BIOCHIMIE, 1975, 57, 1315-1321.

Methylation process of mammalian ribosomal precursor RNA. P i e r r e TIOLLAIS *, Marie-Annick AUGER a n d F r a n c i s GALIBERT.

Laboratoire d'H~matologie Exp~rimentale, Hdpital Saint-Louis, 2, Place du Docteur A. Fonrnier, 75010 Paris. (30-6-1975).

Summary. - - The analysis of RNA from L5178 Y cells, by electrophoresis in 1.7 per cent polyaerylamide gel, after short time labelling with [methyl-all] methionine, sho~s that the three molecular species 47S, 46S and 45S RNA from the ribosomal RNA ¢ 45S fraction>> are methylated. Comparative studies of the kinetics obtained after labelling for short periods with [methyl-all] methionine and [~H] uridine, and brief labelling cxperlments with [methyl-Sill methionine in the presence of aetinomycin D, show that the methylation of ribosomal precursor RNA occurs after the transcription process has been completed, and that methylation and transcription are not coordinated. These results lead us to propose the follo~cing scheme for ribosomal RNA biosynthesis : 1) transcription of ribosomal genes, 2b methylation of 47S I~NA (and possibly 46S RNA), 3) maturation of these molecules which are converted to 45S RNA.

INTRODU,CTION. Methylation of r i b o s o m a l RNA takes place at an early stage of biosynthesis, i n e a k a r y o t i c calls, since the ribosoma~l p r e c u r s o r RNA, in m a m m a l i a n cells, was i n fact heterogeneous and composed of three m o l e c u l a r species, called 47S, 46S and 45S RNA [8]. Similar observafi(~ns have been made recently on a m p h i b i a n ribosom'al p r e c u r s o r RNA [12, 13,

14].

Fro. 1. - - Time course o~ RNA labelling ,with [methyl-3H] methionine for short periods. A L5178Y cell culture (A), containing 7 X 105 cells/ml, was concentrated to 4 × 106 cells/ml, in Fischer medium, containing 5 per cent dialysed calf serum, lacking methionine and supplemented with 0.01 M adenine and 0.01 M guanine. Four aliquots of 4 ml were taken from this culture and incubated for different periods : a) 30 sec.; h) 1 min. ; c) 4 min. ; d) 10 min., with [methyl-~H] methionine (100 ,ttCi/ml ; specific activity : 9 Gi/mmol). A L5178Y cell culture (B) containing 7 X 105 eells/ml was concentrated to 1.5 X 10¢ cells/ml in complete Fischer medium containing 5 per cent dialysed calf serum. 12 ml of this culture were incubated for 1 hour with [14C] uridine (0.2 .t~Ci/ml ; specific activity 25 Ci/mmol). After cooling, each aliquot of culture A was mixed with 3 ml of culture B. RNA 'was extracted and analysed by 1.7 per cent polyacrylamide gel electrophoresis (22 × 0.7 cm gels ; 8 V/cm ; 6 hours migration ; 1 mm slices). O O [methyl-all] methion.in~ • . . . . . • [14C] urk~ine

BIOCHIMIE, 1975, 57, n ° 11-12.

Methglation of ribosomal RNA. cDm

47" S

COrn

1 Min

3H 14

45 S

CDM

cpm

30 Sec

3H

1317

3H Methionine

C

3 H

14 C

Methionine

41 S

45 S *46 S 600

300

(~00

400

41 S

® I

110

110

SLICE NUMBER

SLICE NUMBER

A

B

cpm

3H

4

45 S

~0

47 S

3H

Min

cpm

cnm

14C

3~,

10 Min 45 S

3H

Methionine

14C

Me~hionine

,

i ooo

[l LI

3~ s i! e, :,,

"

~oo

J, 'i,t

II

, 'i

18 s tiI

,'

dA

,: I

,1

I-~

:: Ii e I I, , 'b

,, ~6

I I

,,~ ~,

iII I ~,II

1

110 SLICE NUMBER

C

BIOCHIMIE, 1 9 7 5 ,

cpm

57, n ° I I - 1 2 .

