264

Biochimica et Biophysica Acta, 564 (1979) 264--274 © Elsevier/North-Holland Biomedical Press

BBA 99503

INHIBITORY EFFECT OF METHYLATED DERIVATIVES OF GUANYLIC ACID FOR PROTEIN SYNTHESIS WITH REFERENCE TO THE FUNCTIONAL STRUCTURE OF THE 5'-'CAP' IN VIRAL MESSENGER RNA

KIN-ICHIRO MIURA a,*, YAEKO KODAMA a, KUNITADA SHIMOTOHNA a, TOSHIKAZU FUKUI b, MORIO I K E H A R A b, IWAO NAKAGAWA c and TSUJIAKI HATA c

a National Institute of Genetics, Mishima, 411, b Faculty of Pharmaceutical Science, Osaka University, Yamadakami, Suita, Osaka, 565 and c Department of Life Chemistry, Tokyo Institute of Technology, Nagatsuta, Yokohama, 227 (Japan) (Received January 3rd, 1979)

Key words: Viral mRNA, Protein synthesis inhibitor; Guanylic acid; 'Cap' structure

Summary Guanylic acid modified variously with methyl groups on base or sugar moieties were synthesized chemically and their inhibitory effects on protein synthesis were tested in a wheat germ cell-free system using mRNAs from cytoplasmic polyhedrosis virus and tobacco mosaic virus. The confronting dinucleotide mTGS'pppA that corresponds to the most simple 'cap' structure of an eukaryotic mRNA is a strong inhibitor of protein synthesis, but non-methylated GS'pppA or GS'ppA is not inhibitory. The strong inhibitory effect is observed only by 7-methylguanylic acid (pmTG). Among 11 derivatives of pG, the most effective inhibitors are methylated at the 7-position. Further methylation at the other position sometimes cancels the inhibitory effect. Although pmTG carries a positively charged base, other nucleotides which carry a plus charged base (1-methyladenylic acid and 2-methylthio-7-methylinosinic acid) were not inhibitory. Thus, methylation at the 7-position on guanylic acid is specifically required for the inhibitory effect. Addition of pmTG was inhibitory for the formation of the initiation complex for eukaryotic protein synthesis. These results suggest that the 'cap' component containing 7-methylguanylic acid in viral mRNA participates during protein synthesis, especially in its initial steps. Protein synthesis in a bacterial cell-free system was not inhibited by addi* T o w h o m correspondence should be addressed. Abbreviations: T M V , tobacco mosaic virus; CPV, cytoplasmic polyhedrosis virus: Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid.

265 tion of mTGpppA or pmTG when either TMV RNA or phage MS2 RNA was used as an mRNA.

Introduction Most eukaryotic mRNAs and viral RNAs carry a 'cap' at the 5'-terminal as m~GS'pppN(m) - [1--3]. Since protein synthesis proceeds in the 5' -~ 3' direction of the template mRNA, the 5'-terminal modification may be related to the initial step of protein synthesis. The 'cap' (mTGpp) can be deleted by tobacco phosphodiesterase with no damage to the RNA chain [4]. The ability to synthesize protein and the ability to form the complex for the initiation of protein synthesis in the 'cap'-deleted mRNA are decreased remarkably [5]. Similar results were obtained by other treatments for 'cap' elimination [6,7]. These experiments suggest that the role of the 'cap' is in protein synthesis, especially in the initiation step. Since the 'cap'-eliminated mRNA loses its stability in cell extracts [5,6], the 'cap' may function in protein synthesis through stabilization of mRNA. However, there are some data suggesting that the 'cap' interacts with cellular components required for protein synthesis [9--12]. Several reports have described that the addition of pmTG to the protein synthesizing system causes strong inhibition [13--21]. To determine if pmTG is a specific inhibitor of protein synthesis, various methylated derivatives of guanylic acid were synthesized chemically and their inhibitory effects were studied in wheat germ and Escherichia coli cell-free systems. Materials and Methods

