Partial purification of a ribosomal ribonucleic acid methylase from rat liver nuclei and methylation of undermethylated nuclear ribonucleic acid from regenerating liver of ethionine-treated rat

Biochimica etBiophysicaActa, 740(1983) 29-37

29

Elsevier

BBA91205

PARTIAL PURIFICATION OF A RIBOSOMAL RIBONUCLEIC ACID METHYLASE FROM RAT LIVER NUCLEI AND METHYLATION OF UNDERMETHYLATED NUCLEAR RIBONUCLEIC ACID FROM REGENERATING LIVER OF ETHIONINE-TREATED RAT T H U N G WEN LONG, HIROBUMI TERAOKA and KINJI TSUKADA *

Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, KandasurugadaL Chiyoda- ku, Tokyo 101 (Japan) (Received December 17th, 1982)

Key words: rRNA methylase; RNA methylation; Ethionine treatment; Liver regeneration," (Rat)

S-Adenosylmethionine-dependent ribosomal RNA (rRNA) methylase has been purified approx. 90-fold from rat liver muclei. The partially purified methylase catalyzes the methylation of base and ribose in hypomethylated nuclear rRNA prepared from the regenerating rat liver after treatment with ethionine and adenine. The enzyme has an apparent molecular weight of about 3" 104 and a sedimentation coefficient of 3.0 S. The enzyme is optimally active at pH 9.5 and sensitive to p-chloromercuribenzoate. Thiol-protecting reagents, such as dithiothreitol, are necessary for its activity, and the enzyme requires no divalent cations for its full activity. This enzyme did not efficiently transfer the methyl group to nuclear rRNA from normal rat liver, compared with hypomethylated nuclear rRNA. Methyl groups were mainly incorporated into pre-rRNA larger than 28 S, and the extent of 2'-O-methylation of ribose by this enzyme was greater than that of base methylation in the hypomethylated rRNA. No other nucleic acids, including transfer RNA (tRNA) and microsomal RNA from normal as well as ethionine-treated rat livers, tRNA from Escherichia coli, yeast RNA, and DNA from rat liver and calf thymus, were significantly methylated by this methylase. These results suggest that partially purified rRNA methylase from rat liver nuclei incorporates methyl groups into hypomethylated pre-rRNA from S-adenosylmethionine.

Introduction In mammalian cells, newly synthesized pre-rR N A is methylated at specific sequences before it

is processed to cytoplasmic rRNA [1,2]. Methylation of pre-rRNA occurs on both specific bases and ribose moieties following transcription, and it seems to play an essential role in ribosomal function [3,4]. In particular, 2'-O-methylation of nuclear precursor 45 S rRNA in nucleolus determines the specificity of the maturation processes

* To whom correspondence should be addressed. 0167-4781/83/$03.00 © 1983 Elsevier Science Publishers B.V.

to mature 28 and 18 S rRNA [5-7]. Most methylation of pre-rRNA has been reported to be in the 2'-O-methylation of the ribose [8,9]. However, the isolation and purification of rRNA methylase which modified pre-rRNA by transfer of the methyl group from S-adenosylmethionine have not yet been accomplished so far. In this paper, we prepared the hypomethylated nuclear rRNA from regenerating rat liver after treatment with ethionine and adenine. So, we purified rRNA methylase from rat liver nuclei using this hypomethylated nuclear rRNA as substrate. The partially-purified methylase modified base moiety as well as ribose moiety of pre-rRNA.

