Characterization of messenger RNA from embryonic muscle

BIOCHIMIE, 1972, 54, 187-194.

Characterization of messenger RNA from embryonic muscle. Daniel CAPUT, Denise LUZZATI and F r a n c o i s Gnos. S e r v i c e de P h y s i o l o g i e Cellulaire, l n s t i t u t de Biologie Moldculaire, Paris VII. (13/3/1972).

Summary. - - We have studied the polysomal patterns of three differentiating muscle tissues : neonatal rat heart, embryonic rat skeletal muscle and embryonic calf heart. They all display similar patterns, with a characteristic shoulder in the heavy polysome region (50 to 60 S ribosomes) which probably correspond to polyribosomic complexes participating in myosin synthesis. In fact, we have found that isolated polysomes from rat differentiating muscle synthetize in vitro at least two of the major muscle proteins, myosin and actin. RNA extracted from calf embryonic heart and embryonic muscle contains a 26 S component which can be considered as a presumptive messenger for myosin. This assumption is supported by the fact that the 26 S fraction of rat muscle polysomal RNA both stimulates aminoaeid incorporation in an E. Colt ¢ in vitro • protein synthetizing system as well as binding of f-met-tRNA to E. Colt ribosomes.

Much attention has been focussed, d u r i n g the last t h r e e years, on nmscle p r o t e i n - f o r m i n g systems f r o m avian or m a m m a l i a n origin. This interest is related w i t h the fact that fibrous musclespecific p r o t e i n s are r e l a t i v e l y easy to c h a r a c t e r i z e ; t h e i r p h y s i c o - c h e m i s l r y is well k n o w n and they constitute an a p p r e c i a b l e p o r t i o n of the total p r o t e i n s y n t h e s i z e d by muscle cells at the e m b r y o nic stage of t h e i r d e v e l o p m e n t . Moreover, established m y o b l a s t i c lines from rat h a v e been desc r i b e d w h i c h m a i n t a i n over m a n y g e n e r a t i o n s the p o t e n t i a l i t y to differentiate into m y o t u b e s [1]. Rat myoblasts c u l t u r e thus r e p r e s e n t a m a t e r i a l particularly suitable for i n v e s t i g a t i n g certain b i o c h e m i c a l aspects of the late steps of d i f f e r e n t i a t i o n in a p r e d e t e r m i n e d cell. Recent i s o l a t i o n [2] of temp e r a t u r e - s e n s i t i v e m u t a n t s affected in cell fusion f u r t h e r illustrates the v e r s a t i l i t y and interest of the system. One i m p o r t a n t tool for the study of muscle d i f f e r e n t i a t i o n resides in the possibility to easily c h a r a c t e r i z e m e s s e n g e r RNAs c o r r e s p o n d ing to the most p r o e m i n e n t p r o t e i n species synthesized in the system. Considerable progress has been m a d e in this area w i t h the w o r k of Heyw o o d , R i c h and t h e i r associates [3, 4, 5]. Myosin, actin or t r o p o m y o s i n s p e c i f i c p o l y s o m e s h a v e been c h a r a c t e r i z e d a m o n g the m u l t i r i b o s o m a l c o m p l e x isolated from c h i c k skeletal muscle. E a c h size class of p o l y s o m e s w a s s h o w n to synthesize in vitro one of the m a j o r r e p r e s e n t a t i v e s of m u s c l e fibrous proteins. Moreover, from the large m y o s i n specific cluster, i n c l u d i n g 5.0-60 ribosomes, a 26 S RNA species could be c h a r a c t e r i z e d by sucrose