(1) Synthesis of methylated derivatives of guanylic acid (a) 8-Methylguanosine 5'-phosphate, NLmethylguanosine 5'-phosphate, and N : ~ - d i m e t h y l g u a n o s i n e 5'phosphate were prepared as described previously by Maeda et al. [22] and Ikehara and Hattori [23]. (b) 2-Methylthio-8-methylinosine 5'-phosphate was prepared as follows: FeSO4 (20 mmol in 10 ml of H20) and t-butyl hydroperoxide (15 mmol in 10 ml of H:O) were simultaneously added dropwise to a precooled solution (15°C) of 2-methylthio inosine 5'-phosphate (sodium salt, 5 mmol) in 1 M H2SO4 (100 ml). This mixture was stirred for 1 h and then desalted by absorbing on charcoal, washing with water, and eluting with H20: ethanol : concentrated ammonia (45 : 50 : 5). The eluate was evaporated to dryness. The material was then dissolved in 15 ml of water and applied to a column (3 × 60 cm) of Dowex 50 W-X2(H÷-form). Elution was performed with water and the fractions containing the product were evaporated and lyophilized. The yield was 50%. (c)N2,8-dimethylguanosine 5'-phosphate was prepared as follows: To a solution containing 2 mmol of 2-methylthio-8-methyl inosine 5'-phosphate in 25 ml of water. 6 mmol of N-chlorosuccinimide was added at 40°C. After 1 h, 10 ml of 30% aqueous methylamine was added. This mixture was heated at 120°C for 2 h in an autoclave and applied to a column (1.8 × 20 cm) of anion exchange resin Dowex l-X2 (formate form). After washing the column with

266 water, elution was performed with 0.25 M HCOOH. The fractions containing the product were evaporated to dryness. The yield was 39%. (d) N2,N2,8-Trimethylguanosine 5'-phosphate was prepared as follows: To a solution containing 2.4 mmol of 2-methylthio-8-methylinosine 5'-phosphate in 50 ml of water 7.2 mmol of N-chlorosuccinimide was added at 40°C. After 1 h, 5 ml of 40% aqueous dimethylamine was added. The mixture was heated at 120°C for 4 h in an autoclave and applied to a column (1.8 × 20 cm) of D o w e x 1-X2 (formate form). After washing with water, elution was carried out with a linear gradient from 0 to 0.5 M HCOOH (2 1 each). The fractions containing the product were pooled and evaporated to dryness. The yield was 27%. (e) Methylation reaction of 7-position of guanylic acid and the analogues were performed by two procedures. Method 1. Starting material, nucleoside 5'-phosphate {0.43 mmol) was dissolved in 2 ml of dimethylsulfoxide containing 0.2 ml of methyliodide at r o o m temperature for 5 h with continuous stirring. The reaction mixture was then diluted to 10 ml with acetone, and the resulting precipitate was collected by centrifuge and washed twice with 10 ml of acetone. It was then dissolved in 3 ml of water and applied to a column (1.8 × 23 cm) of DE 52 anion exchange cellulose (bicarbonate form). Elution was carried o u t with a linear gradient from 0 to 0.1 M triethylammonium bicarbonate (1 1 each), pH 7.5. The fractions containing the methylated product were evaporated to dryness. Method 2. Starting material (0.1 mmol) was suspended in 1 ml of dimethylformamide and 0.1 ml of dimethyl sulfate was added at once. This solution was stirred for 20 h at room temperature and then diluted to 20 ml with water and adjusted to pH 9 with 1 M NH4OH. Chromatographic separation of the methylated product was carried o u t as described in Method 1. The yields of 7-methyl derivatives were as follows: By Method 1, 7-methyl derivative of 8-methylguanosine 5'-phosphate (46%), N2,8-dimethylguanosine 5'-phosphate (32%), and N2-methylguanosine 5'-phosphate (43%); by Method 2, N2,N2-dimethylguanosine 5'-phosphate (90%), and N~,N2,8trimethylguanosine 5'-phosphate (54%).

