BIOCHIMIE, 1976, 58, 317-323.

A comparative study by sucrose gradient electrophoresis of messenger ribonucleoprotein complexes. J e a n - P i e r r e LIAUTABD~ a n d K u r t KSHLER. Biologisches I n s t i t u t d e r Universitiit Stuttgart, G e r m a n y .

(28-8-1975).

Summary. ~ The recently developed method o f sucrose gradien.t electrophoresis has been used in the investigation of mRNA containing particles prepared in an undenatured state. The particles con,raining messenger RNA migrate as a homogeneous fraction with a mobility different from that of ribosomal particles, lnformosomes and polysoma] mRNPs have about the same mobility. On the contrary artificial~ complexes, formed by the reaction of cytoplasmic binding factor with RNA, migrate as a heterogeneous fraction. The particles carrying mRNA are drastically and irreversibly affeet.ed by a treatment with EDTA. S.odium d.eoxycholate removes some proteins bu.t seems also to denature them. After treatment by high salt or Sod,turn deoxyeholate the mR'NPs migrate as a homogeneous fraction showing that all particles are equall.y affected.

INTBODUCTIO~N. In e u k a r y o t i c celIs, m e s s e n g e r RNA is associat e d w i t h p r o t e i n s [1, 21. T h e s e m e s s e n g e r ribon u c l e o p r o t e i n parti,cles fmRN~P) are d i v i d e d in t w o f u n c t i o n a l classes. T h e first o n e c o n s i s t s of the m R N P not actually i n v o l v e d in p r o t e i n s y n thesis w h i c h are f o u n d as free p a r t i c l e s in the c y t o p l a s m a n d n a m e d in f o r m o s o m e s [3]. The s e c o n d f u n c t i o n a l class is e x t r a c t e d f r o m the p o l y somes a n d thus is a c t i v e l y e n g a g e d i n p r o t e i n synthesis. T h i s latter t y p e is n a m e d p o l y s o m a l m R N P ( p s - m R N P ) [4]. C h a r a c t e r i s t i c p r o p e r t i e s of m R N P s are t h e i r h e t e r o g e n e o u s s e d i m e n t a t i o n rates a n d a v e r y h o m o g e n e o u s d e n s i t y in CsCl (i.e. Q = 1.39- 1.41) (see [i, 2] for r e v i e w ) . But as s t r e s s e d b y Baltim o r e a n d H u a n g [5] c y t o p l a s m i c p r o t e i n s can a d v e n t i t i o u s l y a d h e r e to RNAs f o r m i n g m R N P artefacts w i t h the s a m e d e n s i t y as g e n u i n e m R N P s [51. T h e s a m e d e n s i t y (o ----- 1.4.0 i n CsC1) can also be o b t a i n e d for an a r t i ~ c i a l p a r t i c l e s m a d e f r o m RNA a n d c y t o c h r o m e C [5].

results b e c a u s e no d i f f e r e n c e can be f o u n d betw e e n mR~NP a n d artificial c o m p l e x e s by p h y s i c a l m e t h o d s . U n t i l n o w it has not b e e n c l e a r l y s h o w n that the so calIed b i n d i n g factor [5] is realIy absent f r o m a n y c y t o p l a s l n i c m R N P . In o r d e r to solve t h i s i m p o r t a n t p r o b l e m w e u s e d an e l e c t r o p h o r e s i s m e t h o d in s u c r o s e grad i e n t s t h a t h a d b e e n d e v e l o p p e d by us to s e p a r a t e the r i b o s o m a l p a r t i c l e s [7]. This m e t h o d s e p a r a t e s the p a r t i c l e s a c c o r d i n g to t h e i r e x t e r n a l c h a r g e a n d t h e r e f o r e d e p e n d s on the p r o t e i n c o m p o s i t i o n of t h e p a r t i c l e s . This m e t h o d has b e e n u s e d to c o m p a r e the d i f f e r e n t p a r t i c l e s a n d to s h o w that c o n v e n t i o n a l t r e a t m e n t s , s u c h as EDTA o r DOC d r a s t i c a l l y affect t h e m RNPs. F r o m t h e s e s t u d i e s it can be conclude~d t h a t m R N P s are d i f f e r e n t f r o m artificial c o m p l e x e s . MATERIALS ANI) METHODS. 1

--

THE FOLLOWING BUFFERS WERE USED

:

I s o t o n i c buffer : 10 mM TRIS-H~C1 (pH = 7.4) ; 140 mM KC1 ; 3 mM MgC12.