density gradient c e n t r i f u g a t i o n ; this RNA h a d the c a p a c i t y to stimulate synthesis of a 200,000 MW m y o s i n subunit w h e n a d d e d to p r e i n c u b a t e d muscle 75 S ribosomes. A 12 S, p r e s u m a b l y actins p e c i f i c m e s s e n g e r has also been d e s c r i b e d [5]. The p r e s e n t w o r k r e p o r t s on the e x i s t e n c e o | s i m i l a r 26 S and 12 S RNA c o m p o n e n t s in polysomes d e r i v e d f r o m d i f f e r e n t i a t i n g rat h e a r t and skeletal m u s c l e and front foetal calf heart, a n d p r o v i d e s p r e l i m i n a r y e v i d e n c e that these RNA's r e p r e s e n t m y o s i n and actin s p e c i f i c mRNA's. MATERIAL AND METHODS. a.R a d i o a c t i v e labeling of e m b r y o n i c call h e a r t slices. The hearts f r o m 3 m o n t h s calf e m b r y o s h a v e been collected at the slaughter house (Sorga), w a s h e d w i t h cold saline and keyt on ice d u r i n g t r a n s p o r t a t i o n (ca. 1/2 h). 0.2-0.3 m m t h i c k slices (about 10 g of tissue) w e r e i m m e d i a t e l y p r e p a r e d and r i n s e d in cold saline b e f o r e i n c u b a t i o n in 20 ml ¢ Ham ~> m e d i u m (c) s u p p l e m e n t e d w i t h 10 mc of pa2. I n c u b a t i o n was c a r r i e d out for t w o h o u r s at 32°C u n d e r aeration. The slices w e r e then r i n s e d t h r e e times w i t h cold saline and h o m o g e neized in MSB [3] (see c o m p o s i t i o n in section <( c >>) w i t h a D o u n c e H o m o g e n i z e r . b - - R a d i o a c t i v e labeling at e m b r y o n i c skeletal muscle. 17 days p r e g n a n t rats w e r e injected w i t h 1 m c H a n r i d i n e (specific activity 10 C / t a m a l e s ) inside 13

188

D. C a p u t , D. L u z z a l i a n d F. Gros.

of the vittelin vein after a surgical i n c i s i o n i n the a b d o m i n a l cavity a c c o r d i n g to C. Petter [6]. After 1 hour, the embryos were r e m o v e d from the u t e r i n e horns, put i n cold saline and t h e i r leg muscles dissected, washed and homogenized as in

samples were layered on gels in 5 p. cent sucrose. The gels were s c a n n e d at 2,80 m.~ in a 24.00 Gifford s p e c t r o p h o t o m e t e r ; r a d i o a c t i v i t y b e i n g determined as already m e n t i o n e d .

(a).

eI n c o r p o r a t i o n of 1',C labeled aminoacids into nascent, p o l y s o m e - b o u n d proteins.

c - - P o l y s o m e extraction, sucrose gradient analysis. In order to p r e p a r e polysomes, the tissues (emb r y o n i c calf heart, rat e m b r y o n i c skeletal muscle of n e a n a t a l rat heart) were m i n c e d a n d gently homogenized in 2 volumes (w/v) of cold MSB buffer [3j (250 mM K'CI: 10 mM MgC12 ; 10 m~M Tris-HC1 pH 7.5) w i t h 20 strokes of a loose fitting Dounce homogenizer. After c e n t r i f u g i n g 10 minutes at 10,00,0 g a n o n - i o n i c detergent ¢ N.P-40 ~> was added to the s u p e r n a t a n t (S 10) at a c o n c e n t r a t i o n of 0.5 p. cent. Polysomes were either fract i o n a t e d by s e d i m e n t a t i o n in a 15-40 p. cent l i n e a r sucrose gradient in MSB buffer or pelleted on a sucrose c u s h i o n a c c o r d i n g to Noll [7~.

Polysomes p r e p a r e d as described i n ¢ c >> were r e s u s p e n d e d i n MS.B buffer c o n t a i n i n g 150 mM KCI and i n c u b a t e d w i t h the following reagents in a total volume of 1 ml : KC1, 150 mM ; MgC12, 5 mM ; Tris-HC1 10 mM ; ~ m e r c a p t o e t h a n o l 5 mM ; ATP 1 mM GTP, 0.2 mM ; CTP, 0.1 mM ; phosp h o e n o l pyruvate, p y r u v a t e kinase, 50 ~g ; SJS° fraction from n e o n a t a l liver, 0.6 mg ; polysomes, 0.5 mg ; tRNA, 100 ~g. 1 ~curie of a complete 1~C labeled a m i n o a c i d m i x t u r e was added (specific activity 50 m c / n l o l e ; final radioactivity, 0.1 m c / ml). After i n c u b a t i o n for 60 m i n u t e s at 37°C, the r e a c t i o n mixture was made 0.5 mM in KC1 (final c o n c e n t r a t i o n ) a n d s e d i m e n t e d for 1 h o u r at 10,5,000 g. The resulting s u p e r n a t a n t was dialyzed o v e r n i g h t against a 20 mM potassium buffer (K4 P207), pH 8.5, c o n t a i n i n g 1 p. cent casaminoacids.

