Papers in the Biological Sciences


Date of this Version



Molecular Biology of the Cell, Vol. 6, 611-625, May 1995


Copyright 1995 by The American Society for Cell Biology


In Saccharomyces cerevisiae the UPF1 protein is required for nonsense-mediated mRNA

decay, the accelerated turnover of mRNAs containing a nonsense mutation. Several lines

of evidence suggest that translation plays an important role in the mechanism of

nonsense mRNA decay, including a previous report that nonsense mRNAs assemble in

polyribosomes. In this study we show that UPF1 and ribosomal protein Li co-localize in

the cytoplasm and that UPF1 co-sediments with polyribosomes. To detect UPF1, three

copies of the influenza hemagglutinin epitope were placed at the C-terminus. The tagged

protein, UPF1-3EP, retains 86% (± 5%) of function. Using immunological detection, we

found that UPF1-3EP is primarily cytoplasmic and was not detected either in the nucleus

or in the mitochondrion. UPF1-3EP and Li co-distributed with polyribosomes fractionated

in a 7-47% sucrose gradient. The sucrose sedimentation profiles for UPF1-3EP and

Li exhibited similar changes using three different sets of conditions that altered the

polyribosome profile. When polyribosomes were disaggregated, UPF1-3EP and Li accumulated

in fractions coincident with 80S ribosomal particles. These results suggest that

UPF1-3EP associates with polyribosomes. L3 and S3 mRNAs, which code for ribosomal

proteins of the 60S and 40S ribosomal subunits, respectively, were on average about

100-fold more abundant than UPF1 mRNA. Assuming that translation rates for L3, S3,

and UPF1 mRNA are similar, this result suggests that there are far fewer UPF1 molecules

than ribosomes per cell. Constraints imposed by the low UPF1 abundance on the

functional relationships between UPF1, polyribosomes, and nonsense mRNA turnover

are discussed.

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