190 SLICE NUMBER D

500

o~

c~

or-A

c~

,F.-I

'~"

|

I

I'

T,.,

.°-O - ' ~ -

®

®

~c

~DO

r~

u~

,i,-I .~

~D -r~

CY~

1

'

I"

I

I

o~4. . . . .

~D°~

L~

~t

Methylation

o[

ribosomal RNA.

32S

%

45 S

c~ 25

~ 28 S 3 ;~

i,

'l

i

h

:,,

L

'1 ut i|

|

q

i

:

,i

i I

i

,i

, ,

.,

'

H [k/dine

k p

i

ll;;

Min

1 8S

':

i!,,

,i

1319

J

t

,

ii

'

'

"'

, ',

,i

I'

14C Fw,. 2 . -

Time course of RNA labelling with [sH] uridine for short periods.

Several identical L5178 Y cell cultures were incubated for different periods ~dth [3HI uridine as described in figure 1. After 2000 cooling each culture was mixed with a L5178 Y cell culture incubated for 1 hour with [14C] uridine as described in figure l. RNA was extracted and analysed by 1.7 p. cent polyacrylamide gel electrophoresis as described in figure 1. a) 30 sec. labelling with [3H] uridine, b) 2 min. labelling, c) 5 rain. labelling, d) 8 min. labelling, e) 25 min. labelling. O O [3H] uridine • • [1~C] aridine

!

100

gLIf£ I~ER

180

E

The results p r e s e n t e d in this p a p e r show that 47S a n d 46S RNA are methylated, a n d h e n c e that m e i h y l a t i o n takes place on the molecules w h i c h are the direct p r o d u c t s of t r a n s c r i p t i o n , 45S b e i n g c o n s i d e r e d as a m a t u r a t i o n product. A c o m p a r i s o n of the k i n e t i c s of labelling RNA w i t h m e t h i o n i n e a n d u r i d i n e shows that methylafion takes p~lace after t r a n s c r i p t i o n on completely synthesized molecules. The fact thai the appar e n t m a t u r a t i o n rate is greater d u r i n g e x p e r i m e n t s using methionine than during experiments using u r i d i n e argues in favour of p o s t - t r a n s c r i p t i o n a l m e t h y l a f i o n . Other i n t e r p r e t a t i o n s are possible, h o w e v e r ; e.g. that the po~l of m e t h i o n i n e is cons i d e r a b l y smaller t h a n that Of u r i d i n e . On the other h a n d , the p r e s e n c e of i n c o m p l e t e l y s y n t h e sized molecules seen i n ,labelling e x p e r i m e n t s w i t h u r i d i n e but not w i t h m e t h i o n i n e (fig. l a a n d 2a) c a n be e x p l a i n e d only b y a p o s t - t r a n s c r i p t i o n a l methyl,ation. This was c o n f i r m e d by the fact that m e t h y l a t i o n takes place n o r m a l l y w h e n t r a n s c r i p tion is blocked by a c t i n o m y c i n D (fig. 3). The slightly d i m i n i s h e d (by 30 p e r cent) i n c o r p o r a t i o n of m e t h y l groups into 47S RNA in the p r e s e n c e of actinomyci.n D res,ul~s from the fact that this speBIOCHIMIE, 1975, 57, n ° 11-12.

cies is not b e i n g synthesized d u r i n g the blockage of t r a n s c r i p t i o n as is the case for the control experiment. The q u a n t i t y of 4,5,S RNA is not affected by the block d u r i n g the two m i n u t e s of the experiment. It was f o u n d at the same level as i n the control. These results are i n c o n t r a d i c t i o n to those of G r e e n b e r g - a n d P e n m a n [1] a n d of Z i m m e r m a n a n d Holler [2] who h a d c o n c l u d e d that methylation takes p l a c e d u r i n g t r a n s c r i p t i o n . The r a d i o : active moqecules observed by~ Green.berg a n d Penman, w h i c h sedimen~ed m o r e : s.lowly t h a n 45S RNA after labelling w i t h m e t h i o n i n e , a n d w h i c h were consi,dered to be i n c o m p l e t e l y synthesized, but already methylated, molecules, could in fact c o r r e s p o n d to produ, cts of m a t u r a t i o n of 45.S BNA. I n fact, as seen in figure 2, ~after 2 or 3 rain of methiouin,e labeHi.ng, R.NA mol.e~utes w h i c h are more ma,ture t h a n 4,5~S RNqA can be seen. Z i m m e r m a n and Holler came to the same c o n c l u s i o n since u n d e r their e x p e r i m e n t a l c o n d i t i o n s (actinomycin D 5 nfin, methio,n.in,e 30 mi,n) the actinomyci.n blocks the m e t h y l a t i o n of 45S RNA. This block is also compatible w i t h p o s t - t r a n s c r i p t i o n a l met h y l a t i o n since, u n d e r these c o n d i t i o n s , the un-

