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 ) .
/
."... A
~1,s.lo
0~
Buffer C :
2
50 10 3 7
mM mM mM m,M
~
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.
Rib.
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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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