[CANCER RESEARCH 27, 646-«S3,April 1967] Utilization of L-Methionine and S-Adenosyl-L-methionine for Methylation of Soluble RNA by Mouse Liver and Hepatoma Extracts1 R. L. HANCOCK The Jackson Laboratory, Bar Harbor, Maine SUMMARY following reaction: ATP + L-methionine = SAM + pyrophos The methyl group of L-methionine or S-adenosyl-L-methionine phate + monophosphate (7). SAM is used in the methylation of sRNA by sRNA methylases in the following reaction: sRNA + is used by nonparticulate mouse liver and hepatoma prepara SAM = methylated sRNA + S-adenosyl-L-homocysteine (10). tions to methylate soluble ribonucleic acid (sRNA). Esclierichia The two enzyme reactions are presented along with a representa coli K12 sRNA was a more active substrate than yeast sRNA. tion of the origin of the unmethylated sRNA (tp-RNA) in Chart Four different lots of E. coli B sRNA gave similar incorporations between lots. "Stripped" and "nonstripped" sRNA had similar 1. An array of RNA methylases exhibiting specificity for par ticular bases and for sRNA from different biologic sources, along amounts of methyl incorporation. Other nucleoside triphosphates with other, less si^ecific RNA methylases, has been described besides adenosine triphosphate were capable of sup]x>rting the incorjioration of methyl groups from L-methionine into sRNA. (11, 15, 16, 17, 22). The most extensively methylated sRNA molecules known have been found in mammary adenocarcinoma The nonspecificity of the nucleoside triphosphate reaction was tissue (3), and recently Mittelman et al. (18) have found RNA shown to be due to an adenosine diphosphokinase reaction and methylase activity to be extremely high in certain viral-induced not to nucleoside tri phosphate: L-methionine nucleosidyl trans- tumor cells. Soluble and ribosomal RNA isolated from HeLa ferase activity. cells cultured in vitro were shown to have a large percentage of Mouse hepatoma BW7756 was found to have lower amounts of methylation activity than mouse liver if L-methionine-14CH3 methylated nucleotides. A large proportion of these methyl groups was believed to be on the ribose moiety (6). was used as the methyl donor, although hepatoma tissue had Studies on the enzymatic pathway of sRNA methylation were over twice as much sRNA methylase activity per se as liver on initiated to examine further the possibility of "hypermethyla- a gm tissue equivalent or protein weight basis. However, em tion" of hepatoma sRNA. In previous studies (12) it was found bryonic liver as well as other embryonic and neoplastic tissues that although mouse liver had a large amount of S-adenosyl- also had increased amounts of sRNA methylase activity over transferase activity, the activity of B\V 7756 mouse hepatoma that of their adult counterparts. was not measurable. The work described in the present report indicates that this mouse hepatoma apparently does possess INTRODUCTION trace amounts of S-adenosyltransferase activity since the labeled The methyl group of methionine is activated through a reac methyl group of L-methionine was found to be transferred to tion catalyzed by adenosine triphosphate: L-methionine S-adeno- sRNA. Furthermore, the methyl group of SAM was used by syltransferase (E.G. 2.4.2.13), hereafter termed S-adenosyltrans- hepatoma cell extracts to methylate sRNA. Experiments with ferase. This enzyme catalyzes the formation of SAM2 by the SAM demonstrate that the BW 7756 hepatoma has twice as much sRNA methylase activity ¡«rgmwet weight or per mg 1This investigation was supported in part by an allocation protein as that found in mouse liver. from USPHS General Research Support Grant SO 1 FR-05545-03 and American Cancer Society Grant IN 19F to the Jackson Lab MATERIALS AND METHODS oratory, Bar Harbor, Maine. 2Abbreviations used are as follows: sRNA, soluble ribonucleic The following chemicals were obtained commercially: hy- acid; rRNA, ribosomal ribonucleic acid; tp-RNA, precursor trans droxylamine HC1 grade I, ATP, salmon sperm DNA, yeast fer ribonucleic acid; t"'-RNA, methylated transfer ribonucleic sRNA, L-methionine, Tris (Sigma, Inc., St. Louis, Mo.) ; Esch- acid; Ar, adenosine; AMP, adenosine monophosphate; ADP erichia coli K12, E. coli B, and yeast ("stripped"3 and "non- adenosine diphosphate; ATP, adenosine triphosphate; dATP, stripped"), rabbit and rat liver sRNA (General Biochemicals, deoxyadenosine triphosphate; UTP, uridine triphosphate; GTP, Inc., Chagrin Falls, Ohio); UTP, GTP, CTP, ITP (Pabst Lab guanosine triphosphate; CTP, cytidine triphosphate; GSH, gluta- oratories, Milwaukee, \Vis.); L-methionine-14CH3 (10.3 me/ thione; SAM, «S-adenosyl-L-methionine; SAH, S-adenosyl-L- homocysteine; SAM-UCH3,jS-adenosyl-L-methionine-uCH3; Tris, mmole) and SAM-14CH3 (50.2 mc/mmole) (New England Nu- tris(hydroxymethyl)aminomethane; ITP, inosine triphosphate; IDP, inosine diphosphate; UDP, uridine diphosphate; NADPH, 3The term "stripped" is used to denote the removal of the reduced nicotinamide-adenine dinucleotide phosphate. amino