Brain nuclear and cytoplasmic ribonucleoproteins carrying DNA-Like RNA: Comparison of their proteins with those from soluble nuclear and cytoplasmic supernatants by polyacrylamide gel electrophoresis

BIOCHIMIE, 1972, 54, 11169-1178.

Brain nuclear and cytoplasmic ribonucleoproteins carrying DNA-Like RNA : comparison of their proteins with those from soluble nuclear and cytoplasmic supernatants by polyacrylamide gel electrophoresis. H. ~-ATRINGE (*) a n d M. JACOB ('*). Centre de Neurochimie du CNRS et Unit~ de Recherches Fondamentales sur la Biochimie de la Cellule Cancdreuse de I'INSERM, Facult~ de Mddecine, 6 7 - S l r a s b o u r g (France). (8/3/1972).

Summary. - - The p r o t e i n s from n u c l e a r a n d cytoplasmic dRNP a n d p o s t - p a r t i c u l a t e s u p e r n a t a n t s from r a t b r a i n were a n a l y s e d by p o l y a e r y l a m i d e gel electrophoresis at pH 4.5 in u r e a and i n the presence of sodium dodecylsulphate. The two m a j o r p r o t e i n s f r o m n u c l e a r dRNP, h a d a m o l e c u l a r weight of 35,000 and -10,000. The soluble n u c l e a r extract c o n t a i n e d the 35,000- 40,000 p r o t e i n s a n d in a d d i t i o n several p r o t e i n s of h i g h e r m o l e c u l a r weight (40,000 to 120,000). It is suggested t h a t native dRNA associates specifically w i t h the 35,000-40,000 nuclear soluble p r o t e i n s to form dRNP. The p r o t e i n p a t t e r n of cytoplasmic dRNP was more complex t h a n t h a t of nuclear dRNP. Most p r o t e i n s were found in a range of m o l e c u l a r weights f r o m 40,000 to 70,000. T h e i r p a t t e r n was i n m a n y ways analogous w i t h t h a t of the soluble cytoplasmic proteins. P r o t e i n s w i t h m o l e c u l a r weights f r o m 32,000 to 40,000 close to those of the m a j o r nuclear dRNP were also present in the cytoplasmic dRNP. These proteins w e r e relatively enriched in cytoplasmic dRNP p r e p a r e d u n d e r e x p e r i m e n t a l conditions w h i c h m a y have been effective in e l i m i n a t i n g c o n t a m i n a t i n g soluble proteins. Therefore, t h e r e are some indications t h a t cytoplasmic dRNP m a y be derived f r o m nuclear dRNP b u t no final conclusions can as yet be drawn. Other m e t h o d s of analysis, such as c o m p a r i s o n of p r i m a r y structures m u s t be p e r f o r m e d in order to resolve this u n c e r t a i n t y .

INTRODUCTION. S e v e r a l y e a r s ago, it w a s s u g g e s t e d [1] t h a t D N A - l i k e R N A ( d R N A ) is t r a n s p o r t e d f r o m t h e n u c l e u s to t h e c y t o p l a s m i n t h e f o r m of r i b o n u cleoprotein particles (dRNP). The cytoplasmic d R N P (or i n f o r m o s o m e s [2]) c o n t a i n d R N A of t h e size of m e s s e n g e r R N A [3, 4]. O n t h e o t h e r h a n d , it has been suggested that the nucleotide sequences of m e s s e n g e r R N A w e r e d e r i v e d f r o m t h e h i g h m o l e c u l a r w e i g h t d R N A [5, 6, 7, 8, 9] f o u n d i n t h e n u c l e u s i n t h e f o r m of d R N P [10]. However it has not yet been shown that the prot e i n s of t h e n u c l e a r a n d c y t o p l a s m i c d R N P a r e related. Furthermore, it has been shown that, w h e n a n y R N A is a d d e d to a c y t o p l a s m i c s u p e r natant, ribonucleoproteins, with the low densities (*) Attachde de Recherche au CNRS. (**) Maitre de Recherche au CNRS.

i n CsCI c h a r a c t e r i s t i c of <> d R N P i s o l a t e d f r o m t i s s u e e x t r a c t s , a r e f o r m e d [11, 12]. T h u s t h e possibility that cytoplasmic dRNP are formed by non-specific binding of soluble proteins to cytop l a s m i c d R N A m u s t b e c o n s i d e r e d . T h a t <> dRNP are formed in this way has been questioned since there are differences in stability between <> a n d <> d R N P [13, 14]. C o m p a r a t i v e a n a l y s i s of t h e p r o t e i n s of n u c l e a r a n d c y t o p l a s m i c d R N P a n d s o l u b l e p r o t e i n s is necessary to decide whether cytoplasmic dRNP a r e r e a l a n d r e l a t e d to n u c l e a r d R N P . I f d R N A is t r a n s p o r t e d f r o m t h e n u c l e u s t o t h e c y t o p l a s m as d R N P , t h e p r o t e i n s of t h e n u c l e a r a n d c y t o p l a s m i c dRNP should he related. On the other hand, if the cytoplasmic dRNP are artifacts, their proteins s h o u l d b e m o r e c l o s e l y r e l a t e d to t h e p r o t e i n s of the cytoplasmic soluble protein pool. The proteins of n u c l e a r d R N P a n d o f c y t o p l a s m i c d R N P , p r i -

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m a r i l y those released from polysomes by EDTA, have already been analysed [14-25] but as yet the p r o t e i n s of free cytoplasmic dRNP have not been studied. Nor have these studies i n v o l v e d comparison w i t h the c o r r e s p o n d i n g pools of n u c l e a r a n d cytoplasmic soluble proteins. This p a p e r is the result of a systematic c o m p a r i s o n of the p r o t e i n s of n u c l e a r a n d cytoplasmic dRNP and soluble fractions.

METHODS. All p r e p a r a t i o n s w e r e p e r f o r m e d at 0-4 ° unless otherwise i n d i c a t e d .