F o r these r e a s o n s t h e ml~N,Ps a r e s o m e t i m e s susp e c t e d to be artefacts [5]. To p r o v e t h a t m R N P s do r e a l l y exist in the cell d i f f e r e n t m e t h o d s h a v e b e e n u s e d [1, 6] b u t w i t h o u t e n t i r e l y c o n v i n c i n g

H y p o t o n i c buffer :

Abbreviations. nRNP ribon'ucleoprateins complexes. containing mRNA. ps-mRNP mRNP from polysomes. Informoc~omes mRNP not engaged in protein synthesis. D0C sodium deoxyehotate. To whom all c~rrespondence should be addressed

H y p e r t o n i c buffer :

10 mM TRIS-HC1 (pH = 7 . 4 ) ; 10 mM K,CA ; 3 mM MgCA2 ; 7 mM M e r c a p t o e t h a n o l ; 0.0cl p e r c e n t Macaloid (as RNase i n h i b i t o r ) . 10 mM TRIS-HC1 (pH ---- 7.4) ; 500 mM KC1 ; 3 mM MgC12 ;

J.-P. L i a u t a r d a n d K. K S h l e r .

318

7 m M M e r c a p t o e t h a n o l ; 0.01 p e r c e n t M a c a l o i d (as R N a s e i n h i b f f o r ) .

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~

CELL

T R I $ - H C 1 ( p H 8:1) ; N~HaC1 ; MgClu ; Mercaptoethanol. CULTURE

AND

The suspension was stirred for three more hours. In some experiments the temperature was r a p i d l y r a i s e d to 43°C b y i m m e r s i n g t h e flask i n a water bath ; the incubation was continued for t w e n t y m i n u t e s l o n g e r . T h e cell s u s p e n s i o n w a s t h e n p o u r e d On 150 m l o,f c r u s h e d f r o z e n b u f f e r e d s a l i n e ( - - 80°C). All f u r t h e r opera~tions a n d s u b s e q u e n t c e n t r i f u g a t i o n s w e r e p e r f o r m e d at 4°C. FRACTIONATION PROCEDURE.

T h e cells w e r e c o l l e c t e d r a p i d l y b y c e n t r i f u g a t i o n a n d r e - s u s p e n d e d i n h y p o t o n i c b u f f e r , recentrifuged once and re-suspended in the same b u f f e r . A f t e r t e n m i n u t e s t h e s w o l l e n cells w e r e broken with ten strokes in a Dounce homogenizer. N.uclei, m i t o c h o n d r i a , and membranes were r e m o v e d b y cen,trifugatio,n [7]. S u c r o s e g r a d i e n t c e n t r i f u g a t i o n s w e r e m a d e as d e s c r i b e d r e c e n , tly [8] o r as i n d i c a t e d i n t h e l e g e n d s . All s e d i m e n t a t i o n coeffi!eients w e r e c a l c u l a t e d a c c o r d i n g to M c E ~ e n [9]. F o r t h e r a d i o a c t i v i t y d e t e r m i n a t i o n TC.A-precip i t a t e d f r a c t i o n s w e r e t i t t e r e d o n glass-fi,bre filters. T h e c o u n t i n g w a s d o n e i n a P a c k a r d s c i n t i l l a t i o n s p e c t r o m e t e r w i t h a n e f f i c i e n c y o f (app r o x i m a t e l y ) 50 p e r c e n t . 4 - - THE SUCROSE G R A D I E N T iELECTROPHORESIS p r o c e d u r e h a s b e e n t h e s u b j e c t of a r e c e n t p a p e r [7]. T h e m o b i l i t y i n d i c a t e d is t h e m i g r a t i o n r e l a t i v e to p u r e RNA. 5 - - R N A PREPARATION. R i b o s o m a l R N A w a s p r e p a r e d b y e x t r a c t i n g 1.25.109 H e L a cells w h i c h h a d b e e n i n c u b a t e d w i t h 0.2 l~Ci/ml f o r t w e n t y h o u r s , a c c o r d i n g to P e r r y et al [10]. R N A e x t r a c t e d h a s a

BIOCH1MIE, 1976, 58, n ° 3.

2102

1 ~H_~_.?"g; _/I'O, e~~4 70_g0s /

.

HARVEST.