(2) Synthesis of a, 7-dinucleoside triphosphates and ~, ~-dinucleoside diphosphates Method for the synthesis of mTGS'pppA, GS'pppA was described previously [24]. GS'ppA was prepared by a modification of the procedure [25], reported previously, using di-n-butylphosphinothioyl bromide via di-n-butylphosphinothioic guanosine 5'-phosphoric anhydride intermediate.

(3) Identification of methylated nucleotides The structures of the methylated nucleotides were varified by ultraviolet absorption spectra, paper chromatography, and paper electrophoresis. Paper chromatography was carried out on T o y o filter paper No. 51A developed with solvent system A, 600 g (NH4)2SO4 in 1 1 of 0.1 M sodium phosphate buffer (pH 6.8) plus 20 ml of n-propyl alcohol. Paper electrophoresis was performed using 0.05 M triethylammonium bicarbonate (pH 7.5) at 900 V/40 cm. The maximum absorption in the ultraviolet spectrum and the mobilities in paper chromatography and paper electrophoresis are given in Table I.

267 TABLE I CHARACTERS

OF THE METHYLATED

DERIVATIVES

OF GUANYLIC ACID

Ultraviolet absorption maximum

Compound Number

Formula

Condition

I

pm8G

II

pm2,SG

III

pm2,2,SG

IV

pm2G

V

pm2,2G

VI

pmTG

VII

pm 7 fiG

VIII

pm2,7,8G

pH 0.1 0.1 pH 0.1 0.1 pH 0.1 0.1 pH 0.1 0.1 pH 0.1 0.1 pH 0.1 0.1 pH 0.1 0.1 pH

7.0 N HC1 N NaOH 7.0 N HC1 N NaOH 7.0 N HC1 N NaOH 7.0 N HC1 N NaOH 7.0 N HCl N NaOH 7.0 N HCI N NaOH 7.0 N HC1 N KOH 7.0

0 . 1 N HC1

IX

pm2fl,7,8G

X

pm2,7G

XI

pm2,2,TG

pG

0.1 pH 0.1 0.1 pH 0.1 0.1 pH 0.1 0.1 pH 0.1 0.1

N KOH 7.0 N HC1 N KOH 7.0 N HC1 N KOH 7.0 N HCI N NaOH 7.0 N HC1 N NaOH

Wavelength (nm) 251 260 260 253.5 263 261 262 269 265 255 259:5 259 261 265 262 258 257 268 257 261 268 (224 ~263 {224 263.5 267 268.5 269 268 257.5 261 269 264 266 273 252 256 258

e (P)

15 12 11 15 12 12 17 15 13 14 13 11 16 16 12 10 12 8 13 12 9 35 15 34 15 8 13 15 7 12 11 9 12 13 8 13 12 11

900 200 600 100 800 200 000 300 400 000 000 000 000 000 000 300 000 300 400 700 400 100 ~ 100 ~ 500 } 300 700 800 000 840 600 600 800 200 000 500 700 200 600

Paper chromatography in s o l v e n t A RF

Paper electrophoresis in p H 7 . 5 r e l a t i v e mobility to pG

0.28

0.97

0.21

0.90

0.15

1.00

0.24

0.95

0.22

0.89

0.64

0.97

0.64

0.94

0.57

0.76

0.51

0.94

0.53

0.90

0.44

0.80

0.46

1.00

(4) Protein synthesizing system in vitro The protein synthesizing system was prepared from a wheat germ extract according to Roberts and Peterson [26]. For one assay, 50 pl of the reaction mixture was used. It contained 20 mM N-2-hydroxyethylpiperazine-N2-2 ethanesulfonic acid (Hepes) (adjusted to pH 7.6 with KOH), 2.5 mM magnesium acetate/100 mM KC1/4 mM dithiothreitol/1 mM ATP/20 gM GTP/ 8 mM creatine phosphate/2 pg of creatine phosphate kinase/an amino acid mixture (3 uM each, except leucine)/2 pCi of [3H]leucine (50 Ci/mmol), 10 gg of RNA and about 5 A260 units of wheat germ extract. The RNA used as messenger was prepared as described previously [5]. An aqueous solution of a