Preparation o[ nuclear dRNP. B r a i n nuclei p r a c t i c a l l y devoided of external m e m b r a n e were p r e p a r e d as described p r e v i o u s l y [10]. Nuclei from 12 rat b r a i n s were s u s p e n d e d i n 10 ml of m e d i m n E (10 mM Tris-HC1 pH 8.0, 25 mM KC1, 2.5 mM MgCI~, 0.1 M NaC1). Sodium deoxycholate (DOE) was added to a final concent r a t i o n of 0.2 p. cent a n d the m i x t u r e was gently homogenized b y h a n d in a Potter Elvehjem homogenizer. 5 ml of m e d i u m E was added a n d the susp e n s i o n was centrifuged in a R40 rotor for 10 rain at 39,000 r e v . / m i n (100,000 g). Most of the chromat i n was f o u n d i n the pellet. 5 ml of the s u p e r n a t a n t (nuclear extract) was l a y e r e d onto 25 ml of a 5-20 p. cent l i n e a r sucrose g r a d i e n t made up i n m e d i u m A (10 mM Tris-HC1 pH 7.4, 25 mM KCI, 2.5 mM MgC12). Centrifugation was c a r r i e d out i n a SW 25.1 rotor for 3 h o u r s at 24,000 r e v . / m i n (59,500 g). F r a c t i o n s c o n t a i n i n g dRNP were located w i t h the aid of an extract labelled for 1 h with H3-uridine, treated a n d centrifuged u n d e r the same conditions. The d e n s i t y of the dRNP in CsC1 after fixation by f o r m a l d e h y d e was 1.40. The dRNP fractions from the g r a d i e n t were pooled for f u r t h e r analysis.

Preparation of nuclear post-particulate supernatant (NS). The n u c l e a r extract (see above) was centrifuged for l h at 56,000 r e v . / m i n (200,000 g). The dRNP and residual c h r o m a t i n sedimented. The supern a t a n t (NS) was collected for analysis.

Preparation of cytoplasmic dRNP (CP). Only free dPfl~P from the cytoplasmic s u p e r n a tant were studied. 12 rat b r a i n s were gently homogenized by h a n d i n 84 ml of 0.3 M sucrose i n med i u m A. U n d i s r u p t e d cells, nuclei and nfitochondria were e l i m i n a t e d b y a 5 rain c e n t r i f u g a t i o n at 5,000 r e v . / m i n (2,500 g) followed by a 5 rain centrifugation at 17,000 r e v . / n f i n (20,000 g). The

BIOCHIMIE, 1972, 54, n ° 9.

s u p e r n a t a n t was centrifuged again for 45 rain at 3%000 r e v . / m i n (100;000 g). The microsomes were f o u n d i n the pellet. Two methods were e m p l o y e d for the o b t e n t i o n of dRNP from the post-microsomal s u p e r n a t a n t .

Method 1. The post-microsomal s u p e r n a t a n t was centrifuged for 2.5 hours at 39,000 r e v . / m i n (10'0,000 g) i n a R 40. rotor. The pellets were susp e n d e d in 6 ml of m e d i u m A. The s u s p e n s i o n was clarified by a 10 m i n c e n t r i f u g a t i o n at 5,000 r e v . / m i n (2,50,0 g) a n d 2 ml of the s u s p e n s i o n were layered on top of a l i n e a r 5-2~0 p. cent sucrose gradient m a d e up in m e d i u m A, a n d c e n t r i f u g e d i n a SW 25 rotor for 5 hours at 24,000 r e v . / m i n (59~500 g). F r a c t i o n s c o n t a i n i n g dRNP were located w i t h an extract labelled for 1 h w i t h H~-uri dine, p r e p a r e d a n d centrifuged u n d e r the same conditions. After f o r m a l d e h y d e fixation, the density of pooled H3-uridine labelled fractions was 1.40. Method 2. The p o s t - m i c r o s o m a l s u p e r n a t a n t was layered on 3.0 ml of 1.3 M sucrose i n med i u m A in a R65 tube a n d centrifuged for 14 hours at 3'9,000 r e v . / m i n (100,000 g). Some m a t e r i a l was c o n c e n t r a t e d on the top of the 1.3 M sucrose layer. The dRNP f r a c t i o n was f o u n d at the bottom of the tube. It is likely that the totality of the cytoplasmic particles is not o b t a i n e d w i t h these methods. Due to the possible c o n t a m i n a t i o n w i t h cytoplasmic soluble p r o t e i n s (see (( Results >>), higher centrifugational speeds were avoided.

Preparation of cytoplasmic post-particulate supernatant ( CS). The s u p e r n a t a n t o b t a i n e d after the 2.5 hours c e n t r i f u g a t i o n i n m e t h o d 1 for the p r e p a r a t i o n of cytoplasmic dRNP (see above) was collected and analyzed. Similar electrophoretie p a t t e r n s were o b t a i n e d w h e n a 5 h c e n t r i f u g a t i o n was c a r r i e d out i n o r d e r to e l i m i n a t e lighter particles w h i c h could contam i n a t e this s u p e r n a t a u t . Since the post-particulate s u p e r n a t a n t c o n t a i n e d about 5,0,0 times more p r o t e i n s than the c o r r e s p o n d i n g dRNP f r a c t i o n it was u n l i k e l y that even half of the total particles could modify signi,ficantly the electrophoretic pattern of the s u p e r n a t a n t proteins.

Preparation of ribosomes (R). A p o s t - m i t o c h o n d r i a l s u p e r n a t a n t p r e p a r e d as for cytoplasmic dt~NP was treated for 15 rain with 1 p. cent D OC. It was centrifuged for i h at 40;000 r e v . / n f i n (105,000 g). The pellet was suspended i n 10 ml m e d i u m A i n 0.3 M sucrose ; 5 ml

Proteins f r o m nuclear and c y t o p l a s m i c d R N P . w e r e l a y e r e d on 3 ml of m e d i u m A in 1.3 M sucrose and c e n t r i f u g e d for 2 h o u r s at 40,000 r e v . / m i n (105,000 g). Some m a t e r i a l c o n t a i n i n g p r o b a bly m i c r o s o m a l m e m b r a n e s floated on top of the 1.3 M layer. The pellet was taken as purified ribosomes.

