IDENTIFICATION AND CHARACTERIZATION OF MUTATIONS IN THE UPF1 GENE THAT AFFECT NONSENSE SUPPRESSION AND THE FORMATION OF THE UPF PROTEIN COMPLEX BUT NOT MESSENGER-RNA TURNOVER

To understand the relationship between translation and mRNA decay, we have been studying how premature translation termination accelerates t he degradation of mRNAs. In the yeast Saccharomyces cerevisiae, the Up f1 protein (Upf1p), which contains a cysteine- and histidine-rich regi on and nucleoside triphosphate hydrolysis and helicase motifs, was sho wn to be a trans-acting factor in this decay pathway, A UPF1 gene disr uption results in the stabilization of nonsense-containing mRNAs and l eads to a nonsense suppression phenotype. Biochemical analysis of the wild-type Upf1p demonstrated that it has RNA-dependent ATPase, RNA hel icase, and RNA binding activities. In the work described in the accomp anying paper (Y. Weng, K. Czaplinski, and S. W. Peltz, Mol. Cell. Biol , 16:5477-5490, 1996) mutations in the helicase region of Upf1p that i nactivated its mRNA decay function but prevented suppression of leu2-2 and tyr7-1 nonsense alleles are identified, On the basis of these res ults, 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 phe notype should be identified, In this report, we describe the identific ation and biochemical characterization of mutations in the amino-termi nal cysteine- and histidine-rich region of Upf1p that have normal nons ense-mediated mRNA decay activities but are able to suppress leu2-2 an d tyr7-1 nonsense alleles. Biochemical characterization of these mutan t 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. M utations in the cysteine- and histidine-rich region of Upf1p abolish U pf1p-Upf2p interaction. On the basis of these results, the role of the Upf complex in nonsense-mediated mRNA decay and nonsense suppression is discussed.