268 guanylic acid derivative was added to the reaction mixture. Following incubation at 30°C for 30 min, 5% (wt./vol.) ice-cold trichloroacetic acid was added, and the mixture was heated at 90°C for 20 min. The acid-precipitable fraction was collected on a glass fiber filter (Whatman GF/A, 24 mm in diameter) and its radioactivity was assayed in a liquid scintillation spectrometer. The in vitro protein synthesizing system was prepared from E. coli strain Q13 according to Nirenberg and Matthaei [27]. Results The effect of adding pmTG to the in vitro protein synthesizing system was studied by incorporation of labeled amino acids into protein in the wheat germ cell-free extract with cytoplasmic polyhedrosis virus m R N A and tobacco mosaic virus RNA as an m R N A . As shown in Fig. 1, protein synthesis with both m R N A s was inhibited by pmTG at concentrations higher than 0.1 mM. The confronting nucleotide structure, mTGS'pppA, the 'capping' structure at the 5'-terminal of eukaryotic m R N A , was also inhibitory. However, GS'pppA and GS'ppA, which do not contain a methyl group at the 7-position of G, were not inhibitory (Fig. 2). Since the methylation of guanosine in the 'cap' of m R N A seems to be required for inhibition of protein synthesis, a variety of methylated derivatives o f guanylic acid were prepared to test their inhibitory effects. The characteristics of these derivatives are listed in Table I. Fig. 3 shows the typical results of an inhibition experiment. Since addition of guanylic acid (pG) to the wheat germ protein synthesizing system shows slight inhibition, guanosine was selected as an example of lack of inhibition. This is expressed as '--' (negative)

100 w "F I-.Z

u3 w II.z >u")

Z

z w

N 5O b--

0 rl

0 or

100'

',

~:~~______~pp

,,

"~ 50

0., ..........

13_

~'~0 i

.

a

i

i

i

a

i

i

~ i

i

......... ,

i

a

i

~

0.5 1 1.5 pmTG C O N C ( m M ) Fig. 1. Effect of pmgG and TMV-RNA (e .....

mTGpppA

•

0

i

A "~

I

I

i

0.5

2

on protein synthesis in the in vitro wheat - e ) w e r e u s e d as m R N A .

INH]BITOR germ system.

F i g . 2 . E f f e c t o f t h e a n a l o g s o f t h e 5 ' - ' c a p ' s t r u c t ,u£e i n a n m R N A o n p r o t e i n system using TMV RNA as mRNA. • ...... •, m7G 5 pppA; o o, G S p p p A ;

110 CONCI (mM)

CPV mRNA

(o

o)

s y n t h e s i s i n w h e a t gexzn A. . . . . . A G 5 ppA.

269 Inhibition

effect 100

~O~o~O---.._o~O

G

LIJ tl.-Z >-. U3 Z

,V, 5o E0 rr n

A~'A--

0

--A

prnTG

i

!

1.0

2.0

+ +

INHIBITOR CONC. (raM) Fig. 3 . T y p i c a l c a s e s i n t h e i n h i b i t o r y e f f e c t o f m e t h y l a t e d d e r i v a t i v e s o f g u a n y l i c a c i d f o r p r o t e i n s y n t h e s i s i n w h e a t g e r m s y s t e m u s i n g T M V R N A as m R N A . • A pmTG; • ...... e, pm2,2,7G;

0

O, G.

for the inhibitory effect in this paper. Remarkable inhibition was shown with addition of pmTG, so that it is denoted as '++'. A little inhibition, less than pmTG was shown by N2,N2,7-trimethylguanylic acid (pm]'2'TG). This weak inhibition is expressed as '+' (positive) for the inhibitory effect. Table II summarizes the inhibition of protein synthesis by the methylated derivatives of guanylic acid. Strong inhibitors (++) carry a methyl group at the 7 position, suggesting that the 7-methyl group is specially important for inhibition of protein synthesis. However, all the 7-methylguanylic acid derivatives did not show strong inhibition; compounds VII and XI showed weak inhibition, and furthermore compound IX did not give any inhibitory effect. In these cases, T A B L E II INHIBITION EFFECT OF THE METHYLATED DERIVATIVES OF GUANYLIC SYNTHESIS IN WHEAT GERM EXTRACT USING TMV RNA AS mRNA Compound