Preparation of proteins. T h e sucrose g r a d i e n t f r a c t i o n s and the postp a r t i c u l a t e s u p e r n a t a n t s w e r e dialyzed for 1 day against 1 mM s o d i u m acetate (pH 6.8) and then lyophilized. Proteins were dissociated with 6 M u r e a a c c o r d i n g to Molnar et al. E16] at least overnight. LiCI did not lead to any f u r t h e r dissociation. Storage for a f e w days in the cold in the presence of u r e a did not m o d i f y the e l e c t r o p h o r e t i c profiles. R i b o s o m e s w e r e also dissociated w i t h 0 M u r e a since the aim of the e x p e r i m e n t w a s to c h e c k the c o n t a m i n a t i o n of the dRNP f r a c t i o n by r i b o s o m e s or t h e i r subunits. A 9J0 rain c e n t r i f u g a t i o n at 39,00'0 r e v . / m i n (100,000 g) w a s then n e c e s s a r y to s e d i m e n t the u n d i s s o e i a t e d r i b o n u c l e o p r o t e i n s . The final s u p e r n a t a n t s w e r e analyzed.

Reduction of the S-S groups. It has been s h o w n [171 that after a t r e a t m e n t w i t h ~ - m e r c a p t o e t h a n o l , the e l e c t r o p h o r e t i c profile of the p r o t e i n s f r o m n u c l e a r dRNP was consid e r a b l y simplified. U s i n g the same e x p e r i m e n t a l c o n d i t i o n s as these authors ( i n c u b a t i o n in the presence of 0.14 M ~ - m e r c a p t o e t h a n o l d u r i n g 3 h o u r s at 37 ° ) w e f o u n d only slight m o d i f i c a t i o n s of the e l e c t r o p h o r e t i c profiles. In p a r t i c u l a r , l o w - m i g r a ting m a t e r i a l (zones A and B in u r e a gels) (see figures) was still present. H o w e v e r , r e d u c t i o n was a c h i e v e d and simplification of the profiles obtain e d w h e n d i t h i o t h r e i t o l (DTT) w a s a d d e d to the l a r g e - p o r e gels and f~-mercaptoethanol was p r e s e n t in the e l e c t r o p h o r e s i s buffers as d e s c r i b e d below. P r i o r t r e a t m e n t Of the p r o t e i n s w i t h D T T 10-8 M at p H 8, 60°C for 10 m i n did not f u r t h e r s i m p l i f y the e l e c t r o p h o r e t i c profile. T r e a t m e n t by S-S r e d u c i n g agents also simplified the e l e c t r o p h o r e t i c profiles of p r o t e i n s f r o m the o t h e r s u b c e l l u l a r fractions.

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W i l l i a m s E26! and Leboy, Cox and Flaks [27] u s i n g 3 a l a n i n e - a c e t i c acid buffer pH 4.5. Modifications w e r e as follows : - - 6 M urea w a s p r e s e n t in gels but not in tray buffers. - - NNN'N' - t e t r a m e t h y l e t h y l e n e d i a m i n e w a s a d d e d to the small-pore gel solution to a final conc e n t r a t i o n of 0.25 p. cent just b e f o r e use. --The final c o n c e n t r a t i o n of a m m o n i u m persulfate w a s 2.5 m g / m l . D i t h i o t h r e i t o l fDTT) was a d d e d to the largep o r e gel solution to a final c o n c e n t r a t i o n of 0.4 raM. -

-

- ~ - m e r c a p t o e t h a n o l was a d d e d to the tray buffer of the a n i o n i c c o m p a r h n e n t to a final conc e n t r a t i o n of 1 p. cent. E l e c t r o p h o r e s i s w a s c a r r i e d out in 7.5 p. cent a c r y l a m i d e gels first at 0.5 m A / g e l for about 60 min, then at 5 m A / g e l for 80 rain. P y r o n i n e was a d d e d to the sample as a t r a c k i n g dye.

Polyacrglamide gel electrophoresis in the presence of SDS. This m e t h o d is d e r i v e d f r o m that of Davis !28] as modified by W a e h n e l d t and Mandel [291. The final c o n c e n t r a t i o n of SDS w a s 0.1 p. cent. D T T was a d d e d to the l a r g e - p o r e gel to a final c o n c e n t r a t i o n of 0.5 mM. 6 - m e r c a p t o e t h a n o l w a s a d d e d to the u p p e r c o m p a r t m e n t tray buffer to a final c o n c e n t r a t i o n of 1 p. cent. E l e c t r o p h o r e s i s was c a r r i e d out in 10 p. cent a e r y l a m i d e gels at 0.3 m A / g e l until the t r a c k i n g dye ( b r o m o p h e n o l blue) r e a c h e d the end of the gel (about 15 h). Molecular w e i g h t s w e r e estimated a c c o r d i n g to S h a p i r o et a!. E30] and W e b e r and Osborne ~31].

Staining, destaining and recording. Gels w e r e s t a i n e d in a 2.5 p. cent solution of Coomassie B r i l l i a n t Blue in a 10 p. cent acetic acid, 50 p. cent m e t h a n o l solution for SDS gels. A fivefold diluted solution w a s used for the staining of ~rea gels. Gels w e r e d e s t a i n e d in a 7.5 p. cent acetic acid, 5 p. cent m e t h a n o l solution. The gels w e r e s c a n n e d w i t h the aid of a Vernon r e c o r d e r (Paris, F r a n c e ) .

Sodium dodecylsulphate (SDS) treatment. SDS w a s a d d e d to a final c o n c e n t r a t i o n of 1 p. cent to aliquots of the samples in u r e a and the m i x t u r e w a s i n c u b a t e d for 1 h at 38 °. The samples were immediately electrophorised.

Polyacrylamide gel eleclrophoresis al pH ~.5 in the presence of urea. The m e t h o d (urea m e t h o d ) is a slight modification of that d e s c r i b e d by Reisfeld, L e w i s and

BIOCHIM1E, 1972, 54, n ° 9.

RESULTS. A. ELECTRt)PHORESIS AT p H 4.5 IN THE PRESENCE OF UREA.