W h e n RNA h a d to be extracted for f u r t h e r analysis the polysomal pellets was s u s p e n d e d in NETS buffer (100 raM NaC1 E D T A ; 10 mM Tris HCl pH 7.5 ; 0.5 p. cent SDS) and, after 5 m i n u t e s of i n c u b a t i o n at 37°C, the s u s p e n s i o n was l a y e r e d on a 5-20 p. cent sucrose gradient in NETS buffer c o n t a i n i n g 0.2 p. cent SDS.

d - - Gel electrophoresis o[ n e w l y s y n t h e s i z e d proteins or of R N A samples. F o r the electrophoretic s e p a r a t i o n of proteins, we have used a modification of W e b e r a n d Osborn t e c h n i q u e [8~, u s i n g 5 p. cent a c r y l a m i d e gels in w h i c h b i s - a c r y l a m i d e was r e p l a c e d by ethylenediacrylate. The electrophoresis buffer was PO4H Na 2 0.1 M pH 7.0, 0.1 p. cent SDS. Eleetrophoresis was c a r r i e d out for 5 hours at 25°C with an i n t e n sity of 8 m A / t u b e . Gels were either s t a i n e d w i t h COolnassie blue or cut into 1 m m thick slices. Slices were solubilized in 0.5 ml M/1 p i p e r i d i n at 6p0~C for 2 hours. Radioactivity was d e t e r m i n e d in a liquid s c i n t i l l a t i n g c o u n t e r (with an efficiency for 14C r a d i o a c t i v i t y close to 30 p. cent) a c c o r d i n g to Choules and Z i m m [9]. RNA electrophoresis was p e r f o r m e d a c c o r d i n g to L o e n i n g [8]. A c r y l a m i d e recristallised i n chloroform a n d acetone purified b i s - a c r y l a m i d e were utilized : the gels c o n t a i n e d a c r y l a m i d e ('final conc e n t r a t i o n 2.4 p. cent) plus b i s - a c r y l a m i d e (5 p. cent of the a c r y l a m i d e c o n c e n t r a t i o n ) . The electrophoresis buffer c o n t a i n e d 4.0 mM Tris ; 30 mM Nail 2 PO 4 pH 7.8; 1 mM EDTA a n d 0.2 p. cent SDS. Samples were electrophoresed for 3 h o u r s (7 volts/cm ; 5 mA/gel) at 24°C) at 24°C. RNA BIOCHIMIE, 1972, 54, n ° 2.

0.1 nfl of the dialyzed solution was m i x e d w i t h 1 p. cent SDS a n d 5 p. cent sucrose, heated at 100°C for 10 m i n u t e s a n d layered on the acrylamide gel.

gStimulation o[ protein s y n t h e s i s in a E. colt S so fraction, by RNA f r o m e m b r y o n i c mllscle. Various sucrose gradient fractions r e s u l t i n g from s e d i m e n t a t i o n of a p h e n o l extracted RNA p r e p a r a t i o n were freed of SDS by a d d i t i o n of cold 1 KC1 ; SDS was r e m o v e d by centrifugation and the RNA c o n t e n t of each f r a c t i o n was p r e c i p i t a t e d w i t h 2.5 volumes of cold ( - - 2 0 ° C ) ethanol in the p r e s e n c e of 10,0 ~g E. colt tRNA as carrier. Each p r e c i p i t a t e d sample was washed 3 times w i t h cold 70 p. cent ethanol a n d assayed for its ability to stimulate 14C a m i n o a c i d i n c o r p o r a t i o n in an E. colt 30S fraction. The r e a c t i o n m i x t u r e c o n t a i n e d the following reagents (in 1 ml) : prei n c u b a t e d $39 extract (Nierenberg and Matthaei), 250 ~1 ; Tris-HCl, pH 7.8, 20 mM ; NH~C1, 50 m M ; Mg acetate, 7.5 mM ; u n i f o r m l y labeled ~4C aminoacid mixture, 1 ~c ; ATP, 5 m M ; GTP, 0.25 mM ; P.E.P. 5 m M ; pyruvate-kinase, 10 ~g ; t e t r a h y d r o folic acid, 30 ~g, CTP a n d UTP, 0.15 mM. After 1 h o u r of i n c u b a t i o n at 37°C samples were precipitated in 10 p. cent TCA c o n t a i n i n g 1 p. cent casein hydrolysate. After 15 nfin h e a t i n g at 9'0°C, the r e s u l t i n g pellets were w a s h e d w i t h 5 p. cent

Characterization of messenger RNA [rom embryonic muscle. TCA c o n t a i n i n g 1 p. c e n t c a s m i n o - a c i d s , d r i e d o n nitrocellulose membrane filters before counting in a scintillation counter.

