Methylation of yeast tRNAAsp by enzymes from cytoplasm, chloroplasts and mitochondria of Phaseolus vulgaris

332

Bmchimica et Biophysica Acta, 374 (1974) 3 3 2 - - 3 4 1 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 98147

M E T H Y L A T I O N O F YEAST t R N A Asp BY ENZYMES FROM CYTOPLASM, C H L O R O P L A S T S AND M I T O C H O N D R I A OF PHASEOLUS VULGARIS

E V E L Y N E G. DUBOIS*, G U Y D I R H E I M E R and J A C Q U E S H. WEIL Institut de Biologie Moldculaire et CeUulaire, Universitd Louis Pasteur,15, rue Descartes, Esplanade, Strasbourg (France)

(ReceivedMay 27th, 1974)

Summary

Pure yeast t R N A Asp was methylated in vitro in the presence of S-adenosyl[Me -I 4C]methionine and enzymatic preparations from plant cytoplasm, chloroplasts or mitochondria. In order to determine the sites methylated by the three different enzymatic preparations, [Me -I 4C] t R N A A~ was hydrolyzed by TI or pancreatic ribonuclease and the resulting oligonucleotides were fractionated by DEAE-cellulose chromatography in the presence of 7 M urea or by high-voltage electrophoresis. The radioactive oligonucleotides were further analyzed using the classical techniques of nucleotide sequence determination. Cytoplasmic t R N A methylases transform into m ~ A the second adenylic residue in the T--d/--C--A--A--U sequence located in the TXp loop. This site is also methylated by the chloroplast and by the mitochondrial enzymes, b u t these enzymes also transform into m ~ A the adenylic residue located at Position 7 from the 5' end of the t R N A A~ molecule. It is the first time that a methylated A is found in this area of a t R N A molecule, between the 5' end and the stem of the hU loop. Our results demonstrate the existence of an organelle~ specific t R N A methylase which is n o t present in the plant cytoplasm.

Introduction In higher plants, three protein-synthesizing systems coexist within the same cell; that of the cytoplasm, of the chloroplasts and of the mitochondria. The organelles have a certain degree of autonomy and contain some specific t R N A s and aminoacyl-tRNA synthetases [1--3]. In t R N A s there are minor nucleotides which result from post-transcriptional enzymatic modifications of the polynucleotide chain; methylation of the bases is one of the most frequent * Asplrante du FNRS, Laboratoire de Cytog~n~tique, Unive~sit4 Catholique de Louvain, Belgique.

333 modifications, and is catalyzed by a variety of tRNA methylases using S-adenosylmethionine as a donor of methyl groups [4]. We have compared the tRNA methylases of the cytoplasm, the chloroplasts and the mitochondria of Phaseolus vulgaris in order to find out whether the presence of specific tRNA methylases could be characterized in plant organelles. Usually tRNAs are fully methylated in vivo and can only be overmethylated in vitro by heterologous enzymes. We have, therefore, decided to study the methylation of yeast tRNA by the enzymes from the various plant cell compartments, but rather than using total yeast tRNA which is a mixture of over 50 different substrates, we have used pure yeast tRNA A~ . The nucleotide sequence of this tRN.~ has been determined by Keith et al. [5] and Gangloff et al. [6] ; it contains only two methylated nucleotides in addition to the T present in the G--T--~--C sequence found in most tRNAs. The use of a pure tRNA of known sequence made it possible to determine the exact sites methylated by the enzymes from the three different cell compartments. Material and Methods

Isolation of plant organelles and preparation of enzymes Chloroplasts were obtained from lyophilized leaves by a non-aqueous technique according to Burkard et al. [7], and mitochondria were obtained from dark-grown hypocotyls as described by Guillemaut et ai. [8]. Cytoplasmic enzymes were prepared upon homogenization of 30 g of dark-grown hypocotyls in a mortar with 5 ml of 5 × concentrated enzyme buffer (the standard enzyme buffer contains 20 mM Hepes (pH 8.5), 1 mM reduced glutathione, 10 mM MgC12 and 10% glycerol). Lyophilized chloroplasts [7] (100 mg) were homogenized in a Potter tissue grinder with 4 ml of standard enzyme buffer. The mitochondrial pellet obtained from 2 kg dark-grown hypocotyls [8] was homogenized in a Potter tissue grinder with 2 ml of 2 × concentrated enzyme buffer. In all cases, the homogenate was centrifuged for 10 min at 27 000 × g and the supernatant was centrifuged for 2 h at 105 000 × g. The upper two-thirds of the supernatant were filtered through a Sephadex G-75 column (1.5 cm × 60 cm). The enzymatic proteins are eluted with the void volume, free of tRNAs and of small molecules which are retarded on the gel. After determination of the protein content of each fraction, those containing the enzymatic proteins were pooled and used immediately in the methylation reactions. The following yields were obtained: 60--70 mg cytoplasmic proteins per 20 g hypocotyls, 15--20 mg proteins per 100 mg lyophylized chloroplasts and 7--10 mg mitochondrial proteins per 2 kg hypocotyls.

