The quantitative determination of metabolites of 6-mercaptopurine in biological materials. VI. Evidence for posttranscriptional modification of 6-thioguanosine residues in RNA from L5178Y cells treated with 6-mercaptopurine

Abstract

Mammalian cells incorporate 6-thioguanosine into their nucleic acids when grown in the presence of 6-mercaptopurine. 35S-labeled total RNA was prepared from L5178Y murine lymphoma cells grown in vitro in the presence of 6-[ 35S]mercaptopurine. Base analyses of this RNA suggested that 6-thioguanosine residues in RNA molecules undergo posttranscriptional modification. Thus, enzymatic peak-shifting analyses using anion-exchange high-performance liquid chromatography were applied to the hydrolysis products released from total RNA preparations by digestion with nuclease P 1 or nuclease P 1 plus nucleotide pyrophosphatase. At least eight 35S-labeled, phosphatase-sensitive compounds structurally different from [ 35S]6thioGMP were found in nuclease P 1 digests. Four of these compounds were susceptible to cleavage with nucleotide pyrophosphatase, thus indicating that they contained phosphoric acid anhydride bonds. Individual RNA species were not separately examined, the radiochromatographic data, however, which were obtained from digests of total RNA preparations, present evidence that 6-thioguanosine 5′-diphosphate and 6-thioguanosine 5′-triphosphate exist as 5′-terminal starting nucleotides (in tRNA and rRNA) and that 6-thioguanosine becomes incorporated into the highly modified dinucleoside triphosphate structures (caps) which commonly block the 5′-termini of eukaryotic poly(A) + mRNA-molecules.