RELATIONSHIP BETWEEN YEAST POLYRIBOSOMES AND UPF PROTEINS REQUIRED FOR NONSENSE MESSENGER-RNA DECAY

In yeast, the accelerated rate of decay of nonsense mutant mRNAs, call ed nonsense-mediated mRNA decay, requires three proteins, Upf1p, Upf2p , and Upf3p. Single, double, and triple disruptions of the UPF genes h ad nearly identical effects on nonsense mRNA accumulation, suggesting that the encoded proteins function in a common pathway, We examined th e distribution of epitope-tagged versions of Upf proteins by sucrose d ensity gradient fractionation of soluble lysates and found that all th ree proteins co-distributed with 80 S ribosomal particles and polyribo somes. Treatment of lysates with RNase A caused a coincident collapse of polyribosomes and each Upf protein into fractions containing 80 S r ibosomal particles, as expected for proteins that are associated with polyribosomes. Mutations in the cysteine-rich (zinc finger) and RNA he licase domains of Upf1p caused loss of function, but the mutant protei ns remained polyribosome-associated. Density gradient profiles for Upf 1p were unchanged in the absence of Upf3p, and although similar, were modestly shifted to fractions lighter than those containing polyriboso mes in the absence of Upf2p. Upf2p shifted toward heavier polyribosome fractions in the absence of Upf1p and into fractions containing 80 S particles and lighter fractions in the absence of Upf3p, Our results s uggest that the association of Upf2p with polyribosomes typically foun d in a wild-type strain depends on the presence and opposing effects o f Upf1p and Upf3p.