30 Materials and Methods

Chemicals Reagents were obtained as follows: S-adenosylL-[methyl-3H]methionine (60 Ci/mmol) and [53H]uridine (2 Ci/mmol) from the Radiochemical Centre (Amersham, U.K.); S-adenosylmethionine, Escherichia coli tRNA (MRE 600), ATP, UTP and 2'-deoxy-guanosine from Boehringer (Mannheim, F.R.G.); E. coli alkaline phosphatase and snake venom phosphodiesterase from Worthington (Freehold, U.S.A.); E. coli RNA polymerase from Alpha Therapeutic Corp. (Los Angeles, U.S.A.); N6-methyladenosine, calf thymus DNA (type 1), yeast RNA (type VI), crystalline bovine serum albumin and S-adenosyl-L-homocysteine from Sigma Chemical Company (St. Louis, U.S.A.); 2'deoxycitidine, 2'-deoxyadenosine and the reagents for polyacrylamide gel electrophoresis from Nakarai Chemicals (Kyoto, Japan); DL-ethionine from Wako Chemicals (Osaka, Japan); CTP and GTP from Yamasa Shoyu (Tokyo, Japan); adenine from Kohjin (Tokyo, Japan); DEAE-cellulose (DE 52) and DEAE-cellulos paper discs (DE 81) from Whatman (Kent, U.K.); DEAE-Sephadex A-25, Sephacryl S-200 superfine and Blue-Sepharose CL-6B from Pharmacia (Uppsala, Sweden); Dowex AG 50W-X8 (Hydrogen form, 100-200 mesh) from Bio-Rad Laboratories (Richmond, U.S.A.); Celite545 diatomaceous earth from John-Manville Products Corp. (New York, U.S.A.). All other chemicals used were of analytical grade. Animals Male Wistar rats (170-200 g body weight) were obtained locally and given laboratory chow and water ad libitum, unless otherwise specified. Preparation of hypomethylated nuclear rRNA substrate Rats were given dosages of Dt-ethionine (200 mg/kg body weight) plus adenine (120 mg/kg body weight) intraperitoneally 24 h before partial hepatectomy and at the operation. Partial hepatectomy refers to the removal of about 70% of the liver as described by Higgins and Anderson [10]. Rats were killed by decapitation 20 h after partial hepatectomy, and livers were removed rapidly and kept on ice. The livers were homogenized in 4 vols.

50 mM Tris-HCl (pH 7.8)/0.25 M sucrose/25 mM KC1/5 mM MgC12 with a glass-Teflon homogenizer. The homogenate was centrifuged at 10 000 × g for 10 min. The sediment was suspended well in 10 vols. of 2.3 M sucrose containing 3.3 mM MgC12 by homogenization. The suspension was centrifuged at 40000 × g for 60 min and the pellets were obtained. Nuclear RNA from the pellets was isolated by the hot SDS-phenol method according to Steele et al. [11]. The nuclear rRNA precursor was purified from the nuclear RNA by methylated Kieselguhr column according to the procedure by Osawa and Shibatani [12].

Assay of rRNA methylase The standard reaction mixture (100 ~1) contained 50 mM glycine-NaOH (pH 9.5)/50 mM KC1/2.6 M dithiothreitol/4 ~M S-adenosyl-L[methyl-3H]methionine (0.25 /.tCi)/40 #g hypomethylated nuclear rRNA as the methyl acceptor. After incubation for 60 min at 37°C, the reaction mixture was chilled in ice bath, and was spotted on DEAE-cellulose paper disc (DE 81). The disc was washed three times by dipping it in 0.2 M ammonium bicarbonate as described by Rubin and Modrich [13], and then in ethanol. After drying the disc at 80°C for 10 min, the radioactivity on the paper was counted in 10 ml of toluene-based scintillation fluid by a Packard Tri-Carb liquid scintillation spectrometer. One unit of the enzyme was defined as that amount of enzyme that catalyzed the transfer of 1 nmol of methyl group into rRNA per 60 min in the reaction mixture. Purification of rRNA methylase Preparation of the crude enzyme fraction. Rat liver nuclei were prepared as described previously [ 14]. The nuclei from 50 g of livers were suspended in 25 ml of 10 mM Tris-HC1 (pH 7.5) containing 30% (v/v) glycerol/5 mM MgC12/5 mM dithiothreitol/0.3 M (NH4)2SO 4. After the sonication of the nuclei in Branson sonifier at full output in 10 s bursts, monitoring for complete nuclear breakage was done (approx. 60 s). The suspension was centrifuged at 105 000 × g for 60 min to obtain the nuclear extracts. The extracts were dialyzed against 10 mM Tris-HCl (pH 7.5)/10% glycerol/2 mM EDTA/4 mM 2-mercaptoethanol (Buffer A) for 4 h.