F o r m o s t e x p e r i m e n t s 3 l i t e r s of H e L a c e l l s g r o w n to a d e n s , t r y of 5.10 ~ c e l I s / m l w e r e e m p l o y e d , (1.5.109 c e l l s r e s p e c t i v e l y ) . W h e n l a b e l i n g with radioactive precursors and pretreatment w i t h a c t i n o m y c i n D w a s d e s i r e d , t h e sus,p'ensions w e r e c o n c e n t r a t e d t e n t i m e s , to 5.106 cells p e r m l ; actinomycin D (0.05 lxg/ml) w a s a d d e d , a n d t w e n t y m i n u t e s l a t e r 0.5 t~Ci/ml [SH] u r i d i n e .

3 --

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FIG. 1. - - Sucrose gradient eleelrophoresis of informosomes of different size classes. F~g. 1 a, b, c : I4eLa cells grown i u suspension culture. were c o n c e n t r a t e d to 3@0 ml w i t h 5.106 cell s/ml. 20 rain. aft,er the a,ddibio,n, of 1 . 5 ~ g aetinromTein- D, 0.5 ~xCi/ml [~Hi] urid.ine were add~dd and the cells incubated four 3 hrs. After the i n c u b a t i o n periods 150 ml of frozen isotonic buffer (--SO.°C) w e r e ad@ed to stop f u r t h e r incorporati;on and~ t h e cells were harvested by een.trifugati~n (4 rain., 4.00 × g). P o s t m i t o c h o n d r i a l supernatan~s w e r e prepared as described in Materials and Methods, and divided in.to three aliquots.. Fig. 1,~a : 1') One t kir,ds (4 ml) was layered onto a cushion (1.5 ml) ¢rf 15 per cent sucrose in h y p o t o n i e buffer and cent r i f u g e d at 22.00,0 r p m for 16, hrs. (4°C, S,pineo rotor SW 50.1), ~he r e s u l t i n g pellet coneain~i,ng p,arti,el,es, r a n gin~g f r o m ~0S-~H)0S (i.e. rib,osomes, pclyso,mes, ribosomal subunits and informosomes). 2,) The o t h e r ~two t h i r d s of the sarnpl'e were layered auto 1.5-30 per cent sucrose gradtients in h:ypotonic buffor and ,centrifuged at 22.5'1)0 r p m for 14 hTs. (4°C, Spinco r o t o r S'W 27), Two fractions c,ontaining the p,a~tieles were collected. Pig. 1 b : Particles w i t h sed,imen,ta,ti,on con:stunt between 70S and 90S (i.o. ribosomes and informosomes). Fig. 1 c : Pa~rticles w i t h sedimenta,ti~n constant b e t w e e n 30S and 60S [i.e. ribt~somal s u b u n i t s and inf, ormosomes). T,hen, the p,articles were eo~neentrated by eenerifugafion a,t 35.061)0 r p m for 16 hrs, (4°C, Sp:ineo r o t o r 50 T,i). All pell,ets were. w a s h e d o,n~ee with, buffer C and. st~ored at --80°C u n t i l use'. Fig. 1 d, e, f : Show e x p e r i m e n t with particles labeled for 40, rain, (in ,order t,o avoid, a e t i n o m y e i n D0 w i t h 1.l) wzCi/ml [aH] uridir~e. The differents s e d i m e n t a t i o n s classes wore p r e p a r e d as descr,ibed above. For the sucrose gradient eleetrop.horesis pellets were resuspended in buffer C at a m a x i m u m concentration of I 2 0 . D . / m l and el eetroph~resed in the eoI,umn at 2t)0 v,olts for 17 hrs. [7]. Absorbanee 260 nm. • ---$ [3H] u r i d i n e radioa,etivity.

Sucrose gradient eleclrophoresis of messenger ribonucleoprotein. specific a c t i v i t y of 0.6~5 ~ C i / m g . Small s a m p l e s w e r e k e p t f r o z e n un.til used. 6 - - , CYTOPLASMIC SUPERNATANT w a s p r e p a r e d f r o m five liters of cells (2.5.10 ~ cells). 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 w a s p r e p a r e d , an,d ribosomes r e m o v e d b y cent.rifugation (rotor 50.1, 160.00 r p m , 16 h o u r s ) . F r o m the s u p e r n a t a n t , cont a i n i n g 2.4 m g / m l p r o t e i n , 1 m l - s a m p l e s w e r e k e p t frozen at ~ 80°C.