Methylation of

t R N A Asp

The reaction mixture contained, in a total volume of 2 ml: 0.5 mmole Hepes buffer (pH 8.5}, 6 #moles glutathione, 40 /~moles ATP, 14 pmoles MgC12, 0.6 mmole ammonium acetate, 60 nmoles S.adenosyl[Me.~4C]. methionine (Radiochemical Centre, Amersham, 55 Ci/M), 4 nmoles pure tRNA A~p prepared according to Keith et al. [9] and 4 mg enzymatic proteins.

334

After 3 h at 30°C, the [Me-' 4C] tRNA Asp was extracted with phenol and passed through a Sepadex G-75 column (30 cm X 2 cm) in the presence of 1 mM NaC1.

Analysis of nucleotide sequences containing methylated bases Hydrolysis of the [Me-' 4 C] tRNA n~ by T, or by pancreatic ribonuclease was performed according to Gangloff et al. [ 10]. Fractionation of the resulting oligonucleotides was achieved either by DEAE-cellulose chromatography in the presence of 7 M urea [11], or by high-voltage electrophoresis [10] on Whatman DE-81 3 MM paper. After DEAE-ceUulose chromatography in the presence of urea, the radioactivity of the fractions was determined according to Pegg [12] ; after electrophoresis, the paper was cut into squares which were counted in a liquid scintillation counter. Further digestion of the oligonucleotides was performed using a mixture of T, and T2 ribonuclease [13] or by the action of phosphomonoesterase followed by the action of snake venom phosphodiesterase after the removal of phosphomonoesterase [9] or by the action of piperidine [ 14 ] followed by action of phosphomonoesterase after elimination of piperidine in a rotary evaporator. Separation of the resulting nucleotides and/or nucleosides was achieved by two-dimensional thin-layer chromatography on Schleicher and Schull F 1440 TLC Cellulose Plastic Sheets, using the following solvents: NH4 OH (25% NH3 )--propanol--water (30 : 60 : 10, by vol.) [15] in the first dimension, and HCl--isopropanol--water (17.6 : 68 : 14.4, by vol. ) in the second dimension [ 16 ]. The radioactive spots were located either by autoradiography, or by dividing the sheet into squares which were cut and counted in a liquid scintillation counter.

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as d a s e r i b e d i n M e t h o d s . After addition o f 2 m l earzier t R N A A s p , it w a s d i g e s t e d w t t h 4 0 0 u n i t s T 1 r l b o n u c l e a s e i n 0 . 1 M T ~ - - H C I b u f f e ~ ( p H 7 . 5 ) i n t o t a l v o l u m e 0 . 2 5 m l f o l 3 h a t 8 7 ° C . The d i p s t w a s f r a e t i o n a t e d o n a D E A E - e e n u l o s e e o l u m n ( 0 . 5 c m X 5 0 e r a ) u s h ~ a l i n e ~ i p ~ U e n t of lqaCl ( 0 - - 0 . S ~ M) i n 7 M u r e a , 0 . 0 2 M T r i s - - H C l b u f f e r ( p H 7 . 5 ) ( t o t a l v o l u m e of the ~ r a d i e n t = 4 0 0 m l ) . A 2 6 0 n m ( ); ~ a d i n a c t i v i t y i n 1 m l of each fraction ( . . . . . . . ).

335

Results

Sites of tRNA Asp methylated by cytoplasmic enzymes After methylation by cytoplasmic tRNA methylases, [Me .14 C] tRNA Asp was isolated and an aliquot was hydrolyzed by T1 ribonuclease. The resulting oligonucleotides were ffactionated by DEAE-cellulose chromatography in the presence of 7 M urea at pH 7.5 (Fig. 1). Some radioactive material (Peaks A and B) is eluted very rapidly from the column (even before the first ultravioletabsorbing peak which contains the mononucleoticles); it consists of S-adenosyl[Me., 4 C] methionine and of degradation products which are present in the hydrolysate (together with ADP which is eluted with the dinucleotides, and ATP which is eluted with the trinucleotides), especially when the tRNA is dialyzed instead of passed through Sephadex G-75 prior to T~ ribonuclease digestion. Only one major radioactivity peak is obtained (Peak C). As it is eluted just before the ultraviolet-absorbing Peak VI, which represents the dodecanucleotide T--~---C--A--A--U--U--C--C--C--C--G of the T ~ loop, it is likely to be due to this dodecanucleotide which has been methylated and has thus acquired an extra positive charge. Peak C was further hydrolyzed by a mixture of T~ and T2 ribonuclease into mononucleotides which were separated by two-dimensional thin-layer chromatography. Autoradiography of the plate showed only one radioactive spot, located at the position of N ~ -methyladenosine 3'-phosphate. As the dode-

