Abstract
Inactivation of poly(A) polymerase (encoded by PAP1) in Saccharomyces cerevisiae cells carrying the temperature-sensitive, lethal pap1-1 mutation results in reduced levels of poly(A)+ mRNAs. Genetic selection for suppressors of pap1-1 yielded two recessive, cold-sensitive alleles of the gene RRP6. These suppressors,rrp6-1 and rrp6-2, as well as a deletion of RRP6, allow growth of pap1-1 strains at high temperature and partially restore the levels of poly(A)+mRNA in a manner distinct from the cytoplasmic mRNA turnover pathway and without slowing a rate-limiting step in mRNA decay. Subcellular localization of an Rrp6p-green fluorescent protein fusion shows that the enzyme residues in the nucleus. Phylogenetic analysis and the nature of the rrp6-1 mutation suggest the existence of a highly conserved 3′-5′ exonuclease core domain within Rrp6p. As predicted, recombinant Rrp6p catalyzes the hydrolysis of a synthetic radiolabeled RNA in a manner consistent with a 3′-5′ exonucleolytic mechanism. Genetic and biochemical experiments indicate that Rrp6p interacts with poly(A) polymerase and with Npl3p, a poly(A)+ mRNA binding protein implicated in pre-mRNA processing and mRNA nuclear export. These findings suggest that Rrp6p may interact with the mRNA polyadenylation system and thereby play a role in a nuclear pathway for the degradation of aberrantly processed precursor mRNAs.
ACKNOWLEDGMENTS
We thank Michael Briggs, Mark Dumont, Beth Grayhack, Eric Phizicky, Fred Sherman, and Terry Platt for helpful discussions and Terry Platt, Biswadip Das, Roy Parker, and the members of our laboratory for comments on the manuscript. We are grateful to Geoff Dance, Dave Goldfarb, Elizabeth Grayhack, Mark Martzen, Pam Silver, Maurice Swanson, Jon Warner, and Nilsen Zanchin for providing antibodies, plasmids, and strains, to Thomas “Trey” Westbrook for constructing pAS2-RRP6, and to Mark Burkard and Sherry Spinelli for help with thin-layer chromatography analysis.
This work was supported by grants from the National Science Foundation (MCB 9603893) and the National Institutes of Health (GM 59898) to J.S.B.