Genetic suppression of precocious transcription termination identifies mutations in essential subunits of the fission yeast cleavage and polyadenylation machinery

The fission yeast phosphate acquisition (<i>PHO</i>) regulon is repressed under phosphate-replete conditions by upstream lncRNA-mediated transcriptional interference. Inositol-1-pyrophosphates control phosphate homeostasis via their action as agonists of precocious <i>PHO</i> lncRNA 3'-processing/termination. Inositol pyrophosphatase-inactivating mutations that increase inositol-1-pyrophosphates elicit derepression of the <i>PHO</i> genes and a severe growth defect in YES medium. Previous studies demonstrated suppression of inositol pyrophosphate toxicosis by targeted deletion or loss-of-function mutations in the nonessential Ssu72, Ppn1, Swd22, and Ctf1 subunits of the fission yeast cleavage and polyadenylation factor (CPF) complex. Here we conducted a selection for spontaneous mutations that suppress the precocious <i>PHO</i> lncRNA termination underlying the sickness of <i>asp1-STF</i> pyrophosphatase mutants. We thereby recovered and characterized novel hypomorphic missense mutations in five essential CPF subunits: Ysh1 (the cleavage endonuclease), Pta1 (an armadillo/HEAT-repeat protein), Pfs2 (a WD repeat protein), Cft1 (a WD repeat protein), and Msi2 (a tandem RRM RNA-binding protein). The suppressor screen also yielded an intron branchpoint mutation in the gene encoding essential CPF subunit Iss1. In addition, we found that <i>asp1-STF</i> toxicosis was suppressed by a missense mutation in the active site of Pla1, the essential poly(A) polymerase subunit of CPF. Genetic crosses revealed a hierarchy of mutational synergies between the essential CPF subunits, the inessential CPF subunits, termination factor Rhn1, the Thr4 "letter" of the RNA polymerase II CTD code, and the Asp1 kinase that synthesizes inositol-1-pyrophosphates. The synthetic lethality of <i>msi2-G252E</i> with <i>ctf1</i>Δ, <i>swd22</i>Δ, <i>ppn1</i>Δ, <i>ssu72-C13S</i>, <i>rpb1-CTD-T4A</i>, and <i>asp1</i>Δ establishes Msi2 as a central agent of 3'-processing/termination, functioning in parallel to inositol-1-pyrophosphates.