31

Acetone fractionation. The dialyzed extract was centrifuged to remove resulting precipitate and was brought to 27% (v/v) concentration with acetone ( - 2 0 ° C ) . The solution was centrifuged to remove the precipitate. Acetone was added to the supernatant to bring the final concentration to 40% (v/v). The suspension was centrifuged to obtain the precipitate. The pellet was dissolved in 20 ml of Buffer A, and the solution was brought to 70% saturation with ammonium sulfate by stirring for 30 min. The precipitate collected by centrifugation was dissolved in 2 ml of Buffer A and dialyzed against 1 liter of the same buffer containing 0.1 M KC1. Sephacryl S-200 gel filtration. The dialysate was applied to a column of Sephacryl S-200 (1.3 x 70 cm) previously equilibrated with Buffer A containing 0.1 M KCI. The enzyme was eluted with the same buffer and 1.5 ml fractions were collected. Active fractions (12 ml) were pooled and dialyzed against Buffer A. DEAE-cellulose column chromatography. The dialysate was applied to a column of DEAE-cellulose (DE 52) (1.3 × 5.0 cm) previously equilibrated with Buffer A. After the column was washed with 3 vols. of Buffer A, the enzyme was eluted with 40 ml linear KCI gradient to 1.0 M in Buffer A. Fractions of 1.6 ml were collected. The active fractions (6.4 ml) were combined and dialyzed against Buffer A. Blue-Sepharose colum chromatography. The dialysate was applied to a column of Blue-Sepharose CL-6B (0.8 × 4 cm) previously equilibrated with Buffer A. The column was washed with 5 ml of the same buffer. The flow-through fractions (9 ml) were collected, dialyzed against Buffer A containing 50% glycerol and stored at - 2 0 ° C . Preparation of methyl-labeled rRNA To determine the methylated sites in hypomethylated and control nuclear rRNA, the standard reaction mixtures were scaled up 10-fold. After incubation, the methyl-labeled r R N A was extracted with water-saturated phenol containing 0.1% hydroxyquinoline. After the centrifugation, 3H-labeled rRNA was precipitated from the aqueous phase by adding 2.5 vols. of ethanol and was left overnight at - 2 0 ° C . Resulting precipitate (8 absorbance units at 260 nm) was washed twice

with 95% ethanol at 4°C and used for the following experiments.

DEAE-Sephadex A-25 column chromatography of alkaline hydrolysate of the methylated rRNA 3H-Labeled nuclear rRNA was hydrolyzed in 0.5 ml of 0 . 3 M KOH at 37°C for 18h. The alkaline hydrolysate was neutralized with 1 N HC104 and centrifuged at 2000 x g at 4°C after being kept in ice for 20 min. The materials were adjusted to 10 ml of 20 mM Tris-HC1 (pH 7.5)/7 M urea/0.1 M NaCI (Buffer B) and applied to a DEAE-Sephadex A-25 column (1 X 25 cm) equilibrated with Buffer B at room temperature. Mono- and oligonucleotides were eluted with 60 ml linear NaC1 gradient (0.1-0.7 M) in Buffer B [15]. The elution positions of mono- and oligonucleotides were determined as described previously [16]. Fractions of 0.6 ml were collected, and an aliquot of each fraction was counted in 10 ml of toluene-Triton X-100 scintillation fluid.

Cation exchange column chromatography on acid hydrolysate of the methylated nuclear rRNA The patterns of methylation in the four major ribonucleotide bases were determined from acid hydrolysate as described by Gantt et al. [17]. 3HLabeled nuclear rRNA was hydrolyzed in 0.5 ml of 1 N HC1 for 30 min at 100°C. The aliquots of the sample containing 2'(Y)-UMP, 2'(3')-CMP, guanine and adenine, added as markers, were applied to a column (0.5 x 60 cm) of AG 50W-X8 cation exchange resin. Flow rate was maintained at 12 m l / h with Gilson minipuls 2. The column was run with ammonium acetate buffer (pH 4.1) (1 M ammonium acetate (1 liter)/glacial acetic acid (350 ml)/water (50 ml)). The eluate was monitored at 254 nm. Fractions of 1 ml were collected and counted in 10 ml of toluene-Triton X-100 scintillation fluid.