P. Tiollais, M . - A . A u g e r a n d F. Galibert.

1320

fact that m e t h y l a t i o n takes place n o r m a l l y even w h e n t r a n s c r i p t i o n is b l o c k e d p r o v e s that lhese processes o c c u r indepen,dently of one another, and that only completely synthesized molecules

labelled 47S RNA m o l e c u l e s s y n t h e s i z e d before the a d d i t i o n of a c t i n o m y c i n D w e r e c o n v e r t e d to m a t u r a t i o n p r o d u c t s (45S I~NA) on w h i c h methylation does not take place.

cpm

2 Min

ld C

2 Min 3H M~thionine

47S

3H

corn

3H Methionine 15 Sec Actinomycin 5,J.g/ml 45 S d7 S =

70 %

FIG. 3, - - $7S and 56S RNA labelling

S ~45

S

with [methyl-3H] methionine in the presence of actinomyein D.

100 %

200

IOC

1.000

a) A L5178 Y cell culture was incubated for 2 rain., with [methyl-a] methionine and mixed with a L51,78 Y cell culture incubated for 1 hour as described in figure 1. I~NA was extracted and analysed by 1.7 per cent polyacrylamide gel electrophoresis as described in figure 1.

500

b) The same experiment was performed Ks in figure ~a, except that actinomycin D (0,05 ~xg/ml) was addled 15 sec. before the [methyl-SH] methionine labelling. © • ....

®

--(3 [methyl-all] • [14C] uridine

I

I

100

100

SLICE NUMBER

More r e c e n t sttLdies u s i n g f i n g e r p r i n t i n g technics after digestion of RCqA by T l - r i b o n u c l e a s e h a v e c h a r a c t e r i z e d the m e t h y l a t e d s e q u e n c e s in the p r e c u r s o r and m a t u r e ri,bosomat R,NA [15, 16]. It has been s h o w n that most of the m e t h y l a t i o n events o c c u r in the nucleolus at the first stage of the m a t u r a t i o n process, but t h e r e is e v i d e n c e for a f e w additiona'l m e t h y l a t i o n events on 28S and l g S after t h e i r departt~re f r o m t h e nueleolus. H o w ever, these affect only t h r e e p o i n t s w i t h i n 18S RN,A and faintly one p o i n t in 28S RNA, w h e r e a s 45S ~ICd%Au n d e r g o e s methylat i o n at some 110 p o i n t s w i t h i n its r i b o s o m a l s t r u t lure. T h e c o n c l u s i o n s d r a w n by the a u t h o r s on the m a t u r a t i o n p a t h w a y for r i b o s o m a l RNA are c o n s i s t e n t w i t h a r e c e n t s t u d y of our l a b o r a t o r y [17] p r e s e n t i n g a c o m p a r i s o n of the f i n g e r p r i n t s of T l - r i b o n u e l e a s e - d i g e s t e d 4.7S, 45S and 41S RNA w i t h f i n g e r p r i n t s of ~8,S a n d 18S RNA. T h e results p r e s e n t e d in this p a p e r lead us to p r o p o s e a d e s c a i p t i v e s c h e m e for the earliest met h y i a t i o n events on r i b o s o m a l p r e c u r s o r R NA. T h e

BIOCHIMIE, 1975, 57, n ° 11-12.

are m e t h y l a t e d , the early stages of b i o s y n t h e s i s of r i b o s o m a l I~NA in e u k a r y o t i e cells can be summ a r i z e d as fol~lows : 1) t r a n s c r i p t i o n of the ribosomal genes giving rise to 47S RNA, then c o n v e r ted to 46S RNA ; 2~) m e t h y l a t i o n of 47,S Riga (and possibly of 46S RNA) ; 3) m a t u r a t i o n of these produets to give 45S RNA.