T h e gels w e r e d i v i d e d into 5 zones : A, B, C, D, E (see figures 1, 2, 3, 4). W i t h i n each zone the p r o t e i n bands ~vere n u m b e r e d in o r d e r of i n c r e a sing m i g r a t i o n . W h e n samples w e r e e l e c t r o p h o resed in the absence of S-S r e d u c i n g agents, nume-

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rous b a n d s w e r e f o u n d in zones A and B, both for n u c l e a r and c y t o p l a s m i c fractions. T h e y w e r e absent, at least for dRNP, u n d e r the e x p e r i m e n t a l c o n d i t i o n s that we have adopted. This is in agreem e n t w i t h the o b s e r v a t i o n s of K r i c h e v s k a y a and Georgiev E17~.

I

A

B

I

C

I

D

J

The absence of p r o t e i n s in zone E s h o w s that c o n t a m i n a t i o n w i t h c h r o m a t i n was u n s i g n i f i c a n t since histones w o u l d h a v e m i g r a t e d in this zone.

,A

B, C

,

D

,

E

E

J= D2

I I

C|

I

Fro. 2. - - Polgacrylamide gel densitograms of proteins from nuclear and cytoplasmic dRNP. .

Fro. 1. - - Po/yacrylamide yet densilograms of proteins from nuclear and cytoplasmic dRNP. Electrophoresis in the presence of urea at pH 4.5. Cytoplasmic dRNP (CP) were prepared by method 1 (see <>). Nuclear (NP) and cytoplasmic (CP) dRNP proteins were eIectrophorised independently or together (CP + NP). Zones are indicated at the top of the figure.

1. Nuciear dRNP (NP). T h e d e n s i t o m e t r i c proliles of p r o t e i n s f r o m 3 d i f f e r e n t p r e p a r a t i o n s of n u c l e a r dRNP are s h o w n in figures 1, 2 and 3. One peak p r o b a b l y c o m p o s i t e as suggested by the presence of shoulders, w a s f o u n d in zone C (C~C3). T h e m a j o r p r o t e i n s m i g r a t e d to zone D. Depend i n g u p o n the p r e p a r a t i o n s , either D 1 or D~. was the m a j o r species as can be seen by c o m p a r i n g the r e c o r d i n g s f r o m figure 1 w i t h those f r o m figures 2 and 3. T h e s i m p l i c i t y of the p r o t e i n profiles of n u c l e a r dRNP analysed by e l e c t r o p h o r e s i s at pH 4.5 in the p r e s e n c e of urea has been a l r e a d y noted [14, 17,

20, 22, 24, 25]. BIOCHIMIE, 1972, 54, n ° 9.

Electrophoresis in the presence of urea at pH 4.5. Cytoplasmic dRNP (CP) were prepared by method 2 (see <>). Nuclear dRNP are from a different preparation as in figure 1. (NP) and (CP) proteins were electrophorised independently or together (NP + CP). Zones are indicated at the top of the figure.

2. CI]toplasmic d R N P (CP). D e n s i t o g r a m s of p r o t e i n s f r o m c y t o p l a s m i c dRNP are s h o w n in f g u r e s 1 and 4 (Method 1) and in figure 2 (method 2). As for n u c l e a r d R N P the p r o t e i n s m i g r a ted m a i n l y in zones C and D, but in this case most of the p r o t e i n s w e r e 'found in zone C. T h r e e m a i n peaks C1, C4 and D 1 w e r e always present. D 1 w a s usually small w i t h m e t h o d 1 (fig. 1 and 4) and h i g h e r w i t h m e t h o d 2 of the prep a r a t i o n (fig. 2). D 2 was always small. Besides the m a j o r p r o t e i n s C1 and C4, t h e r e w e r e p r o b a b l y several o t h e r p r o t e i n s in zone C as suggested by the p r e s e n c e of s h o u l d e r s (C2, C5). Little m a t e r i a l was f o u n d in zone E w h e r e most r i b o s o m a l p r o t e i n s m i g r a t e El4, 16, 18, 21, Matringe and Jacob, u n p u b l i s h e d o b s e r v a t i o n s ] . The e l e e t r o p h o r e t i c p a t t e r n of p r o t e i n s f r o m c y t o p l a s m i c dRNP is a p p a r e n t l y m o r e c o m p l e x

Proteins from nuclear and cytoplasmic dRNP. t h a n t h a t of n u c l e a r d R N P , a l t h o u g h b o t h t y p e s of p r o t e i n s m i g r a t e d in t h e s a m e zones. In o r d e r to d e t e r m i n e w h e t h e r o r not t h e m a j o r b a n d s h a d the s a m e m o b i l i t i e s , n u c l e a r a n d c y t o p l a s m i c d R N P p r o t e i n s w e r e c o e l e c t r o p h o r i s e d (fig. 1 a n d 2). T h e r e c o r d i n g of figure 2, as w e l l as t h o s e f r o m o t h e r e x p e r i m e n t s , n o t s h o w n h e r e , suggest t h a t (CP)C 1 a n d (CP)C 4 a r e d i f f e r e n t f r o m (NP)C2_.~. In c o n t r a s t ( N P ) D 1 a n d (NP)D 2 h a d t h e s a m e m o b i l i t y as (CP)D 1 a n d (CP),D 2.

,A

B, C

,D

,

E

1173

in p a r t i c u l a r C,, 3 w a s a b u n d a n t in (NS) a n d v e r y l o w in (NP).

4. Cytoplasmic p o s t - particulate supernatant (CS) (fig. 4). Most of the p r o t e i n s f r o m (CS) m i g r a t e d in z o n e C. T h e p a t t e r n w a s e v e n m o r e c o m p l e x t h a n that of c y t o p l a s m i c d R N P (,CP). C~ a n d C2, t h e m a j o r p r o t e i n s p e c i e s of (CP) w e r e also p r e s e n t in (GS). In a d d i t i o n , a p e a k C 6 is a l w a y s f o u n d in (CS) in r e l a t i v e l y h i g h p r o p o r t i o n w h i l e in (CP) it is s m a l l o r absent. P r o t e i n s w i t h the m o b i l i t y of D t a n d D 2 w e r e s o m e t i m e s p r e s e n t in (CS) but o n l y as s h o u l d e r s in t h e d e s c e n d i n g s l o p e of C 6.

