GENETIC AND BIOCHEMICAL-CHARACTERIZATION OF MUTATIONS IN THE ATPASE AND HELICASE REGIONS OF THE UPF1 PROTEIN

mRNA degradation is an important control point in the regulation of ge ne expression and has been linked to the process of translation. One c lear example of this linkage is the nonsense-mediated mRNA decay pathw ay, in which nonsense mutations in a gene can reduce the abundance of the mRNA transcribed from that gene. For the yeast Saccharomyces cerev isiae, the Upf1 protein (Upf1p), which contains a cysteine- and histid ine-rich region and nucleoside triphosphate hydrolysis and helicase mo tifs, was shown to be a trans-acting factor in this decay pathway. Bio chemical analysis of the wild-type Upf1p demonstrates that it has RNA- dependent ATPase, RNA helicase, and RNA binding activities. A UPF1 gen e disruption results in stabilization of nonsense-containing mRNAs, le ading to the production of enough functional product to overcome an au xotrophy resulting from a nonsense mutation. A genetic and biochemical study of the UPF1 gene was undertaken in order to understand the mech anism of Upf1p function in the nonsense-mediated mRNA decay pathway. O ur analysis suggests that Upf1p is a multifunctional protein with sepa rable activities that can affect mRNA turnover and nonsense suppressio n. Mutations in the conserved helicase motifs of Upf1p that inactivate its mRNA decay function while not allowing suppression of leu2-2 and tyr7-1 nonsense alleles have been identified. In particular, one mutat ion located in the ATP binding and hydrolysis motif of Upf1p that chan ged the aspartic and glutamic acid residues to alanine residues (DE572 AA) lacked ATPase and helicase activities, and the mutant formed a Upf 1p:RNA complex in the absence of ATP; surprisingly, however, the Upf1p :RNA complex dissociated as a consequence of ATP binding. This result suggests that ATP binding, independent of its hydrolysis, can modulate Upf1p:RNA complex formation for this mutant protein. The role of the RNA binding activity of Upf1p in modulating nonsense suppression is di scussed.