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.
Publication types
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Catalytic Domain
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Cell Nucleus / enzymology*
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Cell Nucleus / genetics
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Exoribonucleases / genetics
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Exoribonucleases / metabolism*
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Exosome Multienzyme Ribonuclease Complex
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Fungal Proteins / chemistry
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Fungal Proteins / genetics
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Fungal Proteins / metabolism*
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Genes, Fungal / genetics
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Genes, Fungal / physiology
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Half-Life
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Molecular Sequence Data
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Mutation / genetics
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Nuclear Proteins / metabolism*
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Pancreatitis-Associated Proteins
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Polynucleotide Adenylyltransferase / genetics
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Polynucleotide Adenylyltransferase / metabolism*
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Protein Binding
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RNA Processing, Post-Transcriptional / genetics
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RNA Stability / genetics
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RNA, Fungal / genetics
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RNA, Fungal / metabolism
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RNA, Heterogeneous Nuclear / genetics
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RNA, Heterogeneous Nuclear / metabolism
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RNA, Messenger / genetics
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RNA, Messenger / metabolism*
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RNA-Binding Proteins*
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Recombinant Fusion Proteins / metabolism
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Saccharomyces cerevisiae / cytology
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Saccharomyces cerevisiae / enzymology
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins*
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Sequence Alignment
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Suppression, Genetic / genetics
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Temperature
Substances
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Fungal Proteins
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NPL3 protein, S cerevisiae
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Nuclear Proteins
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Pancreatitis-Associated Proteins
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REG3A protein, human
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RNA, Fungal
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RNA, Heterogeneous Nuclear
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RNA, Messenger
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RNA-Binding Proteins
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Recombinant Fusion Proteins
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Saccharomyces cerevisiae Proteins
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Polynucleotide Adenylyltransferase
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Exoribonucleases
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Exosome Multienzyme Ribonuclease Complex
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RRP6 protein, S cerevisiae