189

a l r e a d y o b s e r v e d [2] i n t h e c h i c k m u s c l e s y s t e m . It e x h i b i t s a c h a r a c t e r i s t i c (( s h o u l d e r >) c o r r e s p o n d i n g to v e r y l a r g e r i b o s o m a l c l u s t e r s ( c l a s s A) p l u s

RESULTS. I. - -

RAT MUSCLE SYSTEMS.

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Numerous studies have been previously devoted to t h e a n a l y s i s of p o l y r i b o s o m e s f r o m e m b r y o n i c chick muscle and these polyribosomes were shown to i n c l u d e at l e a s t t h r e e m a j o r c l a s s e s . O n e c l a s s of l a r g e p o l y r i b o s o m e s c o n t a i n i n g 50-60 r i b o s o m e s [

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FI6. 1. - - P o l y s o m e s e d i m e n t a t i o n profile of neonatal rat heart muscle. Neonatal rat h e a r t muscles have been homogenized in MSB buffer aeeording to the procedure outlined in ~ Material a n d Methods >>. The p o s t - m i t o e h o n d r i a l s n p e r n a t a n t fraction was layered on a 15-4~0 p. cent sucrose gradient. C e n i r i f u g a t i o n was for '1 h o u r s at 4°12, according to Noll [7]-

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p r o v e d to b e a c t i v e i n t h e s y n t h e s i s of smaller multiribosomal complexes (desiB a n d C) w e r e s h o w n to c a r r y o u t t h e of a c t i n a n d t r o p o m y o s i n [3, 4].

Figure 1 describes a typical polysomal pattern from differentiating rat muscle (neonatal rat h e a r t ) (*). T h i s p a t t e r n v e r y m u c h r e c a l l s t h a t (*) An identical p a t t e r n was c u r r e n t l y o b t a i n e d w i t h skeletal muscle of 18 days r a t embryos. BIOCHIMIE, 1972, 54, n ° 2.

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Fro. 2b. - - Electrophoretic pattern of n e w l y s y n t h e s i z e d proteins f r o m a <> rat liver p o l y s o m a l fraction. This e x p e r i m e n t constitutes a control for e x p e r i m e n t 2a. The total liver polysomal f r a c t i o n derived from the same a n i m a l s as those used in e x p e r i m e n t 2a was incubated u n d e r conditions p e r m i t t i n g radioactive a m i n o acid i n c o r p o r a t i o n into proteins. As a source for soluhle p r o t e i n s or effectors, a $15of r a c t i o n f r o m e m b r y o n i c skeleton muscle c o n t a i n i n g soluble m y o s i n and acting was utilized. Electrophoresis was p e r f o r m e d on parallel gels as fig. 2a.

D. Caput, D. L u z z a t i a n d F. Gros.

190

p r o p o r t i o n of clusters c o n t a i n i n g more t h a n two ribosomes. Trace a m o u n t s of RNAase convert the polysomal fractions chiefly into a 75S peak characteristic of monosomes ; a sulall RNAase resistant fraction is h o w e v e r noticeable.

Although electron m i c r o s c o p i c e x a m i n a t i o n of classes <> a n d ¢ B >> has confirmed the p r o e n d n e n c e of large r i b o s o m a l clusters (50-60 ribosomes) i n A, an a p p r e c i a b l e p o r t i o n of these putative m y o s i n f o r m i n g complexes was also f o u n d to be p r e s e n t i n the ¢ B >> subclass. To characterized the m a j o r p r o t e i n p r o d u c t s synthesized by the rat muscle polysomes, the total polysome fractions (with the exclusion of disomes) was i n c u b a t e d w i t h a complete 14C labeled a m i n o - a c i d mixture, u n d e r the c o n d i t i o n s described i n ¢ Material a n d Methods >> (c). F i g u r e 2a shows the r a d i o a c t i v i t y profile of the n e w l y syn-

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E t h a n o l - p r e c i p i t a t e d s u b f r a c t i o n ¢ A >> d e r i v e d f r o m the sucrose gradient sedimented material described in ¢ 3 a >> w a s s u b m i t t e d t o gel e l e e t r o p h o r e s i s a s d e s cribed in the text. T o t a l OD p r o f i l e (260 n m ) . ©--© © 3H r a d i o a c t i v i t y d i s t r i b u t i o n p r o f i l e of RNA f r a c t i o n <> d e r i v e d f r o m m u s c l e m o n o somes. A--&--A 3H r a d i o a c t i v i t y p r o f i l e of RNA f r a c t i o n <> d e r i v e d f r o m m u s c l e p o l y r i b o s o m e s .