Partition chromatography of 2'-O-methyl ribonucleosides from the methylated rRNA To isolate 2'-O-methyl ribonucleosides, 3Hlabeled nuclear rRNA was enzymatically hydrolyzed and fractionated with a partition column of diatomaceous earth as described by Hall [18]. The alkaline hydrolysate from 3H-methylated nuclear rRNA was neutralized with 1 N HC104, as de-

32 scribed above, and lyophilized. The lyophilized samples were dissolved in 0.1 ml of 50 mM TrisHCI (pH 8.5) containing 5 mM MgCI/10 ~g alkaline phosphatase/20 ~g snake venom phosphodiesterase, as described by Glazer and Peale [19]. After incubation for 2 h at 37°C, the samples were lyophilized again. The lyophilized samples were analyzed by the column. Partition column was prepared as described by Hall [20]. Hydrated sodium borate (Na2B40 7 • 10H20; 38 g) was dissolved in 1 1 of water at 50°C and left standing overnight at room temperature. To the filtered solution, 5 ml of concentrated ammonium hydroxide and 3 1 1-butanol were added. After shaking well, the biphasic solutions were separated to the upper and lower phases. A column (1.2 × 40 cm) was dry-packed with 20 g of Celite-545 which had been mixed thoroughly with 9.2 ml of the lower phase. The lyophilized samples were dissolved in 1 ml of the lower phase (the pH was adjusted to a value between 8.2 and 8.6). After the addition of nucleosides as markers, the solution was mixed thoroughly with 2.15 g Celite-545. The free-flowing mixture was packed on the top of the column. The column was eluted with the upper phase of the solvent system at a flow rate of 30 ml/h. 2'-O-Methyl[3H]uridine as a marker was prepared from [3H]UMP-labeled methylated RNA. The reaction mixture (2 ml) containing 100 mM Tris-HC1 (pH 8.5), 2.5 mM MgC12, 2 mM MnCI 2, 6 mM NaF, 50 mM (NH4)2SO 4, 10 mM dithiothreitol, 0.33 mM ATP, CTP and GTP, 0.15 m M / 1 0 /tCi [3H]UTP, 10 /~M S-adenosylmethionine, 0.5 mg nucleolar chromatin, 0.1 mg partially purified rRNA methylase (DEAE-cellulose step) and 50 U E. coli RNA polymerase (Boehringer) was incubated at 37°C for 60 rain. Alkaline hydrolysis of [3H]RNA product, isolation of alkaline-resistant oligonucleotides, and the enzymatic digestion to 2'-O-methyl[3H]uridine were c a r r i e d o u t as d e s c r i b e d a b o v e . 2 ' - 0 Methyl[3H]uridine was eluted as a single peak between N6-methyladenosine and adenine (see Fig. 6).

Acrylamide gel electrophoresis of methylated rRNA Polyacrylamide gel electrophoresis of methylated rRNA was carried out according to the method of Loening [21] with a constant current of 5 m A / t u b e (0.5 × 6 cm) at 4°C. The methylated

rRNA samples were dissolved in 50 /~1 of electrophoresis buffer (0.04 M Tris base/0.02 M sodium acetate/2 mM EDTA, pH 7.8) containing 5% (w/v) sucrose and layered over the gel. Electrophoresis was performed at 5 mA per gel for I h. The gel was scanned for absorbancy with a Gilson Holochrome recording spectrophotometer. Gels containing radioactivity were subsequently frozen and sliced in 1 mm sections in the bath of hexane by the addition of dry ice. Gel slices were heated at 60°C in 0.5 ml of 10% (v/v) piperidine in scintillation vials for several hours and allowed to dry. Then, 0.5 ml of water was added followed by addition of 10 ml of toluene-Triton X-100 scintillation fluid.