C2

i

A

B

C

!

C4 I

t

D

Q

E

i

¢

¢~

J[I c.

Fro. 3. - - Polyacrylamide gel densitograms of nuclear dRNP (NP) and post-particulate supernatant (NS).

÷

Eleetrophoresis in the presence of urea at pH 4.5. dRNP (NP) and supernatant (NS) proteins were eleetrophorised alone or together (NS + NP). The division into zones is indicated at the top of the fignre.

S c h w e i g e r a n d H a n n i g [32] f o u n d in c y t o p l a s mic dRNP released, either by deoxycholate from m i c r o s o m e s o r by ~ D T A f r o m p o l y s o m e s , a p r o t e i n w i t h t h e s a m e m o b i l i t y in u r e a at p H 4.5 as the main nuclear dRNT proteins.

3. Nuclear

post-particulate

supernatant

(NS)

(fig. 3). H e r e again, m o s t p r o t e i n b a n d s w e r e in z o n e s C a n d D. At least 3 p e a k s ~vere f o u n d in z o n e C : C2, C a a n d C~ ; D 1 a n d D 2 w e r e also p r e sent. C o e l e c t r o p h o r e s i s :of (NP) a n d (NS) s h o w s t h a t (NS)C 2, (NS)D 1 a n d (NS)D 2 h a d t h e s a m e m o b i l i t y as (NP)C~, (NP)D 1 a n d (NP)D 2. O n l y t h e relat i v e p r o p o r t i o n s of t h e s e s p e c i e s w e r e d i f f e r e n t :

BIOCHIMIE, 1972, 54, n ° 9.

i\l

D

Fro. 4. - - Polyacrylamide gel densitograms

of cytoplasmic dRNP (CP) and post-particulate supernatant (CS).

Eleetrophoresis in the presence of urea at pH 4.5. The division into zones is indicated at the top of the figure.

B. ELECTROPHORESIS IN THE PRESENCE OF SDS. T h e gels w e r e d i v i d e d i n t o 4 z o n e s : 1, 2, 3, 4 (fig. 5, 6, 7). I n s i d e e a c h z o n e t h e p r o t e i n b a n d s w e r e d e s i g n e d a, b, c, d, etc. W h e n e l e c t r o p h o r e sis w a s p e r f o r m e d in t h e a b s e n c e of S-S r e d u c i n g agents, n u m e r o u s b a n d s w e r e f o u n d i n z o n e 1. Such bands occasionally appear, even in the pres e n c e of D T T a n d ~ - m e r c a p t o e t h a n o l . T h e i r p r e s e n c e d i d not p r o v o k e a n y a p p a r e n t c h a n g e in t h e e l e c t r o p h o r e t i c p a t t e r n of t h e o t h e r zones. F o r this reason, they were disregarded.

1174

H. M a t r i n g e a n d M. J a c o b .

1. N u c l e a r d R N P (NP). N u c l e a r d R N P a n a l y z e d in figures 5, 6 a n d 7 w e r e f r o m t h e s a m e p r e p a r a t i o n s as t h o s e a n a l y z e d b y t h e u r e a m e t h o d i n figures 1, 2 a n d 3 r e s p e c t i v e l y .

1

2

3

4

S e v e r a l s m a l l b a n d s w e r e p r e s e n t in z o n e 2, b a n d 2b b e i n g t h e m o s t p r o m i n e n t . T h e m a j o r p e a k s w e r e f o u n d in z o n e 3. T h e t w o m a i n p e a k s w e r e 3a a n d 3c, b u t t h e i r r e l a t i v e h e i g h t v a r i e d : 3a w a s t h e m a j o r s p e c i e s i n t h e p r e p a r a t i o n s h o w n in figure 5, 3c w a s h i g h e r t h a n 3a i n figures 6 a n d 7. T h i s c a n be r e l a t e d to c h a n g e s in p e a k s D 1 and D e observed with the urea method and will be d i s c u s s e d later.

,

1

T 2

r

3

4

,

t

I

t rut' I

hi

I

•

I

.

I

f

.

i

"\_

I 'l_J .

IliVv/,

Vv

I°

I

Ill

wv

i

_.

= . . . . .

! cP.csA

I

I'b

I

I

I/U k \

] NP+CpII

Fro. 5. --- Polyacrylamide gel densitograms of proteins f r o m nuclear dRNP (NP), cytoplasmic dRNP (CP) cytoplasmic supernant (CS), and ribosomes (R). Electrophoresis in the presence of SDS. (NP) and (NC) proteins from the same preparation as in figure 1, (CS) f r o m the same preparation as in figure 4. The division in zones is indicated at the top of the figure. BIOCHIM1E, 1972, 54, n ° 9.

I

I

I

I ~-%/i

L.,

.t

I Np)l

Fza. 6 . - Polyacrylamide gel deusitog a s of nuclear and cytoplasmic dRNP. Eleetrophoresis in the presence of SDS. (CP) and (NP) proteins are from the same preparation as in figure 2. The division of the gel into zones is indicated.

The protein pattern was simple with this met h o d , as w i t h t h e u r e a s y s t e m . M u c h m o r e c o m p l e x p a t t e r n s w e r e o b s e r v e d b y F a i f e r m a n el al. [14] a n d N i e s s i n g a n d S e k e r i s [251 u s i n g s i m i l a r m e t h o d s of a n a l y s i s . 2. C y t o p l a s m i c d R N P (CP). T h e d e n s i t o g r a m s f r o m t h e t w o p r e p a r a t i o n s a l r e a d y a n a l y z e d at p H 4.5 in t h e p r e s e n c e of u r e a (.fig. 1 a n d 2) a r e s h o w n in figures 5 a n d 6. Most of t h e p r o t e i n s mi-

1175

Proteins f r o m nuclear and cytoplasmic d R N P . grated in zone 2 w h e r e several closely-migrating b a n d s were found. Sometimes these b a n d s overlapped. The most p r o m i n e n t species w e r e 2d, 2e and 2[. N a r r o w bands, 2a and 2b were always present, 2g a n d 2h were only shoulders in the descending slope of 2f.