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3 H - u r i d i n e l a b e l e d RNA f r o m t h e t o t a l r a t m u s c l e p o l y s o m e s w a s c e n t r i f u g e d on a 5-20 p. c e n t s u c r o s e g r a d i e n t in N e t s b uffer. C e n t r i f u g a t i o n w a s f o r 4 h o u r s at 40,000 r p m in a S W 41 r o t o r . O.D. a t 260 n m . O--O--O (3H) cp m. T h e d a s h e d z o n e (~ A >) c o r r e s p o n d s to t h e p a r t of t h e gradient subfraction that was ethanol-precipitated and f u r t h e r a n a l y z e d b y gel e l e c t r o p h o r e s i s as i n d i c a t e d in Fig. 3b.

BIOCHIMIE, 1972, 54, n ° 2.

thesized p r o t e i n s analyzed on a c r y l a m i d e gels a n d p o s i t i o n of n o n - r a d i o a c t i v e m y o s i n or actin markers r u n on parallel gels. One can c o n c l u d e that 6.0-70 p. cent of the 1~C label is associated w i t h three m a i n fractions, two of w h i c h can be identified-from the positions of the cold p r o t e i n m a r k e r s - w i t h the large m y o s i n s u b u n i t (MW : 200,00~0) and with the actin monom e r (1WW : 65,000). A t h i r d peak c o r r e s p o n d i n g to

Characterization of m e s s e n g e r RNA f r o m e m b r y o n i c muscle.

191

a p r o t e i n of 30,000 MW m a y r e p r e s e n t a t r o p o m y o s i n m o n o m e r . I n a d d i t i o n to t h e s e t h r e e p r o e minent components, other polypeptides, which fail to f o r m w e l l d e f i n e d p e a k s , a r e also o b s e r TABLE I.

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vable. T h e y m i g h t c o r r e s p o n d e i t h e r to p r o t e i n s of d i f f e r e n t n a t u r e , to n a s c e n t fibrous p r o t e i n s c h a i n s o r to s o m e d e g r a d a t i o n p r o d u c t s . W h e n class <~A ~ o r ¢ B ~> p o l y s o m e s w e r e s e p a r a t e l y p r o c e s s e d a n d t h e i r in vitro s y n t h e s i z e d

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gradient fractions of muscle polgsomal RNA. For details see the text. Each gradient subtraction was precipitated in cold ethanol, redissolved in a suitable buffer and assayed for its ability to stimulate anainoacid incorporation in the total protein fraction of an E. coli $3o extract. Endogenous activity (without addition of exogenous messenger) corresponded to 1 00,0 epm ; it was subtracted from the counts obtained after addition of the RNA fractions to draw the stimulation profile. O.D. profile. © -© Stimulation profile. BIOCHIMIE, 1972, 54, n ° 2.

I lO Tubes Fro. 5. - - Sucrose gradient distribution profile of total polgsomes from fetal calf heart. The post-mitochondrial supernatant fraction from minced, 2 hours p~2 labeled calf heart tissues was layered on a 10-40 p. cent sucrose gradient under the conditions described in • Material and Methods ~. Centrifugation was for zb0 rain. at 40,000 rpm (SW 41 rotor). OD profile at 260 rim. o--o--o Aeid precipitable ps2 radioactivity.

D. Caput, D. Luzzati and F. Gros.

192

p r o t e i n p r o d u c t s analyzed, the same results w e r e o b t a i n e d as those just d e s c r i b e d w i t h the total p o l y s o m a l fraction. E v e n t h o u g h these results w e r e in a g r e e m e n t w i t h e l e c t r o n m i c r o s c o p e observations on these fractions, a p o s s i b i l i t y existed that the r a d i o a c t i v i t y p r e s e n t at the m a r k e r positions of the t h r e e m a j o r fibrous p r o t e i n s d i d originate f r o m p a s s i v e t r a p p i n g . F i g u r e 2b shows a c o n t r o l e x p e r i m e n t in w h i c h rat liver p o l y o m e s w e r e i n c u b a t e d w i t h r a d i o a c t i v e a m i n o a c i d s in the p r e s e n c e of a muscle $150 s u p e r n a t a n t fraction.

