Cordycepin-hypersensitive growth links elevated polyphosphate levels to inhibition of poly(A) polymerase in Saccharomyces cerevisiae

• Published in: Nucleic acids research Vol. 36; no. 2; pp. 353 - 363
• Main Authors: Holbein, Sandra, Freimoser, Florian M, Werner, Thomas P, Wengi, Agnieszka, Dichtl, Bernhard
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.02.2008; Oxford Publishing Limited (England)
• Subjects: Cyclin-Dependent Kinases - genetics; Cyclins - genetics; Deoxyadenosines - pharmacology; Gene Deletion; Nucleic Acid Enzymes; Polyadenylation; Polynucleotide Adenylyltransferase - antagonists & inhibitors; Polynucleotide Adenylyltransferase - metabolism; Polyphosphates - metabolism; Repressor Proteins - genetics; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae Proteins - genetics
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkm990

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. Δpho80 and Δpho85 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 kD in the low nanomolar range. Poly P also bound mammalian poly(A) polymerase, however, with a 10-fold higher kD compared to yeast Pap1p. Genetic tests with double mutants of Δpho80 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 Δpho80 and Δpho85 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 Δpho80. Based on these observations, we suggest that binding of poly P to poly(A) polymerase negatively regulates its activity.