acid from transfer RNA, usually done by treating the RNA Received August 31, 1966;accepted November 16, 1966. with an alkaline hydrolysis procedure. G46 CANCER RESEARCH VOL. 27 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1967 American Association for Cancer Research. Methylation of Soluble RNA tDNA that the specific activity of the resultant SAM is not known because of SAM present in the supernatant fraction, and there fore these incorporations should not be considered to represent the absolute moles of methyl groups being attached to sRNA tp-RNA molecules. The routine reaction mixture for studies using S-ADENOSYL - SAM-14CHs as a methyl source was as follows: 100 /¿molesof SAM « ATP+ L-METH. TRANSFERASE Tris (pH 7.5), 50 /¿molesofGSH, 30 /¿molesofMgCl2, 5 mg of RNA E. coli K12 sRNA, 0.5 /¿cof SAM-»CH3 (0.499 mc/mmole), METHYLASE and 1.0 ml of 100,000 X g (approximately 250 mg equivalents SAH of tissue) in a total volume of 2 ml. The reaction mixture was incubated for 12 min at 37°C."Soluble RNA methylase activity" t "'-RNA is used in this writing to denote the rate of the sRNA methylase reaction per se, that is, the transfer of methyl groups from SAM CHART 1. Pathway for sRNA methylation. Nonmethylated transfer RNA (tRNA) is synthesized upon a specific DNA tem to sRNA. plate by RNA polymerase. This precursor transfer RNA (t"-RNA) receives its methyl groups from S-adenosyl-L-methionine (SAM) RESULTS via sRNA methylase(s) action resulting in /S-adenosyl-L-homo- cysteine (SAH) and a methylated species of transfer RNA (t'n- Characteristics of the sRNA-methylating Reaction. RNA). SAM is synthesized by ATP:L-methionine (L-meth) The following studies were concerned with the overall reaction S-adenosyl transíerase. ATP, adenosine triphosphate; GTP, gua- that is made up of the S-adenosyltransf erase and sRNA methyl nosine triphosphate; UTP, uridine triphosphate; CTP, cytidine ase reactions. The sRNA methylase activity determined may triphosphate. represent several specific sRNA methylases, as has been found to be the case in bacteria (16, 17). High-speed supernatant frac clear Corp., Boston, Mass.); polyinosinic acid and polyadenylic tions (100,000 X g for 1 hour) of female 129/Rr mouse liver were acid (Miles Chem. Co., Elkhart, Ind.); crystallized bovine albu assayed for their ability to incorporate the methyl groups from min (Pentex, Inc., Kankakee, 111.);yeast D-glucose-6-phosphate, L-methionine-14CH3or SAM-14CH3(Table 1). It was found that NADP oxidoreductase, ATP, and D-hexose-6-phosphotransferase E. coli sRNA served as an excellent substrate (see section on (Boehringer and Soehne, Mannheim, Germany). ]K>lymerspecificity). The reaction rate was linear for a 15-minute The 129/Rr and C57L/J strains of mice and BW 7756 hepa- period (Chart 2A), and incoqjoration of methyl groups was de toma, indigenous to C57L/J mice, were purchased from the Jack pendent upon E. coli K12 sRNA, although a small amount of son Laboratory. AXC and Buffalo strains of rats, and Buffalo endogenous activity was present in all enzyme preparations rats carrying the R3B (3rd generation) and AXC rats carrying (Table 2). Such endogenous activity was undoubtedly due to the the transplantable H35 hepatoma (30th generation) were kindly presence of mouse liver sRNA in crude supernatant fractions. donated for this work by Dr. Melvin D. Reuber of the Depart The reaction rate was linear over a range of sRNA concentra ment of Pathology at the NIH, Bethesda, Maryland. tions from 0 to about 7 mg. Although larger incoq>orations could Race III New Zealand white rabbits were procured from the have been obtained with 10 mg of sRNA, a concentration of 5 Hamilton Station section of the Jackson Laboratory. mg of sRNA was selected for reasons of economy (Chart 2B). iS-Adenosyltransferase was assayed according to the method The reaction rate increased linearly with increasing amounts of of Cantoni and Durell (8) and the sRNA methylase activity by enzyme up to about 25 mg of protein (Chart 2(7)- The optimal the method of Srinivasan and Borek (22). Protein determinations concentration of reduced GSH was 0.025 M. The portion of the were done by the spectral method of Warburg and Christian reaction catalyzed by S-adenosyltransferase reaches substrate (23) and the biuret method (9). All enzyme preparations were saturation at about 20 m\i of L-methionine (12). As can be seen from fresh tissues. Unless otherwise stated, the enzyme prepara in Chart 2£>,theoverall reaction appears to become saturated tions were obtained from female 129/Rr mouse liver. The en with L-methionine at the 0.1-mM range and therefore certainly zyme preparation was made by homogenizing 1 gm of fresh liver for 8 strokes in a glass homogenize!1in 4 volumes of a solution TABLE 1 containing 0.25 M sucrose and 0.01 M MgCl».The homogenate Mouse Liver Soluble RNA (sRNA) Methylase Activity was centrifuged for 15 minutes at 10,000 X g and then at 100,000 time "CHi6 X g for 1 hour and used immediately.