The majority of yeast UPF1 co-localizes with polyribosomes in the cytoplasm.
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 L1 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 L1 co-distributed with polyribosomes fractionated in a 7-47% sucrose gradient. The sucrose sedimentation profiles for UPF1-3EP and L1 exhibited similar changes using three different sets of conditions that altered the polyribosome profile. When polyribosomes were disaggregated, UPF1-3EP and L1 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.
Atkin
A L
AL
Laboratory of Genetics, University of Wisconsin, Madison 53706, USA.
Altamura
N
N
Leeds
P
P
Culbertson
M R
MR
D001483:Base Sequence; D002458:Cell Fractionation; D003593:Cytoplasm / Q000737:chemistry; D000939:Epitopes / Q000032:analysis; D005656:Fungal Proteins / Q000032:analysis* / Q000235:genetics; D005816:Genetic Complementation Test; D006389:Hemagglutinins, Viral / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D011132:Polyribosomes / Q000737:chemistry*; D020365:RNA Helicases; D012331:RNA, Fungal / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D012269:Ribosomal Proteins / Q000032:analysis / Q000235:genetics; D012270:Ribosomes / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D000939:Epitopes; D005656:Fungal Proteins; D006389:Hemagglutinins, Viral; D012331:RNA, Fungal; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; C074151:NAM7 protein, S cerevisiae; D020365:RNA Helicases