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Fig. 2. E l e c t r o p h o r e t i c s e p a r a t i o n o n D E A E - c e l / u i o s e p a p e r o f a panczeatic r i b o n u c l e a s e digest o f t R N A A s p m e t h y l a t e d in vitro b y c y t o p l a s m i c e n z y m e s using S.adenosyl[Me- 1 4 C ] m e t h i o n / n e . M e t h y l a t e d t R N A A S p w a s re-extracted and, after a d d i t i o n o f 1 m g o f c a r r i e r t R N A A s p , w a s i n c u b a t e d w i t h 0 . 1 m g pancreatic r / b o n u c l e a s e f o r 2 h a t 3 7 ° C in a t o t a l v o l u m e o f 0 . 2 n,3 in 0 . 1 M t r i c t h y l a m m o n i u m b l c a r bonate buffer (pH 7.5). The digest was submitted to a co-electrophoresis on DE-S1 paper for 16 h at 700 V in 7% f o r m i c acid, t o g e t h e r w i t h the p r o d u c t s o f p a n c r e a t i c r i b o n u c l c a s e d i g e s t i o n o f t R N A T r p . T h e ultraviolet-absorbing s p o t s w e r e l o c a l i z e d and 1 c m X 1 . 5 c m pieces w e r e cut and c o u n t e d in a liquid scintillation c o u n t e r . T h e o l i g o n u c l e o t i d e s c o n t a i n e d in the ultraviolet-absorbing s p o t s had b e e n determ i n e d p r e v i o u s l y b y G a n ~ I o f f et aL [ 1 0 ] f o r t R N A A s p , a n d b y Keith et al. [ 1 9 ] f o r t R N A T r p , and are indicated in Table I. I n the e l e c t r o p h o r c s l s r e p r e s e n t e d here, S p o t 1 5 o f t R N A A s p a n d S p o t 1 7 o f t R N A T r p are n o t visible, as t h e c o r r e s p o n d i n g m o n o n u c l e o t i d e s had m i ~ a t e d t o o f a r t o w a r d s the a n o d e .

336

canucleotide of the TxP loop contains only two A's (which are next to each other), we proceeded to confirm that the A--A--Up sequence had been methylated. Another aliquot of [Me-14C] t R N A As° was hydrolyzed by pancreatic ribonuclease, and the resulting oligonucleotides {among which the A--A--U from the T ~ loop was present) were submitted to a co-electrophoresis on DE-81 paper, together with the products of the pancreatic ribonuclease digestion of yeast t R N A T~p which is known to contain a A--m ~ A--Up sequence in its T ~ loop [ 1 7 ] . As shown on Fig. 2, the only radioactive peak in the digestion products of t R N A A~p migrates exactly at the same position as the trinucleotide A--m I A--Up from t R N A Trp . This allowed us to confirm that the A--A--U sequence in the To~ loop of t R N A A~p had been methylated, but as a m ~ A--A--Up fragment would have the same electrophoretic mobility as the A--m ~ A--Up fragment, further analysis had to be performed in order to determine which A was methylated. The unlabelled ultraviolet-absorbing trinucleotide A--m 1 A--Up from t R N A Trp, and the labelled (but not ultraviolet-absorbing)trinucleotide from t R N A A~p were eluted, combined and submitted to the action of phosphomonoesterase to remove the phosphate group at the 3' end. After the removal of phosphomonoesterase, they were submitted to the action of snake venom phosphodiesterase which, in the case of the fragment from t R N A Trp , yields A, pm ~ A and pU. The resulting nucleoside and the t w o nucleosides 5'-phosphate were separated by two-dimensional thin-layer chromatography. Determination of the radioactivity on the sheet showed that only the p m ~ A spot was radioactive. It can, therefore, be concluded that in t R N A Asp only the second adenylic residue in the A--A--U sequence of the T ~ loop is methylated by the cytoplasmic t R N A methylases (Fig. 7). TABLE I FRAGMENTS OBTAINED AFTER PANCREATIC RIBONUCLEASE DIGESTION OF tRNAASP[10] A N D t R N A T r P [ 1 9 ] A N D ELECTROPHORESIS OF THE DIGESTS ON DEAE-CELLULOSE PAPER The conditions for digestion and eleetrophoresis are described in the legend of Fig, 2. t R N A Asp

