holbein-cordycepinhypersensitive-2008.pdf (4.99 MB)
Cordycepin-hypersensitive growth links elevated polyphosphate levels to inhibition of poly(A) polymerase in Saccharomyces cerevisiae
journal contributionposted on 2008-01-01, 00:00 authored by S Holbein, F Freimoser, T Werner, A Wengi, Bernhard DichtlBernhard Dichtl
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.