The Biosynthesis of Transfer Ribonucleic Acid in the Developing Rat Brain and in Cultured Glial Cells

Abstract

The biosynthesis of tRNA was investigated in cultured astroglial cells and the 3-day-old rat brain in vivo. In the culture system astrocytes were grown for 19 days and were then exposed to [3H]guanosine for 1.5-7.5 h; 3-day-old rats were injected with [3H]guanosine and were killed 5-45 min later. [3H]tRNA was extracted, partially purified, and hydrolyzed to yield [3H]guanine and [3H]methyl guanines. The latter were separated from the former by high performance liquid chromatography and their radioactivity determined as a function of the time of exposure to [3H]guanosine. The findings indicate that labeling of astrocyte tRNA continued for 7.5 h and was maximal, relative to total RNA labeling, at 3 h, while in the immature brain tRNAs were maximally labeled at 20 min after [3H]guanosine administration. The labeling pattern of the individual methyl guanines differed considerably betweren astrocyte and brain tRNAs. Thus, [3H]1-methylguanine represented up to 35% of the total [3H]methyl guanine radioactivity in astrocyte [3H]tRNA, while it became only negligibly labeled in brain [3H]tRNA. Conversely, brain [3H]tRNA contained more [3H]N2-methylguanine than did astrocyte [3H]tRNA. Approximately equal proportions of [3H]7-methylguanine were found in the [3H]tRNAs of both neural systems. The [3H]methylguanine composition of brain [3H]tRNA was followed through several stages of tRNA purification, including benzoylated DEAE-cellulose and reverse phase chromatography (RPC-5), and differences were found between the [3H]methylguanine composition of RPC-5 fractions containing, respectively, tRNAlys and tRNAphe. The overall results of this study suggest that developing brain cells biosynthesize their particular complement of tRNAs actively and in a cell-specific manner, as attested by the significant differences in the labeling rates of their methylated guanines. The notion is advanced that cell-specific tRNA modifications may be a prerequisite for the successful synthesis of cell-specific neural proteins.

Effect of methionine sulfoximine on methylation of guanine residues in astroglial transfer ribonucleic acids

“…Astrocyte Culture. The culture procedure was as described previously (8), the cells being grown in plates 75 cm 2 containing 10 ml of growth medium (7). Briefly, forebrains of 3 to 4-day-old rats were dissected under sterile conditions, minced, and trypsinized in 0,25% trypsin in Earle's balanced salt solution (BSS).…”

“…A systematic study of the effect of MSO on cell viability was not intended in this study, tRNA Extraction. The extraction procedure has been described in detail previously (7). Briefly, the medium was decanted and the cells rinsed with phosphate-buffered saline containing, in g/liter: CaCI2 and MgCIz, 0.1; KCI and KHzPO4, 0.2, NaC1, 8; and NazHPO4, 215.…”

“…Following dialysis to remove NaCI and lyophilization, the tRNA was hydrolyzed according to Klagsbrun (14). Separation of the guanine from l-methyl-, 7-methyl-, NZ-methyl, and NZ-dimethyl guanine was accomplished using HPLC (7,15).…”

“…tRNA Aminoacylation. The rat liver or brain aminoacyl-tRNA synthetases were prepared as described (7). Aminoacylation of astrocyte tRNA was carried out in a reaction volume of I ml containing, in order of addition: 1 ~Ci [3H]lysine or phenylalanine; 0.4 M Tris HCI, pH 7.5; 20 mM (for lysine) or 50 mM (for phenylalanine) KC1; 10 mM (for lysine) or 20 mM (for phenylalanine) MgClz; 1 mM (for lysine) or 4 mM (for phenylalanine) ATP; 0.6 mM CTP; 2 mM 13-mercaptoethanol; 0.3-0.5 A26o units of tRNA; and 0.4-0.8 mg of aminoacyl-tRNA synthetase protein.…”