analysis of total p o l y s o m a l RNA f r o m 18 days rat e m b r y o s muscle, labeled for one h o u r w i t h aH uridine, fails h o w e v e r to r e v e a l RNA chains w i t h s e d i m e n t a t i o n constants d i s t i n c t f r o m 1 8 S and 28 S r i b o s o m a l I~NA's (fig. 3). That this situation is p r o b a b l y due to the m a s k i n g of m y o s i n and actin messengers by h i g h l y l a b e l e d r i b o s o m a l RNA can be s h o w n as follows : f r a c t i o n s d e s i g n a t e d as

t28

/000

800

0.6 •800 dt 600

06

1 0.~,

$O0 e~

,~oo

400 _ 200

200

I 20

I I0

Tubes

Fro. 6a. - - Sucrose gradient analysis of p3~ labeled calf heart polysomal RNA. OD, 260 nm. • --•

•

p~2

epm.

For the details, see the text.

The n e w l y s y n t h e s i z e d p r o t e i n p r o d u c t s w e r e processed for a c r y l a m i d e gel analysis the same w a y as p r e v i o u s l y . It is clear that v e r y little r a d i o a c t i vity is detectable at the p o s i t i o n s w h e r e the cold actin and m y o s i n m a r k e r s migrate. Since in vitro i n c u b a t e d r a t muscle p o l y s o m e s a p p e a r to m a n u f a c t u r e mostly the m a j o r classes of fibrous p r o t e i n s n o r m a l l y s y n t h e s i z e d in vivo, one could e x p e c t cells f r o m w h i c h these p o l y s o m e s are d e r i v e d to p r e f e r e n t i a l l y synthesize the messengers specific for these proteins. Sucrose g r a d i e n t BIOCHIMIE, 1972, 54, n ° 2.

I

I

20

--

Tube~

I

- v |

tO

Fro. 6b. - - Sucrose gradient analysis of ps~ labeled RNA derived from calf heart monosomes. OD, 260 nm. o--o--$ p32 distribution profile.

<> on Fig. 3a, a n d c o r r e s p o n d i n g to the lightest edge of the 28 S c o m p o n e n t , w e r e pooled, p r e c i p i t a t e d in cold ethanol and f u r t h e r a n a l y z e d by a c r y l a m i d e gel e l e c t r o p h o r e s i s (fig. 3b). A RNA c o m p o n e n t m i g r a t i n g faster than 28 S RNA and c o r r e s p o n d i n g to 26 S m a t e r i a l , was c l e a r l y detectable. W h e n a s i m i l a r e x p e r i m e n t was r e p e a t e d w i t h ethanol p r e c i p i t a t e d A f r a c t i o n s p r e p a r e d f r o m R,NA e x t r a c t e d f r o m <> o r <> of the same p o l y s o m a l gradient, no

C h a r a c t e r i z a t i o n of m e s s e n g e r R N A f r o m e m b r y o n i c m u s c l e . 26 S RNA c o m p o n e n t could be detected. I n addition, the polysomal extracted <<28 S )> RNA exhibits a m u c h higher specific r a d i o a c t i v i t y t h a n 28 S ribosomal RNA strongly suggesting that the former p r o b a b l y represents the myosin-specific messenger, s i m i l a r to the (< 26 S >> fraction p r e v i o u s l y described by Heywood i n e m b r y o n i c chick muscle

[5]. An a d d i t i o n a l c r i t e r i o n for the p r e s e n c e of a 26 S RNA w i t h a p r o b a b l e m y o s i n messenger function w o u l d reside i n the d e m o n s t r a t i o n that total polysomal RNA from rat e m b r y o n i c muscles includes a fraction w i t h this s e d i m e n t a t i o n value that can stimulate in vitro p r o t e i n synthesis. Hence, various g r a d i e n t s u b f r a c t i o n s of polysomal RNA from e m b r y o n i c rat muscle were tested for their ability to stimulate a m i n o a c i d i n c o r p o r a t i o n in a p r e i n c u b a t e d E. colt $30 extract. As s h o w n on Fig. 4, the capacity to stimulate a m i n o a c i d incorp o r a t i o n was located i n some relatively well defined fractions (I, II a n d III). F r a c t i o n I s e d i m e n t e d at about 2,6 S. The n a t u r e of the p r o t e i n formed i n response to this muscle 2.6 S RNA is c u r r e n t l y investigated. F u r t h e r evidence for messenger activity of this 26S c o m p o n e n t is p r o v i d e d b y a f - Met- tRNA b i n d i n g s t i m u l a t i o n e x p e r i m e n t (Table I). While the 26 S RNA s u b f r a c t i o n very significantly stimulates f-Met-tRNA b i n d i n g to 70 S E. colt ribosomes, s u p p l e m e n t e d w i t h crude initiation factors, no such s t i m u l a t i o n is observable w i t h 30 S fraction from the same gradient.