a classical p u l s e - l a b e l i n g e ~ p e r i m e n t as s h o w n in figures 1 d, e, a n d f r e s p e c t i v e l y . T h e r e f o r e in spite of t h e i r h e t e r o g e n e o u s sedimen,tation rates mRN,Ps b e h a v e h o m o g e n e o u s l y in e l e c t r o p h o r e s i s . T h i s p r o b a b l y reflects a s i m i l a r i t y i n s t r u c t u r e and composition : protein mottles exposing fewer i o n i c sites a l l o w the s e p a r a t i o n f r o m o t h e r p a r t i cles of t h e r i b o s o m e f a m i l y . -~

1. ELECTROPHORETIC MOBILITY OF INFORMOSOMES (FREE m R N P s ) OF DIFFERENT SIZE CLASSES. In o r d e r to d i s t i n g u i s h n o n - r i b o s o m a l f r o m r i b o s o m a l p a r t i c l e s , H e L a cells w e r e p r e t r e a t e d w i t h l o w doses of a c t i n o m y c i n D a n d l a b e l e d for t h r e e h o u r s w i t h [3H] u r i d i n e . In t h i s case o n l y m R N A a n d 5S-RNA are labeled [11]. T h e total s p e c t r u m o'f RNA c o n t a i n i n g p a r t i c l e s as r e v e a l e d b y zonal c e n t r i f u g a t i o n w a s a n a l y s e d in t h r e e steps by s u c r o s e g r a d i e n t electro,phoresis. By t h i s m e a n s the total r a n g e of p a r t i c l e s b e t w e e n 40S a n d 400iS is s u b d i v i d e d into t w o m a i n f r a c t i o n s : the p o t y s o m e s ( m o b i l i t y : 0.4.2) a n d the m o n o r i b o somes ( m o b i l i t y : 0.6.5) (fig. 1 a). Most of the r a d i o a c t i v i t y w a s f o u n d u n d e r the p o l y s o m e p e a k w i t h a s h o u l d e r in the c e n t r a l valley b e t w e e n the t w o ; t h e r e is a l m o s t n o r a d i o a c t i v i t y in the m o n o r i b o somes f r a c t i o n . T h e r a n g e of p a r t i c l e s sizes from 70'S to 9O,S (fig. 1 b) reveals the m o n o r i b o s o m a l p e a k (mobidity : 0 . 6 5 ; the r a d i o a c t i v i t y p e a k does not c o i n c i d e w i t h it, b u t is r a t h e r l o c a t e d at the site of t h e s h o u l d e r i n figure 1 a ( m o b i l i t y : 0.58). T h e smalI~r sizes p a r t i c l e s (30S to 60S) are resolved into (fig. 1 c) the free 60S r i b o s o m a l s u b u n i t s ( m o b i l i t y : 0.7,1) a n d t h e free 40S-subunits (mobility : 0.48) (see r e f e r e n c e 7 for the i d e n t i f i c a t i o n o,f the f r a c t i o n s ) . T h e r a d i o a c t i v e m R N A c o n t a i n i n g R N P s a g a i n in the c e n t r a l p o s i t i o n (.fig. 1 c) ( m o b i l i t y : 0.57). The p a r t i c l e s c o n t a i n i n g m R N A are free in t h e c y t o p l a s m b e c a u s e t h e y h a v e the same m o b i l i t y w h e n t a k e n f r o m the 70S to 90S r e g i o n s as well as f r o m the 30iS to 60S region. F u r t h e r m o r e the m o b i l i t i e s o,f these p a r t i c l e s are l o w c o m p a r e d to t h a t of d e p r o t e i n i z e d RNA t h a t m i g r a t e s out o,f the d i a g r a m (fig. 1) u n d e r the same c o n d i t i o n s (7]. T h e results s h o w n in figures 1 a, b, a n d c are not d u e to the a c t i o n of a c t i n o m y c i n D ; essentially the same r e s u l t s are o,btained, if one designs BIOCHIMIE, 1 9 7 6 , 58, n ° 3.

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7 - - ARTIFICIAL RCNPs w e r e p r e p a r e d b y a.dding d i f f e r e n t amoun.ts of c y t o p l a s m i c p r o t e i n to 6 ~g of e i t h e r 18S or 2,8S ri~bosonlal RNA. RESULTS.