1

2 b •

3

4

more complex p a t t e r n was f o u n d b y Morel et al.

[241. Ribosomal p r o t e i n s (R) p r e p a r e d u n d e r the same c o n d i t i o n s as dRNP p r o t e i n s (see Methods) were m a i n l y found i n zones 3 a n d 4. 2 b a n d s m i g r a t e d i n zone 2 at the level of 2 [ - 2 g . The almost complete absence of p r o t e i n s from zone 4 of (CP) excludes a c o n t a m i n a t i o n of cytoplasmic dRNP by whole ribosomes or subunits.

3. Nuclear post-particulate

d

supernatant

(NS).

The p r o t e i n s from the n u c l e a r post-particulate s u p e r n a t a n t migrated i n zones 1, 2 a n d 3 (fig. 7). I n zone 2, w e observed two m a j o r peaks, 2b, a n d 2d, and smaller ones, 2a, 2c, 2g. Peaks 3a, a n d 3c w e r e p r e s e n t in the same relative p r o p o r tions as i n the c o r r e s p o n d i n g dRNP (NP). The min o r species from zone 2 of (NP) had the same m o b i l i t y as the more a b u n d a n t species of (NS).

NS 4. Cytoplasmic post- particulate supernatant (CS) (fig. 5). Most of the (CS) p r o t e i n s migrated i n zone 2. 2a, 2b and 2c were very n a r r o w a n d reproducible bands. 2d, 2e, and 2f were the most prom i n e n t species. They were better resolved t h a n were the honmlogous p r o t e i n s from (CP). 2g was always f o u n d as a d i s t i n c t b a n d w h e r e a s it was only a shoulder of 2[ in (CP). I n zone 3 we f o u n d 4 bands. 3a was always smaller t h a n 3b and 3e. There was a shoulder at 3[. These last b a n d s were not detected in (CP) w h e r e a s 3d, p r e s e n t in (CP) was never found in (CS).

NP

Fit;. 7. -- Polyacrylamide gel densitograms of nuclear dRNP (NP) and post-particulate supernatant (NS). Electrophoresis in the p~esence of SDS. (NP) from the same preparation as in figure 3.

I n zone 3, we f o u n d regularly 3a, 3b a n d 3d. The c o m p a r i s o n b e t w e e n figures 5 a n d 6 shows that the ratio of 3a a n d 3d to the p r o t e i n s of zone 2 was variable. The electrophoresis in figures 5 a n d 6 c o r r e s p o n d to dRN~P p r e p a r e d by methods 1 a n d 2 a n d the differences could well be due to the method of p r e p a r a t i o n . W h e n electrophoresis was c a r r i e d out in the p r e s e n c e of urea, we also observed that peak D 1 w a s m o r e p r o m i n e n t w i t h method 2 that w i t h m e t h o d 1. Only 2 p r o t e i n s were f o u n d in dRNP released from reticulocyte polysomes by EDTA [23] but a

BIOCHIMIE, 1972, 54, n ° 9.

5. Estimation of the molecular weight of the proteins. The Rf of m o l e c u l a r weight m a r k e r proteins was m e a s u r e d and c o m p a r e d to that of various p r o t e i n s from our p r e p a r a t i o n s . Bovine serum a l b u m i n (M.W. 67,000) migrated b e t w e e n 2a a n d 2b, o v a l b u m i n (M.W. 45,000) between 2g a n d 2h, c h y m o t r y p s i n o g e n (~M.W. 25,00~0) b e t w e e n 3d a n d 3e and m y o g l o b i n (M.W. 17,800) close to the front of the gel. The molecular weights of the m a j o r p r o t e i n s (3a a n d 3e) from n u c l e a r dRNP were a p p r o x i m a t e l y 40,000 a n d 35,000. This is in agreement w i t h the results of K r i c h e v s k a y a a n d Georgiev [17! a n d Niessing and Sekeris [25]. The n u c l e a r post-particulate s u p e r n a t a n t c o n t a i n s major p r o t e i n s of up to 120,000 m o l e c u l a r weight. The m o l e c u l a r weights of the m a j o r p r o t e i n s of cytoplasmic dRNP a n d soluble p r o t e i n s were between 50,000 and 63,0,00. The p r o t e i n s from dRNP released from reticulocytes polysomes by EDTA have been shown to c o n t a i n p r o t e i n s of s i m i l a r m o l e c u l a r weight [23, 24] a n d in a d d i t i o n a protein b a n d at 130,000 [23]. 80

H. Malringe and M. Jacob.

1176

ral other species of higher molecular weight i n the n u c l e a r post-particulate s u p e r n a t a n t .

DISCUSSION. The results o b t a i n e d w i t h p o l y a c r y l a i n i d e gel electrophoresis must be i n t e r p r e t e d w i t h c a u t i o n since p r o t e i n s m a y have s i m i l a r electrophoretic mobilities w i t h o u t b e i n g identical. F o r our comparative study, we have therefore used two differ e n t electrophoretic systems w h i c h separate proteins on the basis of different properties. Thus the p r o b a b i l i t y that p r o t e i n s of s i m i l a r electrophoretic m o b i l i t y are i d e n t i c a l is m u c h higher t h a n w i t h a single system. I n o r d e r to facilitate the discussion, the m a i n results are s u m m a r i z e d in table 1.

The most likely i n t e r p r e t a t i o n of the results is that native dRNA associates specifically w i t h D 1 (3c) and D 2 (3a) from the soluble p r o t e i n pool, to form dRN~P. The m i n o r species of zones (C) a n d (2) from dRNP m a y r e p r e s e n t either simple contam i n a t i o n w i t h s u p e r n a t a n t proteins, or a b i n d i n g of m a j o r soluble p r o t e i n s to RNA, less specific than the b i n d i n g of D 1 (3c) and D2 (3a). There are a p p a r e n t l y m a n y analogies between the p r o t e i n p a t t e r n s of cytoplasmic dRNP and post-particulate s u p e r n a t a n t . In SDS electrophorests (fig. 5) we observe that 2a, 2b, 2d, 2e, 2f, 2h are c o m m o n a n d p r e s e n t i n about the same proportions. 2g, 3a, 3b, are also f o u n d i n both the dRNP and the s n p e r n a t a n t but i n different p r o p o r t i o n s ; 3d is f o u n d only i n the dRNP a n d 3e, 3[,

The p r o t e i n s from dB~NP migrate more slowly than ribosomal p r o t e i n s at p H 4.5 (urea method). It can be tentatively assmned that D~ a n d D2, the m a j o r peaks from (N,P) i n urea gels c o r r e s p o n d to (NP) 3c a n d (NP) 3a in SDS gels. This is f u r t h e r

TABLE

Electrophoretic system

I.