II.

--

EMBRYONIC

CALF

HEART

MUSCLE.

A s i m i l a r search for a m y o s i n specific messenger was u n d e r t a k e n w i t h a n o t h e r muscle system, the calf e m b r y o n i c heart. A t y p i c a l polysomal dist r i b u t i o n p a t t e r n extracted from 3 m o n t h s emb r y o n i c calf heart is s h o w n on Figure 5, w i t h its heavy shoulder in the <> region. To characterize the n e w l y synthesized RNA from such a system, calf heart slices were i n c u b a t e d for two h o u r s in the presence of aup. RNA derived from the polysomal region or from 75 S monosomes was analyzed on sucrose g r a d i e n t (Fig. 6a and b). It is clear that, i n this system, c o n t r a r y to what is observable in rat e m b r y o n i c muscle, there is little or no synthesis of ribosomal RNA (fig. 6a). Moreover, 2 hours 32p labeled RNA shows a very different h y d r o d y n a m i c b e h a v i o u r d e p e n d i n g on w h e t h e r it derives from the <> of <> fractions. It is only i n this latter case that a clear 2,6 S s e d i m e n t i n g c o m p o n e n t could be detected in a d d i t i o n to a 22 S fraction. No sup labeled RNA's of such high s e d i m e n t a t i o n cons-

BIOCHIMIE, 1972, 54, n ° 2.

193

rant could be observed after the polysome fraction had been p r e t r e a t e d w i t h trace a m o u n t s of ribonuclease. DISCUSSION. Advantages i n h e r e n t to the muscle system for the p r e p a r a t i o n of polysomal structures a n d study of their b i o s y n t h e t i c activity have already been outlined by Heywood and Rich [3-4-5] : low level of free r i b o n u c l e a s e in the tissue, lack of ribosomal a t t a c h m e n t to the e n d o p l a s m i c r e t i c u l u m , easiness in the c h a r a c t e r i z a t i o n of high m o l e c u l a r weight fibrous p r o t e i n s a m o n g the n e w l y synthesized products. Aside from these general features, w h i c h make tim muscle system suitable for translation studies, the rat skeletal muscle cells can be cultured in vitro a n d their differentiation from y o u n g myoblasls can easily be followed i n cell cultures [9, 11, 12, 13]. The p r e s e n t w o r k points t o w a r d s the remarkable analogies existing i n the polysomal p a t t e r n of d i f f e r e n t i a t i n g muscle from w i d e l y different origins : rat n e o n a t a l heart e m b r y o n i c rat skeletal muscle as well as e m b r y o n i c calf heart, all c o n t a i n ribosomes engaged in more or less the same type of f u n c t i o n a l complexes as e m b r y o n i c chick muscle. W o r t h of notice is the p r e s e n c e of very heavy clusters (50-60 ribosomes) w h i c h are p r o b a b l y involved i n m y o s i n synthesis. The various systems studied thus p r e d o m i n a n t l y m a n u f a e t u r e the three m a j o r fibrous p r o t e i n m o n o m e r s : the large myosin s u b u n i t as well as actin a n d t r o p o m y o s i n monomers. I n the three instances has a 26 S RNA w i t h messenger properties been characterized. Although it is only in the case of the chick system that this RNA has been s h o w n to direct in vitro m y o s i n synthesis ESJ, there is strong suggestion that the 2,6 S RNA from tile two other systems studied i n this work fulfils the same role. Studies are c u r r e n t l y engaged to characterizedthe p r o t e i n s of w h i c h synthesis is stimulated in an in vitro E. colt systems as well as in ascites t m n o r cell S30 extracts. A p r o b l e m of m a j o r interest is w h e t h e r genes r e s p o n s i b l e for the synthesis of fibrous p r o t e i n s are already t r a n s c r i b e d at the early stage of myoblastie differentiation, i.e. specially before a n y increase in the rate of m y o s i n a n d actin synthesis. The possibility of getting 26 S RNA w i t h a very high specific r a d i o a c t i v i t y from both the rat a n d calf systems should p e r m i t use of D N A - R N A a n n e a l i n g studies to tackle this problem.

