Identification and characterization of mutations in the UPF1 gene that affect nonsense suppression and the formation of the Upf protein complex but not mRNA turnover.
To understand the relationship between translation and mRNA decay, we have been studying how premature translation termination accelerates the degradation of mRNAs. In the yeast Saccharomyces cerevisiae, the Upf1 protein (Upf1p), which contains a cysteine- and histidine-rich region and nucleoside triphosphate hydrolysis and helicase motifs, was shown to be a trans-acting factor in this decay pathway. A UPF1 gene disruption results in the stabilization of nonsense-containing mRNAs and leads to a nonsense suppression phenotype. Biochemical analysis of the wild-type Upf1p demonstrated that it has RNA-dependent ATPase, RNA helicase, and RNA binding activities. In the work described in the accompanying paper (Y. Weng, K. Czaplinski, and S. W. Peltz, Mol. Cell. Biol. 16:5477-5490, 1996) mutations in the helicase region of Upf1p that inactivated its mRNA decay function but prevented suppression of leu2-2 and tyr7-1 nonsense alleles are identified. On the basis of these results, we suggested that Upf1p is a multifunctional protein involved in modulating mRNA decay and translation termination at nonsense codons. If this is true, we predict that UPF1 mutations with the converse phenotype should be identified. In this report, we describe the identification and biochemical characterization of mutations in the amino-terminal cysteine- and histidine-rich region of Upf1p that have normal nonsense-mediated mRNA decay activities but are able to suppress leu2-2 and tyr7-1 nonsense alleles. Biochemical characterization of these mutant proteins demonstrated that they have altered RNA binding properties. Furthermore, using the two-hybrid system, we characterized the Upf1p-Upf2p interactions and demonstrated that Upf2p interacts with Upf3p. Mutations in the cysteine- and histidine-rich region of Upf1p abolish Upf1p-Upf2p interaction. On the basis of these results, the role of the Upf complex in nonsense-mediated mRNA decay and nonsense suppression is discussed.
Weng
Y
Y
Department of Molecular Genetics and Microbiology, Robert Wood Johnson Medical School, University of Medicine, Piscataway 08854, USA.
Czaplinski
K
K
Peltz
S W
SW
D000595:Amino Acid Sequence; D001483:Base Sequence; D003545:Cysteine; D004265:DNA Helicases; D017931:DNA Primers; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D006639:Histidine; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010443:Peptide Chain Termination, Translational; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D020365:RNA Helicases; D012333:RNA, Messenger / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D013489:Suppression, Genetic; D014158:Transcription, Genetic
D017931:DNA Primers; D005656:Fungal Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; C074151:NAM7 protein, S cerevisiae; D004265:DNA Helicases; D020365:RNA Helicases; D003545:Cysteine