Fraction studied

Bands of eleetrophoretic mobility corresponding to

I NS

NP

[

Urea

+

CtC2C:+

CP

+++

+++ ÷++

C4

C~C~ + Dl

D~ SDS

++÷ +

+ +++

÷

2a2b2e 2d2e 2f2g2h 3a

+÷+

3b 3e 3d

+

3e

or

or

r+++

+++

+++ ÷++ +++

Or

++-

-

÷+ ++÷ +++

!+++ +++ +

cs

++ +++ +++

or

-

++

-_ -

%

+ + -

÷

The observations (fig. 1 to 7) described under <> are summarized in this table. The number of (q-) is a function of the area of the peaks. (=k_) indicates traces of material. When different proportions of the various peaks were obtained, the two possibilities are shown.

s u p p o r t e d by the fact that, w h e n the relative prop o r t i o n s of (NP)D 1 a n d (NP)D e v a r i e d (fig. 1, 2, 3), those of (NP)3c a n d (.N.P)3a varied i n the same d i r e c t i o n (fig. 5, 6, 7). It is likely that (NS)D 1 a n d (NS)D e (fig. 3) corr e s p o n d to the same species as (N~P)D1 a n d (N,P)D 2 since we find also i n S.D,S electrophoresis peaks (NS) 3c a n d (NfS) 3a c o r r e s p o n d i n g to (NP) 3c a n d (NP) 3a. There are, i n addition, sere-

BIOCH1M1E, 1972, 54, n ° 9.

only in the s u p e r n a t a n t . Two i n t e r p r e t a t i o n s of the results can be p r o p o s e d : 1) I n the cytoplasm, dRNA b i n d s non-specifically soluble p r o t e i n s as suggested b y H u a n g a n d Baltimore [12]. 2) Heavy soluble p r o t e i n s or aggregates of soluble p r o t e i n s may s e d i m e n t together w i t h the dRNP. I n d e e d w h e n the dRNP were s e d i m e n t e d t h r o u g h a sucrose layer (method 2) w h i c h could be a better b a r r i e r for free p r o t e i n s t h a n sucrose gradients

Proteins from nuclear and cytoplasmic dRNP. t h e r e w a s a r e l a t i v e i n c r e a s e of t h e p r o t e i n s of z o n e D ( u r e a ) o r 3 (SDS) (fig. 1 a n d 2, fig. 5 a n d 6). T h e r e f o r e , it is p o s s i b l e t h a t p e a k s D 1 ( u r e a ) a n d 3a, 3 d (SDS) a r e t h e t r u e d R N P p r o t e i n s . U n f o r t u n a t e l y i t is n o t f e a s i b l e to s e p a r a t e u n d e n a t u r e d p a r t i c l e s f r o m p r o t e i n s o n t h e b a s i s of t h e i r c h a r a c t e r i s t i c d e n s i t i e s i n CsC1 s i n c e f o r m a l d e h y d e f i x a t i o n is r e q u i r e d f o r t h i s t y p e o f a n a l y s i s . A n o t h e r e x p e r i m e n t a l a p p r o a c h w i l l b e 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 c y t o p l a s m i c d R N P e x i s t in vivo. As f a r as t h e t r a n s p o r t of d R N A f r o n t t h e n u c l e u s to t h e c y t o p l a s m is c o n c e r n e d , t h e c o n c l u s i o n s d e p e n d o b v i o u s l y u p o n t h e i n t e r p r e t a t i o n of the results concerning cytoplasmic dRNP. If we a s s u m e t h a t t h e i r m a j o r p r o t e i n s a r e t h o s e also found in abundance in the soluble pool (the prot e i n s of z o n e 2 i n SDS gels o r z o n e C i n u r e a gels), there would be no relationship between the proteins from nuclear and cytoplasmic dRNP. A comp l e t e e x c h a n g e of p r o t e i n s c o u l d o c c u r d u r i n g t h e p a s s a g e of t h e n u c l e a r m e m b r a n e a n d t h e d R N A w o u l d n o t b e t r a n s p o r t e d i n t h e f o r m of a w e l l d e f i n e d p a r t i c l e . O n t h e o t h e r h a n d , if (CP) D1, D., ( u r e a ) o r (CP) 3a, 3d (SDS) a r e t h e t r u e d R N P proteins, other conclusions can be drawn. In urea e l e c t r o p h o r e s i s (CP) D~, D 2 h a v e t h e s t o n e m o b i l i t y as (NP) Da, D e. T h e y p r o b a b l y c o r r e s p o n d i n SDS e l e c t r o p h o r e s i s to (CP) 3a, 3 d a n d (NP) 3a, 3c. I t c a n n o t b e e x c l u d e d t h a t (CP) 3 a a n d (NP) 3a are different protein species, but the experim e n t s b r i n g f o r t h a n a r g u m e n t i n f a v o r of a r e l a tionship between nuclear and cytoplasmic dRNP. It h a s b e e n s h o w n [23, 243 t h a t t h e d R N P i s o l ated from reticulocytes polysomes by EDTA contain proteins whose molecular weights are in the s a m e r a n g e as t h o s e of t h e d R N P p r o t e i n s f o u n d i n z o n e 2 of o u r SDS gels a n d c o r r e s p o n d i n g to m a j o r s o l u b l e p r o t e i n s . If w e a s s u m e t h a t f r e e d R N P a r e a t t a c h e d as s u c h to t h e r i b o s o m a l s u b u n i t s f o r t h e f o r m a t i o n of p o l y s o m e s , t h e s e e x p e r i m e n t s w o u l d b e i n f a v o r of t h e e x i s t e n c e of d R N P w h e r e R N A is a s s o c i a t e d w i t h t h e m a j o r c y t o p l a s m i c s o l u b l e p r o t e i n s . O n t h e c o n t r a r y , if t h e d R N P p r o t e i n s p l a y stricto s e n s u a c a r r i e r r o l e , t h e y c o u l d b e e l i m i n a t e d d u r i n g t h e p r o c e s s of p o l y s o m e f o r m a t i o n a n d t h e d R N A c o u l d a s s o c i a t e to o t h e r p o l y s o m a l p r o t e i n s . T h e r e f o r e , t h e r e s u l t s of L e b l e u et al. [23! a n d M o r e l et al. [24! d o n o t e x c l u d e t h e h y p o t h e s i s of t h e t r a n s p o r t of n u c l e a r d R N A to t h e c y t o p l a s m i n t h e f o r m of d R N P , a l t h o u g h t h e y also a g r e e w i t h t h e p o s s i b i l i t y of a c o m p l e x a l i o n of d R N A w i t h s o l u b l e c y t o p l a s m i c p r o t e i n s .

