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

Nucleic Acids Res. 2008 Feb;36(2):353-63. doi: 10.1093/nar/gkm990. Epub 2007 Nov 22.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cyclin-Dependent Kinases / genetics
  • Cyclins / genetics
  • Deoxyadenosines / pharmacology*
  • Gene Deletion
  • 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

Substances

  • Cyclins
  • Deoxyadenosines
  • PHO80 protein, S cerevisiae
  • Polyphosphates
  • Repressor Proteins
  • Saccharomyces cerevisiae Proteins
  • Cyclin-Dependent Kinases
  • PHO85 protein, S cerevisiae
  • Polynucleotide Adenylyltransferase
  • cordycepin