Preparation of nucleic acids Rat liver tRNA was isolated from the ' p H 5 enzymes' fraction by extraction with phenol according to the method of Moldave [22]. RNA from rat liver microsomes was isolated by the SDS-phenol method as described by Moldave [23]. D N A from rat liver nuclei was prepared by the method of Savitsky and Stand [24].

Analytical gel filtration Analytical gel chromatrography of the purified enzyme was carried out at 4°C on a column of Sephacryl S-200 (1.3 × 70 cm) equilibrated with 10 mM Tris-HC1 (pH 7.5) containing 5 mM 2mercaptoethanol/2 mM EDTA/0.1 M KC1. The apparent molecular weight of the enzyme was determined according to Andrews [25]. Marker proteins with the following molecular weight were used: catalase ( M r 240 000); lactate dehydrogenase ( M r 140000); cytochrome c ( M r 12 300).

Sucrose density gradient centrifugation The purified enzyme was sedimented in a 4.8 ml 5-20% (w/v) sucrose gradient in 10 mM Tris-HC1 (pH 7.5) containing 5 mM 2-mercaptoethanol/2 mM EDTA/0.1 M KC1 at 40 000 rev./min, and at 2°C for 20 h in a Hitachi SW50 rotor. Lactate dehydrogenase (s20,w = 7 S), bovine serum albumin (s20,w = 4.4 S) and cytochrome c (s20,w = 1.7 S) were used as external marker proteins.

Protein determination The protein concentration was determined by the method of Bradford [26] using crystalline

33

bovine serum albumin as standard. × 2 E

Results

f

..9 u

Purification of nuclear rRNA methylase Table I summarized the typical purification of the rRNA methylase. The enzyme was purified from the crude enzyme extracts by about 90-fold with a yield of 12%. The partially-purified enzyme was stable in Buffer A containing 50% glycerol at - 2 0 ° C for at least a month. Under the standard assay conditions, the methylase activity was found to be proportional to the amounts of purified enzyme.

Fig. 1. rRNA methylase activity as a function of pH. rRNA methylase activity was determined by using buffers indicated at a final concentration of 50 mM. O, Tris-maleate.for pH 6-7; o, Tris-HCl for pH 7-9; zx, glycine-NaOH for pH 9-10.

General properties

with an apparent K i value of 5/~M (Fig. 2B).

The pH optimum for the nuclear rRNA methylase was assayed at pH 6.0-10.0 under the standard assay conditions. The enzyme was rather active at the alkaline range and showed optimum pH at about 9.5 (Fig. 1). The enzyme activity at pH 7.5 was about 50% of the maximal activity. Divalent cations were not required for the enzyme activity. No significant inhibition of the activity was observed in the range of concentration from 2 to 12 mM Mg 2+. The enzyme was completely inhibited by 0.2 mM p-chloromercuribenzoate, and dithiothreitol, a thiol-protecting reagent, was essential for the full activity of rRNA methylase, indicating that SH groups may be involved in the enzyme activity. Double-reciprocal plots of initial rate of the methylase at varying concentrations of S-adenosylmethionine (0.5-7.5 #M) exhibited a series of straight lines, giving a K m value of 6.6 /~M (Fig. 2A). S-Adenosylhomocysteine, the reaction product, strongly inhibited rRNA methylase activity

o

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6

i

7

i

8 pH

I

9

1

10

Molecular weight of the rRNA methylase To determine the molecular weight of the enzyme, the purified enzyme was subjected to analytical gel filtration on a column of Sephacryl S-200. The molecular weight of the enzyme was estimated to be about 30 000. By sucrose density gradient centrifugation, the sedimentation coefficient of the enzyme was shown to be 3 S.

