A nuclear 3'-5' exonuclease involved in mRNA degradation interacts with Poly(A) polymerase and the hnRNA protein Npl3p.
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.
Burkard
K T
KT
Department of Microbiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14618, USA.
Butler
J S
JS
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002467:Cell Nucleus / Q000201:enzymology* / Q000235:genetics; D005095:Exoribonucleases / Q000235:genetics / Q000378:metabolism*; D063326:Exosome Multienzyme Ribonuclease Complex; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal / Q000235:genetics / Q000502:physiology; D006207:Half-Life; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism*; D000073718:Pancreatitis-Associated Proteins; D011062:Polynucleotide Adenylyltransferase / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics; D020871:RNA Stability / Q000235:genetics; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012332:RNA, Heterogeneous Nuclear / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D013489:Suppression, Genetic / Q000235:genetics; D013696:Temperature
D005656:Fungal Proteins; C077734:NPL3 protein, S cerevisiae; D009687:Nuclear Proteins; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D012331:RNA, Fungal; D012332:RNA, Heterogeneous Nuclear; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D011062:Polynucleotide Adenylyltransferase; D005095:Exoribonucleases; D063326:Exosome Multienzyme Ribonuclease Complex; C112610:RRP6 protein, S cerevisiae