*

Number

of methyl

group

I n h i b i t i o n e f f e c t **

Position

I II III IV V VI VII VIII IX X XI

2

7

8

0 1 2 1 2 0 0 1 2 1 2

0 0 0 0 0 1 1 1 1 1 1

1 1 I 0 0 0 I 1 1 0 0

--+ + -÷+ + ++ -++ +

* T h e n o t a t i o n s o f c o m p o u n d s are l i s t e d in T a b l e I. * * S e e Fig. 3 a n d t e x t . + + , s t r o n g i n h i b i t i o n ; + , w e a k i n h i b i t i o n ; - - , n o i n h i b i t i o n .

ACID FOR PROTEIN

270 the inhibitory effect of the 7-methyl group would be cancelled by steric hindrance of configurational change by other methylation around the 7-methyl group. The 7-methyl guanine residue carries positive charge differently from other purine bases. To determine the effect of the positive charge in inhibition of protein synthesis, other similar nucleotides were prepared. Neither Nl-methyl adenylic acid nor 2-methylthio-7-methylinosinic acid showed any inhibition in a wheat germ protein synthesizing system as shown in Fig. 4a and b. Thus, the methylation itself at the 7 position in guanosine seems to be specifically required for inhibition rather than its positive charge. As seen in Fig. 4a and b, guanylic acid and adenylic acid are inhibitory for protein synthesis although the rates are lower than pmTG. These may be inhibitory for the steps at which ATP or GTP acts. The confronting nucleotide to pmTG at the 5'-terminus of m R N A is sometimes methylated in its base moiety and at the 2'-position in its ribose moiety. As shown in Fig. 4c, addition of 2'-O-methylguanylic acid and 6-methyladenylic acid to the wheat germ system did n o t cause eminent inhibition for protein synthesis. These results indicate that the strong inhibition of protein synthesis was caused by 7-methylguanylic acid residue in the confronting nucleotide structure. It has been known that the 7-methylguanylic acid-deleted m R N A treated with tobacco pyrophosphatase loses its ability to form the initiation complex of protein synthesis [5]. It was also shown that addition of pmTG to the intact CPV m R N A caused inhibition of the formation of the initiation complex. These results were confirmed here, b u t it was also shown that the addition of adenylic acid does n o t inhibit this step (Fig. 5). The 7-methylguanylic acid-blocking structure, so-called 'cap' structure, is c o m m o n l y involved in eukaryotic m R N A and viral RNA, whereas it is not found in prokaryotic m R N A (Refs. 28 and 29 and our unpublished observations). It was of interest to learn if the inhibition of protein synthesis by 7-methylguanylic acid residue is specific for the eukaryotic system. An in vitro protein synthesizing system was prepared from E. coli. Using phage MS2 RNA

b

c pmlA

L~ 100I

100

100, pGm

tlA I I.--

"o . . . . .

•

pr~'-... "o

u3 Z 50 LIJ b-O n.(3_

50

pm7G

0

0 2 INHIBITOR CONC (mM)

1

Fig. 4. Effect o f a d d i t i o n o f p u r i n e RNA b, o

as

mRNA.

4

u 1-methyl

A AMP,

GMP.

• AMP;

ol

~

I

2

INHIBITOR C O N C (raM)

nucleotide

7-methyl • ......

4

pm7G

on a,

c, ~

p r o t e i n s y n t h e s i s in w h e a t germ s y s t e m using T M V -

X

X 2-methylthio-7-methyl --~

6-methyl

AMP,

• ......

IMP,

o ......

• 2'-O-methyl

o GMP.

GMP;

271 ,jsos

a

8

80S

N 'C)

1

6

E a~

h O

~- 0 I.> l.c.)

18o_

C

d

n