Substrate specificity As shown in Table II, methyl-acceptance activities of RNAs and DNAs from various sources were examined. Administration of ethionine into animals caused both tRNA and nuclear rRNA extensively to undermethylate [5,27-29]. Hepatic nuclear rRNA from ethionine-treated rats (hypomethylated nuclear rRNA) was efficiently methylated by the partially-purified methylase, compared with that from normal rat as well as tRNA from ethionine-treated rat liver. Other nucleic acids in-

TABLE I PURIFICATION OF rRNA METHYLASE FROM RAT LIVER NUCLEI Step

Total protein (nag)

Total activity (U)

Specific activity (U/mg)

Yield (%)

Crude extract

45.0 8.1 i.5 O.15 0.059

15.3 6.7 3.0 2.2 1.8

0.34 0.82 2.0 14.7 30.5

100 44 20 14 12

Acetone fractionation Sephacryl S-200 DEAE-cellulose Blue-Sepharose

34

TABLE II 30

SUBSTRATE SPECIFICITY F R O M RAT LIVER NUCLEI

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6

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20

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40

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,

60

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rRNA

METHYLASE

Methyl-acceptance activity of nucleic acids (40 #g) was determined under the standard assay conditions. Methyl-group incorporation is expressed as percentage relative to the level of methylation in the presence of ethionine-treated rat liver nuclear rRNA as an acceptor.

80 Acceptors

Fig. 2. Effect of S-adenosylmethionine concentration on reaction rate (A) and inhibition of transmethylation by S-adenosylhomocysteine (B). rRNA methylase was assayed under the standard assay conditions, except that S-adenosylmethionine and S-adenosylhomocysteine concentrations were variable as indicated. Insets show the double-reciprocal plots of the initial velocity as a function of S-adenosylmethionine or S-adenosylhomocysteine concentration. Ado-Met, S-adenosylmethionine; Ado-Hcy, S-adenosylhomocysteine.

cluding microsomal rRNA from normal and ethionine-treated rat liver, tRNA from normal rat liver, E. coli tRNA, yeast RNA, and DNA from rat liver and calf thymus were inactive as methylgroup acceptors. The addition of 0.1 /~g RNAase abolishes methylation of hypomethylated nuclear rRNA, whereas 0.5/~g DNAase was without effect (Table II). To analyze the distribution of labeled methyl groups incorporated into hypomethylated and control nuclear rRNA species, the methylated nuclear rRNAs were analyzed by polyacrylamide gel electrophoresis and their methyl acceptance patterns were examined. Fig. 3 shows the polyacrylamide gel electrophoresis profile of nuclear 3H-methylated rRNA, which had been prepared from the hypomethylated nuclear rRNA and Sadenosylmethionine by the methylase. A large proportion of radioactivity incorporated into hypomethylated nuclear rRNA was detected in RNA molecular species larger than 28 S. In control nuclear rRNA, the methylation of these molecular species was found to be considerably lower than that of methyl-deficient pre-rRNA. This result is in line with previous observations [4,30]. Determination of the methylated sites in hypomethylated nuclear rRNA by rRNA methylase The 3H-methylated nuclear rRNA, which had been prepared from hypomethylated rRNA using

Methyl-group incorporation

Ethionine-treated rat liver Nuclear rRNA + RNAase (0.1 /Lg) + DNAase (0.5/xg) Microsomal rRNA tRNA Normal rat liver Nuclear rRNA Microsomal rRNA tRNA

27 0 2

E. coli tRNA Yeast RNA Rat liver nuclear D N A Calf thymus DNA

is

100 0 98 0 17

2 2 0 0

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': II

.~

v

,, ,

,~t

,,

i '

,

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0 2 4 6 Distance from origin(cm)

Fig. 3. Electrophoresis of rat liver nuclear rRNA labeled with S-adenosyl-L-[methyl-3H]methionine. rRNA was incubated with rRNA methylase in ! ml of the reaction mixture as described in Materials and Methods. ( ), 3H-labeled hypomethylated nuclear rRNA; ( . . . . . . ), 3H-labeled control rRNA. Electrophoresis was carried out as described in Materials and Methods. Rat liver microsomal rRNA (28 and 18 S) and E. coli tRNA (4 S) were added as internal markers.

