1 lncRNAs interferes with the promoters of the flanking pho84, pho1, and tgp1 genes, respectively.