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Sites o f t R N A A sp methylated by chloroplast or mitochondrial e n z y m e s When yeast tRNA A s~ was methylated in the presence of chloroplast enzymes and hydrolyzed by T1 ribonuclease, the elution profile of the resulting oligonucleotides, after DEAE-cellulose chromatography in the presence of 7 M urea at pH 7.5, showed two radioactive peaks (Fig. 3). One peak is eluted just before the ultraviolet-absorbing peak corresponding to the dodecanucleotide of the T ~ loop, as in the case o f the methylation of tRNA Asp by cytoplasmic enzymes; another radioactive peak is located before the ultraviolet-absorbing peak which corresponds to the tetranucleotides. Another aliquot of [Me -1 4C]tRNAASp was hydrolyzed by pancreatic ribonuclease and the resulting oligonucleotides were fractionated by DEAEcellulose chromatography in the presence of 7 M urea at pH 7.5. As shown on Fig. 4, there is only one radioactive peak which is eluted before the ultravioletabsorbing peak corresponding to the dinucleotides, and, therefore, consists of one or more methylated trinucleotides. 20 ~8

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Fig. 5. Electzophoretic separation o n DEAE-cellulose paper o f a pancreatic ribonuclease digest of tRNAASp m e t h y l a t e d in vitxo b y c h i o r o p l u t e n z y m e s . The conditions for digestion and elcetrophoresis a r e identical to those described in the legend o f Fig. 2. The oligonueleotides c o n t a i n e d in the ultravioletabsorbing spots, d e t e r m i n e d previously b y Gangloff et al. [ 1 0 ] , are indicated in Table I . Fig. 6. Electrophoretic separation o n DEAE-eenulose paper of a T 1 ribonucIease digest of the slowly migrating radioactive fragment obtained after the elect~ophoresis represented o n Fig. 5. The slowly migrating radioactive fragment obtained after the eleetrophoresis represented o n Fig. 5 was eluted w i t h a molar solution of t r i e t h y l a m m o n l u m bicarbonate [ 1 4 ] and digested with T 1 ribonuclease. The digest was submitted to a co-electrophoresis, together with t w o k n o w n fragments (m I A - - U p and m ! A---Gp) f~om rabbit liver t R N A Phe [ 1 8 ] , at 1 5 0 0 V, for 3 h in 2.5% formic acid--8.7% acetic acid buffer (pH 1.9). In addition to the t w o dinucleotides from t R N A Phe, t w o othe~ ul~aviolet-absorbing spots are visible; t h e y correspond to Up and Gp generated b y T 1 ribonuelease a c t i o n o n G--Up (Spot 8 o f Fill. 5) a dinucleotide migrating together w i t h the s l o w l y migrating radioactive trinucleotlde (Fig. 5) and which is therefore also cut and eluted.

Another aliquot of the pancreatic ribonuclease digestion products was submitted to high-voltage electrophoresis on DE-81 paper (Fig. 5); two radioactive spots were obtained. The fast migrating one is identical to that obtained with tRNA A~ methylated by cytoplasmic enzymes and is due to the [Me.14 C] trinucleotide A--m 1 A--Up. The slowly migrating peak migrates at the rate of the ultraviolet-absorbing dinucleotide G--Up (Spot 8) and is most probably due to the methylation of one of the two trinucleotides A--G--Up (Spot 6) or G--A--Up (Spot 7). This radioactive peak was eluted, hydrolyzed by piperidine and then by phosphomonoesterase into nucleosides. The only radioactive spot obtained upon two~iimensional thin-layer chromatography was methyladenosine. In order to determine whether this methylated adenylic residue originates from the methylation of the trinucleotide A--G--Up or of the trinucleotide G--A--Up, another aliquot of the slowly migrating peak (after electrophoresis) was hydrolyzed with T~ ribonuclease. Under these conditions m ~ A ~ U p should yield m ~ A--Gp + Up, while G--m ~ A--Up should yield Gp + m ~ A--Up. The products of ~ ribonuclease digestion were submitted to high-voltage electrophoresis on DE-81 paper, together with the two non-radioactive dinucleo-