35 I

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20 40 60 80 FroctiOn number (0.6 m l / f r o c t i o n )

0

100

Fig. 4. DEAE-Sephadex chromatography of alkaline hydrolysate of 3H-labeled hypomethylatednuclear rRNA. Hydrolysis of 3H-labeledhypomethylatednuclear rRNA in 0.3 N KOH and DEAE-Sephadexcolumn chromatographywere performed as described in Materials and Methods. Mono, Di, Tri and Tetra show the elution positions of each nucleotide. S-adenosyl-L-[3H]methionine and the enzyme, was digested in 0.3 N K O H at 37°C for 18 h. The alkaline hydrolysate was fractionated on a DEAE-Sephadex A-25 column in the presence of 7 M urea. The incorporated methyl groups were detected with 65% recovery of the radioactivity in oligonucleotides and 35% in mononucleotides (Fig. 4). These results suggest that the rRNA methylase from rat liver nuclei can methylate both the ribose and base moiety [31]. Next, we examined the site-specificity of methylation on base and ribose in the undermethylated nuclear rRNA. The patterns of methylation in the four major bases were observed on cation exchange chromatography of acid hydrolysates from the SH-methylated nuclear rRNA (Fig. 5). The 1st and 2nd peaks of radioactivity correspond to the

b

x

E

d

I

Z J_ 0

10 20 30 40 50 Froction number (1 m l / f r o c t i o n )

Fig. 5. Elution profile of methylatedpurine bases and pyrimidine nucleotides from 3H-labeled hypomethylated nuclear rRNA on cation exchange chromatography,a, 2'(3')UMP derivatives; b, 2'(3')CMP derivatives; c, guanine; d, adenine.

x

E

a u v

E

°c 25 50 75 100 125 150 175 FrQction number ( l m l / f r Q c t i o n )

Fig. 6. Isolationof 2'-O-methylribonucleosidesfrom 3H-labeled hypomethylated nuclear rRNA on partition column chromatography. Deoxyadenosine (a), deoxyguanosine (c), deoxycytidine(b), Nr-methyladenosine(d) and adenosine(f) were used as internal markers and 2'-O-methyl-[3H]uridine(e) as an external marker with Nr-methyladenosineand adenosine. pyrimidine nucleotides, 2'(Y)UMP derivatives followed by 2'(3')CMP derivatives. The remaining 2 radioactive peaks were methylated derivatives of guanine and adenine in the order of elution as described by Gantt et al. [17]. T-O-Methylribonucleosides from the 3H-methylated rRNA were separated by partition column chromatography (Fig. 6). The radioactivities in the fractions corresponding to the 2'-deoxyadenosine, T-deoxycytidine and 2'-deoxyguanosine appeared to be 2'-O-methyladenosine, 2'-O-methylcytidine and 2'O-methylguanosine, respectively (see Ref. 20). The radioactivity between N6-methyladenosine and adenosine was determined to be 2'-O-methyluridine (see Methods). A radioactive peak eluted just before N6-methyladenosine always appeared, but it was not identified. Since Hall [20] described that 2'-O-methylguanosine was eluted at two positions on the chromatograph, the radioactive compound may be additional 2'-O-methylguanosine. From these results, it seems that all four 2'-O-ribose sites of undermethylated nuclear rRNA were methylated by the partially-purified rRNA metho ylase. Discussion

The present study has demonstrated that ethionine, a hepatocarcinogen, effectively pro-