D. Caput, D. L u z z a t i and F. Gros.

194 Acknowledgements.

W e w o u l d like to t h a n k Dr. L. B e n e d e t t i f o r p e r f o r m i n g t h e e l e c t r o n m i c r o s c o p e e x a m i n a t i o n of o u r p o l y s o m e f r a c t i o n s , Dr. P e t t e r f o r h i s h e l p a n d i n s t r u c tions in pregnant rat intravitellin vein injections and T. T h i b a u t f o r h i s h e l p i n t h e b i n d i n g e x p e r i m e n t s . T h e t e c h n i c a l a s s i s t a n c e of G. D r u g e o n in gel e l e c t r o p h o r e s i s of m u s c l e p r o t e i n s is g r a t e f u l l y a c k n o w l e d g e d . T h i s w o r k s u p p o r t e d b y f u n d s f r o m : t h e f o n d s de d d v e l o p p e m e n t d e la R e c h e r c h e S c i e n t i f i q u e et T e c h nique, the Commissariat h l'Energie Atomique, the C e n t r e N a t i o n a l de la R e c h e r c h e S c i e n t i f i q u e , t h e L i g u e N a t i o n a l e F r a n g a i s e C o n t r e le C a n c e r , a n d t h e F o n d a t i o n p o u r la R e c h e r c h e Mddicale F r a n q a i s e . R~SUM~. N o u s a v o n s d d t e r m i n d . le profil d e s p o l y s o m e s de t r o i s t i s s u s m u s c u l a i r e s en e o u r s de d i f f d r e n c i a t i o n : le cceur de v e a u e m b r y o n n a i r e . L e s profils o b s e r v d s p o u r ces trois~ t i s s u s s o n t tr~s s i m i l a i r e s , e n p a r t i e u l i e r ils p r d s e n t e n t u n d p a u l e m e n t e a r a c t d r i s t i q u e d a n s la r d g i o n d e s p o l y s o m e s <> (50 h 60 r i b o s o m e s ) q u i c o r r e s p o n d e n t tr~s p r o b a b l e m e n t h d e s c o m p l e x e s p o l y r i b o s o m i q u e s e n g a g d s d a n s la s y n t h ~ s e d e s m o l ~ cu'les de m y o s i n c . E n f a R , n o u s a v o n s t r o u v 6 q u e les p o l y s o m e s p r o v e n a n , t d e ~ i s s u s m u s c u l a i r e s de r a t s y n thdtisen,t (< in v i t r o • d e s q u a n t i t d s d d t e e t a h l e s de m y o s i n e et d ' a e t i n e . L e s R N A e x t r a i t s de p o l y s o m e s de eceur de v e a u e m b r y o n n a i r e a i n s i q u e de m u s c l e s s q u e l e t t i q u e s d ' e m -

BIOCHIMIE, 1972, 54, n ° 2.

b r y o n s de r a t c o n t i e n n e n t u n c o m p o s a n t s d d i m e n t a n t h 26 S q u e l ' o n s u p p o s e ~tre le m e s s a g e r de la m y o s i n e . Cette s u p p o s i t i o n e s t d t a y d e p a r le f a i t q u e la f r a c t i o n <<26 S >> p r o v e n a n t de R N A p o l y s o m i q u e s de m u s c l e s e m b r y o n o a i r e s de r a t est c a p a b l e h la fois de s t i m u l e r l ' i n c o r p o r a t i o u d ' a c i d e s a m i n d s d a n s des p r o t d i n e s l o r s q u ' o n I ' a j o u t e h u n s y s t ~ m e de s y n t h ~ s e p r o t d i q u e <>, A. P. vol. II. 8. W e b e r , K., O s b o r n , M. (19.69) J. Biological Chem., 244, 4406. 9. C h o u l e s , C. L. • Z i m m , B. H. (19'65) Anal. Biochemistry, 336, 13. 10. L o e m i n g , U. E. (1967) Biochem. J., 102, 251. 11. Yaffe, D. (1969) Current Topics in Dcv. Biol., 4, 37. 12. R i c h l e r , C. ~ Yaffe, D. (1970~ Dev. Biol., 2 3 , 1 . 13. Yaffe, D. (1971) Exptl. Cell. Res., 66, 33.