Duf89 abets lncRNA control of fission yeast phosphate homeostasis via its antagonism of precocious lncRNA transcription termination

Abstract

Fission yeast phosphate homeostasis genepho1is actively repressed during growth in phosphate-rich medium by transcription incisof a long noncoding (lnc) RNA from the 5′ flankingprt(nc-pho1)gene. Pho1 expression is: (i) derepressed by genetic maneuvers that favor precocious lncRNA 3′-processing and termination, in response to DSR and PAS signals inprt; and (ii) hyperrepressed in genetic backgrounds that dampen 3′-processing/termination efficiency. Governors of 3′-processing/termination include the RNA polymerase CTD code, the CPF (cleavage and polyadenylation factor) complex, termination factors Seb1 and Rhn1, and the inositol pyrophosphate signaling molecule 1,5-IP8. Here, we present genetic and biochemical evidence that fission yeast Duf89, a metal-dependent phosphatase/pyrophosphatase, is an antagonist of precocious 3′-processing/termination. We show that derepression ofpho1induf89Δ cells correlates with squelching the production of full-lengthprtlncRNA and is erased or attenuated by: (i) DSR/PAS mutations inprt; (ii) loss-of-function mutations in components of the 3′-processing and termination machinery; (iii) elimination of the CTD Thr4-PO4mark; (iv) interdicting CTD prolyl isomerization by Pin1; (v) inactivating the Asp1 kinase that synthesizes IP8; and (vi) loss of the putative IP8sensor Spx1. The findings thatduf89Δ is synthetically lethal withpho1-derepressive mutationsCTD-S7Aandaps1Δ—and that this lethality is rescued byCTD-T4A, CPF/Rhn1/Pin1 mutations, andspx1Δ—implicate Duf89 more broadly as a collaborator in cotranscriptional regulation of essential fission yeast genes. Theduf89-D252Amutation, which abolishes Duf89 phosphohydrolase activity, phenocopiedduf89+, signifying thatduf89Δ phenotypes are a consequence of Duf89 protein absence, not absence of Duf89 catalysis.