title: Cordycepin-hypersensitive growth links elevated polyphosphate levels to inhibition of poly(A) polymerase in Saccharomyces cerevisiae.
abstract: To identify genes involved in poly(A) metabolism, we screened the yeast gene deletion collection for growth defects in the presence of cordycepin (3'-deoxyadenosine), a precursor to the RNA chain terminating ATP analog cordycepin triphosphate. Deltapho80 and Deltapho85 strains, which have a constitutively active phosphate-response pathway, were identified as cordycepin hypersensitive. We show that inorganic polyphosphate (poly P) accumulated in these strains and that poly P is a potent inhibitor of poly(A) polymerase activity in vitro. Binding analyses of poly P and yeast Pap1p revealed an interaction with a k(D) in the low nanomolar range. Poly P also bound mammalian poly(A) polymerase, however, with a 10-fold higher k(D) compared to yeast Pap1p. Genetic tests with double mutants of Deltapho80 and other genes involved in phosphate homeostasis and poly P accumulation suggest that poly P contributed to cordycepin hypersensitivity. Synergistic inhibition of mRNA synthesis through poly P-mediated inhibition of Pap1p and through cordycepin-mediated RNA chain termination may thus account for hypersensitive growth of Deltapho80 and Deltapho85 strains in the presence of the chain terminator. Consistent with this, a mutation in the 3'-end formation component rna14 was synthetic lethal in combination with Deltapho80. Based on these observations, we suggest that binding of poly P to poly(A) polymerase negatively regulates its activity.
authors.affiliation: Institute of Molecular Biology, University of Zürich, Zürich, Switzerland.
authors.forename: Sandra
authors.initials: S
authors.lastname: Holbein
authors.forename: Florian M
authors.initials: FM
authors.lastname: Freimoser
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Werner
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Wengi
authors.forename: Bernhard
authors.initials: B
authors.lastname: Dichtl
mesh_terms: D018844:Cyclin-Dependent Kinases / Q000235:genetics; D016213:Cyclins / Q000235:genetics; D003839:Deoxyadenosines / Q000494:pharmacology*; D017353:Gene Deletion; D026723:Polyadenylation; D011062:Polynucleotide Adenylyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011122:Polyphosphates / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics
chemical_list: D016213:Cyclins; D003839:Deoxyadenosines; C066824:PHO80 protein, S cerevisiae; D011122:Polyphosphates; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D018844:Cyclin-Dependent Kinases; C058605:PHO85 protein, S cerevisiae; D011062:Polynucleotide Adenylyltransferase; C058120:cordycepin