Acknowledgments. We are gretcful to Professenr M. Boiron for encouragement and helpful discussion. R,~suMfi. 11 a 6t6 pr6e6demment montr6 que la ¢ fraction 45S >> des RNA ribosomiques immatures, chez les eucaryotes, 6tait eonstitu6e de trois esp6ces mol6eulaires de 47S, 46S et 45S. La m6thylation de ces trois RNA a pu ~tre raise en 6vidence eu analysant les RNA des celt.ules murines L5178 Y par 61ectrophor6sc en gel d'e polyaerylamide h 1,7 p. cent, apr6s un marquage court par la m6thion.ine [m~thyl-3H]. Des 6~udes comparatives des ein6tiques de marquage des RNA apr6s incubation pendant des temps courts en presence de

Methylation of ribosomal RNA. m6thionine [m6thyl-3Hl et d'uridine-[aH], ainsi que des experiences de marqu~age court par la m6thionine [m6thyl-aH] en pr6senee d ' a c t i n o m y c i n e D ont montr6 que la m 6 t h y l a t i o n des RNA r i b o s o m i q u e s i m m a t u r e s se p r o d u i t lorsque la t r a n s c r i p t i o n est termin6e, et que m 6 t h y l a t i o n et t r a n s c r i p t i o n ne sont pas eoordonn6es. Ces r6sultats nous conduisent h proposer le schema suivant p o u r la biosynth~se des RNA ribosomiq u e s : 1) Transcription des g~nes r i b o s o m i q u e s ; 2) M6thylation du RNA 47S (et 6ventuellement du RNA 46S) ; 3) Maturation de ees molecules pour d o n n e r le RNA 45S. REFERENCES. 1. Greenberg, H. ,~ P e n m a n , S. (1966) J. Mol. 2~1, 527-535. 2. Z i m m e r m a n , E. F. ,¢ Holier, B. (1967) J. MoL 2,3, 371-387. 3. Wagn.er, E. K., P e n m a n , S. ~ Ingram, V. M. J. Mol. Biol., 23, 149-161. 4. Weinberg, R. A. ~ P e n m a n , S. (1970) J. Mot. 47, 169-178.

BIOCHIMIE, 1975, 57, n ° 11-12.

Biol., Biol., (1967)

Biol.,

1321

5. Weinberg, R. A., Loening, U., Willems, M. P e n m a n , S. (1967) Proe. Nat. Acad. Sci. USA, 58, 1088-109'5. 6. Jeanteur, P., Amaldi, F. ~ Attardi, G. (1968) J. Mol. Biol., 33, 757-775. 7. Jeanteur, P. ,¢ Attardi, G. (1969) J. Mol. Biol., 45, 305-324. 8. Tioilais, P., Galibert, F. • Boiron, M. (1971) Proc. Nat. Acad. Sci. USA, 68, 1117-1120. 9. Auger, M. A. & Tiollais, P. (1973) Biochimie, 54, 163-169. 10. Tiollais, P., Galibert, F. & Boiron, M. (1971) Eur. J. Bioehem., !18, 35-45. 11. Tiollais, P., Galibert, F., Lepetit, A. & Auger, M. A. (1972) Biochimie, 54, 339-354. 12. Rogers, M. & Klein, G. (1972) Bioc~em. J., 130, 281-288. 13. Loening, U. E., Grierson, D., Rogers, M. & Sartirana, M. (1972) F E B S Syrup., 23, 395. 14. Scheer, U., Trendelenburg, M. & Franke, W. (1973) Exptl. Cell Res., 80, 175-190. 15. Maden, B. E. H., Salin, M. & Summers, D. (1972) Nature NeJm Biol., 237, 5-9. 16. Salim, M. ~ Maden, B. E. H. (1973) Nature, 244, 334-336. 17. Galibert, F., Tiollais, P. ~ Eladari, M. E. (1975) Eur. J. Bioehem., 55, 239-245.