The homologous methylation of tRNA in rat brain

Abstract

Although several reports describing the properties of cerebral tRNA methyltransferases and of the regional variations in their activities with age have appeared 4,~, 9,10,20,2z, the activity of these enzymes has always been measured in terms of the number of [14C-methyl] or [3H-methyl] groups transferred from appropriately labeled S-adenosyl-L-methionine onto tRNAs purified from non-neural sources4, 6,9,1°,2°,22. Studies on cerebral methylation of tRNA in vivo have been fewT,S, 21 and thus the specificity spectrum of the tRNA methyltransferases of neural tissue as well as the role these enzymes play in the processes of maturation and modification 16 of neuron all and glial tRNAs is wholly unknown. While exploring the consequences of a prolonged depletion of cerebral Sadenosyl-L-methionine 17 on cerebral methylations is, we noted a significant transfer of [14C-methyl] groups from S-adenosyl-L-[14C-methyl]methionine onto tRNAs of the same brain extracts from which we prepared the tRNA methyltransferases, i.e., we observed homologous tRNA methylation, a reaction described only once before in a mammalian system, viz. in rat liver~. In the present communication we provide evidence that the homologous methylation of brain tRNA involves principally two positions of its guanine residues. Male Sprague-Dawley rats were decapitated and the cerebral cortex excised and weighed. A 33 ~ (w/v) homogenate prepared in medium A of Pegg 14 was centrifuged at 104,000 × g for 60 min and the supernatant used directly as a source of methyltransferases. S-adenosyl-L-[14C-methyl]methionine (58 mCi/mmole) was fi'om New England Nuclear, Boston, Mass., and E. eoli B. from Schwarz-Mann, Orangeburg, N.Y. [14C-methyl] transfer was determined both by the 'rate' and the 'total extent' assays, as described by Pegg 14. The rate of methylation is expressed as nmoles of [~4C-methyl]/mg of protein/30 rain, while the 'total extent' methylation is expressed as nmoles of [14C-methyl]/mg of protein/60 rain, each being measured under appropriately optimized conditions 15. Incubations were at 37 °C. Endogenous methylation is defined as the methylation occurring in the absence of E. coli B. tRNA, while total methylation is defined as the methylation occurring in its presence. The * To whom requests for reprints should be sent.

The postnatal methylation of transfer ribonucleic acid in brain. Evidence for the methylation of precursor transfer ribonucleic acid

“…Hence prokaryotic tRNA species, which are less abundantly methylated than their eukaryotic counterparts (Randerath et al, 1974), have been used as substrates, and, more recently, experimentally hypomethylated eukaryotic tRNA species have also served this purpose (Jank & Gross, 1974;Friedman, 1977). However, since we recently demonstrated the occurrence of homologous tRNA methylation in the brain of the immature rat (Cummins et al, 1975;Salas et al, 1976), it became necessary to investigate to what extent such methylation involves tRNA of mature size as against larger-precursor tRNA species (Munns & Sims, 1975;Majima et al, 1977). The present report presents evidence that indicates that precursor tRNA serves as substrate for a significant portion of the tRNA-methylating reactions taking place in the 3-day-old rat brain.…”

Methionine Sulfoximine In Vivo Modifies the tRNALys Pool of Developing Rat Brain

“…In the present study, we continue to characterize the changes in the tRNA pool of rat brain during early postnatal development, a time when the many tRNA-processing and -modifying enzymes, actively at work (Elahi and Sellinger, 1979), are particularly vulnerable to a variety of environmental influences (Cummins et a]., 1975;Salas et al, 1976Salas et al, , 1977Sellinger et al, 1977;Dainat et al, 1978Dainat et al, , 1979Sellinger and Der, 1981). Our results regarding the tRNA content of rat brain (Table 1) in the 3-to 18-day postnatal period show values of approximately 0.15 mglg (1 A,,, unit = 45 p g of tRNA), matching those previously reported by Harris and Maas (1974).…”

“…Over the past few years, work from our laboratory has shown that immature brain tRNAs are subject to the action of homologous tRNA methyltransferases (Cummins et al, 1975) which convert them into mature tRNA molecules postnatally (Salas et al, 1976;Elahi and Sellinger, 1979), and that this process continues for as long as a month and a half. The analysis of [3H]methyl-tRNAs formed by 3and 18-day-old rat brain revealed 10 different [3H]-N-methyl bases in the newly formed [3H]tRNA Dainat et al, 1978;Elahi and Sellinger, 1979).…”

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

“…Evidence from our laboratory has demonstrated the existence and the time course of formation of precursor tRNA molecules in the 3-day-old rat brain (Elahi and Sellinger, 1979). We have also shown that homologous methylation of brain tRNA (Cummins et al, 1975) persists for up to 1 month after birth (Salas et al, 1976) and that many of the cerebral tRNA methyltransferases continue to alter their substrate recognition characteristics and hence their specificity for as long as 40 days postnatally Dainat et al, 1978;Dainat and Sellinger, 1980).…”