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Fig. 7. Clover-leaf stru cture of yeast tRNAASp [5--6]. Comparison of the sites m e t h y l a t e d by cytoplasmic, chloroplast and m i t o c h o n d r i a l enzymes (our experim e nt s ) and by animal enzymes [12,20].

tides m ~ A--Up and m 1A--Gp obtained by Keith et al. [18] during the nucleotide sequence determination of tRNA Ph e from rabbit liver. As shown on Fig. 6, the radioactivity coincides with m 1 A--Up. It can, therefore, be concluded that the second site on yeast tRNA Asp which is methylated by chloroplast tRNA methylases is an adenylic residue at Position 7 from the 5' end of the molecule (Fig. 7). Mitochondrial tRNA methylases recognize the same positions as the chloroplast enzymes and also transform into m I A the A of the TkO loop and the A at Position 7 from the 5' end. Discussion It is interesting to compare our results with those of Pegg, who has recently used yeast tRNA Asp as a substrate to study some animal tRNA methylases; he has shown that rat liver enzymes [12], and enzymes from mouse colon, rat kidney and turnouts of these tissues [20] all methylate yeast t R N A Asp on the guanylic residue at position 26 from the 5' end of the molecule (Fig. 7}. Although rat liver tRNA methylases are able to transform into A--m ~A--Up a A--A--Up sequence in the T~I' loop o f Escherichia coli tRNA Met and tRNA Gl" [19] ; these extracts do not m e t h y l a t e this sequence present in yeast tRNA ~ p . But we have shown that plant cytoplasmic, chloroplast and mitochondrial t R N A methylases are able to transform into m ~A the second A of a A--A--Up sequence in the T~t' loop. In fact m ~A occurs quite frequently in the trinucleotide A--A--Up o f the T~I' loop; it is present in 16 of the 42 tRNA sequences presently k n o w n [21] especially in the only plant tRNA whose sequence has been determined, namely wheat germ tRNA Phe [22]. In contrast, methylation of yeast tRNA A~ at Position 7 from the 5' end,

340

by t R N A methylases from plant chloroplasts or mitochondria occurs at a very unusual position; in fact it is the first time that a methylated residue is found in this area of a t R N A molecule, between the 5' end and the stem of the hU loop. It should be pointed o u t that this A is methylated only by the enzymes of the organelles, but not by the cytoplasmic t R N A methylases. The existence of an organelle-specific t R N A methylase, which is n o t present in the cytoplasm of the same organism, is of particular interest with respect to the problem of the origin of the tRNAs found in the organelles. In this laboratory, chloroplast- and mitochondria-specific tRNAs have been characterized [3,7,8,23,24], and it has been shown for instance that some chloroplast-specific tRNAs L e u, which can only be aminoacylated by chloroplast (or by E. coli) leucyl-tRNA synthetase (and n o t by the cytoplasmic enzyme), nevertheless hybridize with nuclear (but not with chloroplast) DNA and thus seem coded by nuclear genes [25]. That an organelle-specific t R N A coded by nuclear genes (as .the cytoplasmic tRNAs) is not recognized by the cognate cytoplasmic aminoacyl-tRNA synthetase, could be explained if, u p o n entering into the organelle, the t R N A is modified by organeUe-specific enzymes (such as t R N A methylases) and if the resulting modified t R N A is so different from its cytoplasmic counterpart that it is no longer recognized by the cytoplasmic aminoacyl-tRNA synthetase. Some authors have reported that the overall degree of methylation is lower in mitochondrial tRNAs, as compared to that of the corresponding cytoplasmic tRNAs [ 2 6 , 2 7 ] , b u t recent studies have shown that mitochondria have at least one methylase which is absent from the cytoplasm [28] and that mitochondrial tRNAs contain higher proportions of m I A and m 2 G than the corresponding cytoplasmic tRNAs [28,29]. The study of plant t R N A methylases from the different cell compartments deserves to be continued, as it may help understand some of the relations between the cytoplasm, the mitochondria and the chloroplasts, especially as far as the protein synthesizing systems of the three compartments are concerned. Acknowledgements W e thank P. Guillemaut and A. Steinmetz for their help in isolating the mitochondria and chloroplasts, respectively.W e are grateful to Mrs M. Schlegel, J. Gangloff, J. Bonnet for their help in the purification of yeast t R N A Asp . W e are indebted to G. Keith w h o gave us fragments of yeast t R N A Trp and of rabbit liver t R N A e n e, and to J. Weissenbach and Miss C. Werner for their help in the analytical part of this work.

References 1 2 3 4 5 6 7 8

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