Turnover of polyadenylated messenger RNA in fission yeast. Evidence for the control of protein synthesis at the translational level

Abstract

Polyadenylated RNA was isolated from fission yeast (Schizosaccharomyces pombe) total RNA using oligo(dT)-cellulose, and was studied as a model for messenger RNA. The half-life of poly adenylated RNA was measured by two independent methods. (a) The rate of labelling of polyadenylated RNA during incubation of cells with [5-3H]uridine was measured. A half-life of 40-45 min was found by comparing the experimental data with theoretical curves calculated for labelling of RNAs with various half-lives. The influence of precursor-pool specific activity on RNA labelling kinetics is considered. (b) Cells were labelled with [5-3H]uridine then further RNA synthesis was inhibited by addition of 8-hydroxyquinoline. The rate of loos of radioactivity from polyadenylated RNA indicated a half-life of 50 min. The half-life found by these two methods is about one-third of the cell doubling time, and is much longer than previous estimates by indirect methods of yeast messenger RNA half-life. Both experimental methods provided evidence for the existence of tas a half-life of 40-50 min; a much smaller population is probably turning over more rapidly. After inhibition of RNA synthesis by 8-hydroxyquinoline, the rate of total protein synthesis declined much more rapidly than the polyadenylated RNA content of the cells. However, 60 min after inhibition of RNA synthesis there was a small rise in the rate of portein synthesis. These data are interpreted as evidence for mechanisms controlling protein synthesis which operate at the level of messenger RNA translation.