Biochem. J. (1979) 177, 381-384 Printed in Great Britain
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The Postnatal Methylation of Transfer Ribonucleic Acid in Brain EVIDENCE FOR THE METHYLATION OF PRECURSOR TRANSFER RIBONUCLEIC ACID By ELAHE ELAHI* and OTTO Z. SELLINGERt Laboratory of Neurochemistry, Mental Health Research Institute, University of Michigan Medical Center, Ann Arbor, MI 48109, U.S.A.
(Received 17 October 1978) Incubation of 3-day-old rat brain with L-[methyl-3H]methionine resulted in the rapid labelling of low-molecular-weight cytoplasmic RNA. Electrophoresis in 15% polyacrylamide gels provided evidence for the methylation of precursor tRNA molecules, and high-performance liquid chromatography demonstrated N2-methylguanine to be the predominant methylated base formed during the first 2min of labelling. In most studies of tRNA methylation in vitro, eukaryotic tRNA species have proved to be poor substrates for homologous tRNA methyltransferases (Nau, 1976). 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. A preliminary report of some of the findings has appeared (Sellinger et al., 1978).
Experimental Labelling and isolation of low-molecular-weight cytoplasmic RNA The cerebral cortices of 3-day-old Sprague-Dawley rats were dissected out rapidly and minced on ice into fragments of approx. 1 mm3. The mince from two cortices was transferred to a 50ml Erlenmeyer flask containing 15 ml of incubation medium preincubated * Present address: Department of Biochemistry and Biophysics, School of Medicine, University of California, San Francisco, CA 94143, U.S.A. t To whom requests for reprints should be addressed. Vol. 177
at 37°C in an atmosphere of 02/CO2 (19:1). The incubation medium was similar to that used by Judes et al. (1973) and contained, in addition, 1.36g of sodium formate/litre and IOmCi of L-[methyl-3H]methionine (11.25-15.OCi/mmol; New England Nuclear, Boston, MA, U.S.A.; or Research Products International, Elkgrove Village, IL, U.S.A.). The pH of the medium was adjustedto 7.4 with 7.5 % (w/v) NaHCO3. After incubation for 2, 5, 10 or 45 min, the flasks were placed on ice and 10ml of ice-cold Hanks solution (Microbiological Associates, Walkerville, MD, U.S.A.), supplemented with 1 mM-L-methionine, was added. Tissue fragments were collected by centrifugation at 5900g for 7min, the pellet was washed three times with the methionine-supplemented Hanks solution, and then homogenized in 3.5 vol. of 0.25 M-sucrose, containing 35 mM-Tris/HCl, pH 7.4, 25 mM-KCl and 5 mM-MgCl2. The homogenate was centrifuged at 17300g for 10min, and the supernatant was recentrifuged at 160000g for 90min, yielding the high-speed supernatant, which was made 1 % (w/v) in sodium dodecyl sulphate. An equal volume of phenol/m-cresol/water (7:1:2, by vol.) was added, and the RNA extracted by vigorous shaking for 10min at 65°C and 15min at room temperature. The phenol phase was re-extracted at room temperature with 0.5-lvol. of 0.1M-sodium acetate. The combined aqueous layers were reextracted until no interface was detected, and the RNA was precipitated three times by addition of 0.1 vol. of 20% (w/v) potassium acetate, pH5.5, followed by 2.5 vol. of 95 % ethanol.
Electrophoresis Electrophoresis was performed in 10 and 15% (w/v) polyacrylamide gels, which were stained with Methylene Blue (Peacock & Dingman, 1967).
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381
The Postnatal Methylation of Transfer Ribonucleic Acid in Brain EVIDENCE FOR THE METHYLATION OF PRECURSOR TRANSFER RIBONUCLEIC ACID By ELAHE ELAHI* and OTTO Z. SELLINGERt Laboratory of Neurochemistry, Mental Health Research Institute, University of Michigan Medical Center, Ann Arbor, MI 48109, U.S.A.
(Received 17 October 1978) Incubation of 3-day-old rat brain with L-[methyl-3H]methionine resulted in the rapid labelling of low-molecular-weight cytoplasmic RNA. Electrophoresis in 15% polyacrylamide gels provided evidence for the methylation of precursor tRNA molecules, and high-performance liquid chromatography demonstrated N2-methylguanine to be the predominant methylated base formed during the first 2min of labelling. In most studies of tRNA methylation in vitro, eukaryotic tRNA species have proved to be poor substrates for homologous tRNA methyltransferases (Nau, 1976). 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. A preliminary report of some of the findings has appeared (Sellinger et al., 1978).
Experimental Labelling and isolation of low-molecular-weight cytoplasmic RNA The cerebral cortices of 3-day-old Sprague-Dawley rats were dissected out rapidly and minced on ice into fragments of approx. 1 mm3. The mince from two cortices was transferred to a 50ml Erlenmeyer flask containing 15 ml of incubation medium preincubated * Present address: Department of Biochemistry and Biophysics, School of Medicine, University of California, San Francisco, CA 94143, U.S.A. t To whom requests for reprints should be addressed. Vol. 177
at 37°C in an atmosphere of 02/CO2 (19:1). The incubation medium was similar to that used by Judes et al. (1973) and contained, in addition, 1.36g of sodium formate/litre and IOmCi of L-[methyl-3H]methionine (11.25-15.OCi/mmol; New England Nuclear, Boston, MA, U.S.A.; or Research Products International, Elkgrove Village, IL, U.S.A.). The pH of the medium was adjustedto 7.4 with 7.5 % (w/v) NaHCO3. After incubation for 2, 5, 10 or 45 min, the flasks were placed on ice and 10ml of ice-cold Hanks solution (Microbiological Associates, Walkerville, MD, U.S.A.), supplemented with 1 mM-L-methionine, was added. Tissue fragments were collected by centrifugation at 5900g for 7min, the pellet was washed three times with the methionine-supplemented Hanks solution, and then homogenized in 3.5 vol. of 0.25 M-sucrose, containing 35 mM-Tris/HCl, pH 7.4, 25 mM-KCl and 5 mM-MgCl2. The homogenate was centrifuged at 17300g for 10min, and the supernatant was recentrifuged at 160000g for 90min, yielding the high-speed supernatant, which was made 1 % (w/v) in sodium dodecyl sulphate. An equal volume of phenol/m-cresol/water (7:1:2, by vol.) was added, and the RNA extracted by vigorous shaking for 10min at 65°C and 15min at room temperature. The phenol phase was re-extracted at room temperature with 0.5-lvol. of 0.1M-sodium acetate. The combined aqueous layers were reextracted until no interface was detected, and the RNA was precipitated three times by addition of 0.1 vol. of 20% (w/v) potassium acetate, pH5.5, followed by 2.5 vol. of 95 % ethanol.
Electrophoresis Electrophoresis was performed in 10 and 15% (w/v) polyacrylamide gels, which were stained with Methylene Blue (Peacock & Dingman, 1967).
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