Remereiements. Ce t r a v a i l fait partie de la Th6se de Doctorat 6sSciences de M no H. Matringe. I1 a pu ~tre accompli BIOCHIMIE, 1972, 54, n ° 9.

1177

grfiee h l'aide mat6rielle de la DGRST (contrat n ° A 659 0283). Nous r e m e r e i o n s Monsieur le Professeur P. Mandel p o u r sa c o n s t a n t e sollicitude, ses suggestions et s e s critiques au cours de ce travail.

RI~SUMI~. Les prot6ines des dRNP nucl6aires et c y t o p l a s m i ques, ainsi que celles des s u r n a g e a n t s post-particulaires c o r r e s p o n d a n t s ont 6t6 analys6es p a r 61ectrophor~se sur gel de p o l y a e r y l a m i d e soil h pH 4,5 en presence d'ur~e, soil en pr6sence de dod6cylsulfate de sodium. Les deux prot6ines m a j e u r e s des dRNP nucl~aires ont des poids mol6culaires de 35 000 et 40 000. Ces m~mes prot6ines sont pr6seutes h t'~tat soluble d a n s le noyau, mats le s u r n a g e a n t nucl~aire post-particulaire c o n t i e n t en outre plusieurs prot~ines de poids mD16culaire plus 61ev6 (40 000 h 120 000). Nous sugg~t o n s que le dRNA n a t i f s'associe sp6cifiquement aux prot6ines 35 000 et 40 000 p o u r f o r m e r les dRNP. Le profil 61ectrophor~tique des prot~iues des dRNP e y t o p l a s m i q u e s est plus complexes que celui des dRNP nucl6aires. On trouve plusieurs prot6ines m a j e u r e s d ' u n poids mol6culaire v a r i a n t de 40 000 h 70 000. Leur profil ~lectrophor6tique est tr~s proche de celui d e s prot6ines c y t o p l a s m i q u e s solubles. Mats on trouve 6galement dans les dRNP c y t o p l a s m i q u e s des prot~ines qui ont la m~me m i g r a t i o n 61ectrophor6tique que les prot6ines m a j e u r e s des dRNP nucl~aires ; l e u r proportion relative croit dans des conditions off l ' ~ l i m i n a t i o n de prot6ines solubles est p r o b a b l e m e n t i m p o r t a n t e . Ces experiences a p p o r l e n t donc des a r g u m e n t s en f a v e u r d'uue r e l a t i o n entre dRNP nucl~aires et cytop l a s m i q u e s , mats it f a u d r a faire appel h d ' a u t r e s types d'analyse, telle que la c o m p a r a i s o n des structures primaires, pour le p r o u v e r de mani~re d6finitive. ZUSAMMENFASSUNG.

Die P r o t e i n e der Nukleus- u n d der Z y t o p l a s m u s dRNP, sowie diejenigcn der e n t s p r e c h e n d e n p o s t p a r t i kul~iren Uberstiinde sind d u r c h E l e k t r o p h o r e s e an Poly~tkrylamidgel e n t w e d e r bet pH 4,5 in Gegenwart yon Harnstoff oder in Gegenwart von Natriu:mdodezylsulfat a n a l y s i e r t worden. Die b e i d e n H a u p t p r o t e i n e der Nukleus-dRNP h a b e n Molekulargewiehte yon 35.000 u n d 40.000. Dieselben P r o t e i n e sind in 15sliehem Z u s t a n d im Nukleus v o r h a n d e n , aber der postpartikuHire U b e r s t a n d enth~ilt a u s s e r d e m m e h r e r e P r o t e i n e m i t h 6 h e r e m Molekulargewicht (40.000 bis 120.000). Es 1/isst v e r m u t e n , dass die n a t i v e dRNA sich spezifiseh m i t den P r o t e i n e n 35.000 u n d 40.000 u m die dRNP zu b i l d e n assoziiert. Das e l e k t r o p h o r e t i s c h e Profil der P r o t e i n e der Zytop l a m u s - d R N P ist k o m p l e x e r als dasjenige der NukleusdRNP. Man findet m e h r e r e H a u p t p r o t e i n e m i t einem Moleknlargewieht yon 40.000 bis 70.000. l h r elektrophoretisches Profil ist d e m j e n i g e n der 16slichen zytop l a s m i s e h e n Proteine sehr nahe. Man findet a b e t in den Z y t o p l a s m u s - d R N P ebenso Proteine, welehe dieselbe e l e k t r o p h o r e t i s e h e V~'anderung wie die H a u p t p r o t e i n e der Nukleus-dRNP a u f w e i s e n ; i h r relatives Verhiiltnis n i m m t u n t e r B e d i n g u n g e n zu, u n t e r welehen die A b t r e n n u n g der 16sliche P r o t e i n e b e d e u t e n d ist. Diese Versuche b r i n g e n also Beweisgriinde z u g u n s t e n

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