36 duces undermethylated nuclear rRNA in regenerating rat liver. By using the hypomethylated nuclear rRNA as a substrate, rRNA methylase was purified 90-fold from rat liver nuclei (Table I). An optimum pH for the enzyme activity is shown to exist in the alkaline range. The methylase activity did not require Mg 2+. This seems comparable with DNA methylase [32,33]. In contrast, tRNA methylase from several eukaryotes are reported to be stimulated by Mg 2+ [34-36]. rRNA methylase required dithiothreitol, a thiol-protecting reagent, for the full activity, and was reversibly inhibited by p-chloromercuribenzoate. An apparent K m value for S-adenosylmethionine was 6.6 t~M, which is comparable with that for tRNA methylase so far reported [37]. S-Adenosylhomocysteine, the reaction product was a potent inhibitor for rRNA methylase. The poor methyl-acceptance activity of normal nuclear rRNA is presumably because those methylation sites have been saturated in vivo (Table II). The poor degree of methylation of the ethionine-treated tRNA thus obtained could clarify that the methylase is involved in the modification of specific sites in ribosomal precursor (see Ref. 38). Ethionine-treated microsomal rRNA preparation was inactive as a methyl acceptor (Table II), suggesting that there is really no hypomethylated rRNA in the cytoplasm. The undermethylation of pre-rRNA has been reported to result in a blockage of ribosome production [5-7,30]. During the cycloleucine treatment in mammalian cells, however, a low amount of undermethylated 28 and 18 S rRNA can be processed from undermethylated 45 S RNA and transferred to the cytoplasm [6]. In the presence of ethionine as well, there may be a considerable amount of undermethylated 28 and 18 S rRNA in the cytoplasm. If this were the case, no methyl-acceptance activity in the ethionine-treated microsomal rRNA would suggest a possibility that the methylase could not recognize methylation sites of undermethylated 28 and 18 S rRNA after processing. Obara et al. [39,40] have reported that rRNA (cytosine-5)-methyltransferase was purified 5000fold from the nucleolar extracts of mouse Ehrlich ascites tumor cells with the use of E. coli RNA as the methyl acceptor. The enzyme preferentially methylates E. coli tRNA as well as rRNA. AI-

though the enzyme has been purified from nucleoli of Ehrlich tumor cells, it remains obscure that the enzyme is specific for rRNA base-methylation in vivo. Recently it has been reported that the incorporation of methyl groups into RNA in the isolated nuclei was stimulated 3-6-fold by the addition of cellular protein extract [41]. The increased methylation of RNA was mostly due to the methylation of 2'-hydroxy group of ribose and base of nuclear RNA larger than 28 S. In the present study, we did partially pu~fy rRNA methylase from rat liver nuclei, and show that this enzyme modified base as well as ribose of undermethylated nuclear rRNA. At present it is not known whether a single species of the methylase modifies both base and ribose of nuclear pre-rRNA or not. The methylation of hypomethylated nuclear rRNA by the enzyme was found in ribose moiety more than in base moiety (Fig. 4). Although these results are similar to those found in vivo [4,41], we have not yet obtained the evidence that the methylated nucleotides in the hypomethylated nuclear rRNA in vitro are the same as those methylated in vivo. When methylated bases in nuclear rRNA were analyzed by cation exchange resin column, the major radioactive components were eluted with 2'(3')CMP derivatives and methylated adenine bases, the minor one with 2'(3')UMP derivatives and methylated guanine bases (Fig. 5). The methylation pattern seen was similar to that found on HeLa "cell rRNA, where the site of mononucleotide methylation was primarily on 6,6-dimethyladenosine and 5-methylcytosine for 18 S rRNA, and on 5-methylcytosine and 6-methyladenosine for 28 S rRNA [4,38]. Complete resolution of the four 2'-O-methylribonucleosides has been achieved on a partition column chromatography. The partially-purified enzyme recognized all four 2'-O-ribose sites in methyl-deficient nuclear pre-rRNA from liver treated with ethionine and adenine, and 2'-O-ribose sites of pyrimidine nucleosides were predominantly methylated compared with those of purine nucleosides (Fig. 6). r R N A of several vertebrates yield similar fingerprints of methylated oligonucleotides [42], and the frequencies of methylation sites in different parts of nucleotide sequences are somehow related to the ribosome function [38]. So, further

37 s t u d i e s are n e c e s s a r y to d e t e r m i n e w h e t h e r t h e s e sites u n d e r p o t e n t u n d e r m e t h y l a t i o n are i n v o l v e d in the p r o c e s s i n g o f p r e - r R N A . Acknowledgement T h i s w o r k was s u p p o r t e d in p a r t b y a G r a n t - i n A i d for C a n c e r R e s e a r c h f r o m the M i n i s t r y o f Education, Science and Culture of Japan.

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