We examined Saccharomyces cerevisiae mitochondrial RNA for polyadenylate. Using hybridization to [3H]polyuridylate as the assay for adenylate sequences, we found adenylate-rich oligonucleotides approximately 8 residues long. Longer polyadenylate was not detected. Most of the adenylate-rich sequence is associated with the large mitochondrial rRNA. The remainder is associated with the 10-12S group of transcripts.In mammalian cells polyadenylation has been associated with the production of functional cytoplasmic mRNA from nuclear precursors (3,8,30) as well as with its stabilization (14,22,26,38). Polyadenylated RNA has been shown to be present in animal cell mitochondria (21, 31). Although it is quite likely that nucleolytic processing of mammalian mitochondrial RNA occurs (32), the role polyadenylation may have in this process is unknown.The unicellular eucaryote Saccharomyces cerevisiae also contains 3' polyadenylated cytoplasmic mRNA. However, there is disagreement in the literature regarding the presence of polyadenylate [poly(A)] in S. cerevisiae mitochondrial RNA (6,17,19,29,33). It is clear from the recent dramatic advances in the analysis of mitochondrial DNA and its transcripts that the production of several mitochondrial RNA species in S. cerevisiae involves extensive posttranscriptional RNA processing (4,18,24,39). Therefore, we reexamined S. cerevisiae mitochondrial RNA for the presence of poly(A), with an emphasis on procedures which permit the detection of short adenylate sequences. We found that S. cerevisiae mitochondrial RNA contains an oligonucleotide sequence approximately 8 nucleotides long that is composed of at least 80% adenylate residues. The bulk of this adenylate-rich oligonucleotide is associated with the large 21S rRNA. Smaller amounts are associated with the 15S RNA and the 10-12S RNA species. Longer poly(A) was not detected in mitochondrial RNA under conditions where cytoplasmic poly(A) is readily observed. MATERIALS AND METHODSCefl culture. S. cerevisiae IL8-8c (at his-trp-CAPr Ery'), used throughout these studies, was a generous Preparation of RNA. Mitochondria were purified by an adaptation of the procedure of Kovac et al. (23). A routine preparation started with the cells from 2 liters of culture. Cells were harvested by centrifugation at 9,000 x g for 10 min and washed by two cycles of resuspension in 500 ml of water and centrifugation. Cells were converted to spheroplasts by treatment at 30C for 30 min with 20 mg of Zymolyase 60,000 in 200 ml of t M sorbitol-20 mM sodium phosphate (pH 7.0) or with 2 ml of Glusulase in 200 ml of 1 M sorbitol.Spheroplasts were recovered and washed free of soluble materials by sedimentation at 1,500 x g for 10 min through a 0.5-volume layer of 2 M sorbitol. The spheroplasts were resuspended in 1 liter of growth medium containing 0.5% galactose and 1 M sorbitol for osmotic stabilization and incubated for 1 h at 30°C to ensure recovery from the metabolic perturbation created during cell wall digestion. The culture was quickly chilled by pouring into ...
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