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Fro. 2. - - Electrophoresis of polysomes dissociated by EDTA, comparison with EDTA treated informosomes. A three liter suspension culture was ]abeled in the presence of aetinomycin D as indica{ed in Materials and Methods. A postmitochondrial supernatant was repared', layered on a 15-3'0 per cent sucrose gradient in ypotonic buffer and centrifuged at 26.000 rpm for two hrs. (4°C, a-oto.r S,'vV 2.7). Tl~e fractions contain~ing polysomes and p~trticles in the 40S-80S range, were kept respectively, and c(meentrated bv cenirifugation (4°C, rotor Ti 50, 30.000 rpm, 15 hrs). "they were then resnspended in buffer C containing 50 mM EDTA, pH 8.1, eleetrophoresis was run at 150 v ol~s for 17 hours. The arrow on the top of the di.agram recal.1 the mobil i t y of native ribosome (Rib.) and depratein~ized RNA (RNA). --Absorbance at 26ff nm. - - O ~ [3HI uridine radioactivity from polysomes. @@@ [3H] uridine radioactivity from -~0S-80S range fraction.

Polysomal mRNPs (ps-mRNPs). So far, p s - m R N P s h a v e m o s t c o n v e n i e n t l y been o b t a i n e d f r o m p o l y s o m e s by t r e a t m e n t w i t h EDTA. As a l r e a d y s h o w n b y H e n s h a w ' s E12], by P e r r y ' s [4] a n d BlobeI's [13] groups, mI~NA rem a i n s b o u n d to p r o t e i n s w h e n d i s l o d g e d f r o m its r i b o s o m a l associates. T h e l i b e r a t e d c o m p l e x , p s - m R N P , h a s an R N A / p r o t e i n r a t i o a n d a d e n s i t y close to the ones of the i n f o r m o s o m e s (free mRN,Ps) in the c y t o p l a s m [41. H o w e v e r , o n l y in e x c e p t i o n a l cases h a v e E D T A - t r e a t e d p a r t i c l e s c o n s e r v e d f u n c t i o n a l qualities [14, 15]. P o l y s o m e s d i s s o c i a t e d in b u f f e r s contain.ng RDTA w e r e subj e c t e d to s u c r o s e g r a d i e n t e l e c t r o p h o r e s i s . We

320

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h a v e n o t i c e d t h a t all c o n s t i t u e n t s m i g r a t e v e r y r a p i d l y , so t h a t e l e c t r o p h o r e s i s w a s r u n at l o w e r v o l t a g e (1'50 v o l t s ) ; (fig. 2) u n , d e r s t a n d a r d c o n difi,ons t h e m a t e r i a l w o t d d h a v e t r a v e r s e d t h e chamber and reached the buffer reservoir. From actinomycin D treated polysomes, which were subsequently dissociated by EDTA, one broad p e a k of m a t e r i a l is o b t a i n e d ( r e v e a l e d b y its 260 n m a b s o r b a n c e w i t h a m o b i l i t y a r o u n d 1.0) (fig. 2). It c o m p r i z e s b o t h r i b o s o m a l s u b u n i t s , u n resolved. A radioactive mRNA carrying fraction, n o t c o i n c i d i n g w i t h t h e f o r m e r , is also o b t a i n e d ( m o b i l i t y : 0.8,6). C o m p a r i n g w i t h e a r l i e r r e s u l t s (fig. 1 a n d o u r p r e c e d i n g r e p o r t ) [7] w e c o n c l u d e t h a t ]SDTA t r e a t m e n t clla'nges t h e e l e , c t r o p h o r c t i c b e h , a v i o r of 405 a n d 60,S s u b u n i t s . T h e c h a n g e is f a i r l y drastic: the particles migrate even faster than f r e e R N A f p h e n o l - d e p r o t e i n i z e d ) . I n o r d e r to c o n vince ourselves that the radioactive material

and K. Kdhler. s h o w n i n fig. 2 i n d e e e d r e p r e s e n t e d R N A - p r o t e i n complexes we doubly labeled polyso.mes with [1~C] u r i d i n e an.d [3H] l e u c i n e f o r a n e x t e n d e d time. Ribasomal RNA and protein label are superi m p o s e d . A l t h o u g h p s - r n R N P s e e m to b e c o m p l e t e (as also p r o v e n b y m a n y a u l h o r s ) , t h e y p r e s u m a b l y h a v e also b e e n a f f e c t e d .drastica3ply. E D T A - t r e a t e d i n f o r m o s o ~ n e s o.f size c l a s s e s b e t w e e n 305 a n d 90,S h a v e d r a s t i c a l l y c h a n g e d t h e i r m o b i l i t y a n d t h e r e s u l t s r e s e m b l e s t h i s f r o m p s - m R N P (see

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A comparative study by sucrose gradient electrophoresis of messenger ribonucleoprotein complexes.

The recently developed method of sucrose gradient electrophoresis has been used in the investigation of mRNA containing particles prepared in an unden...
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