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Synthesis of α- and β-globin directed by messenger ribonucleoprotein from rabbit reticulocytes
251
Biochimica et Biophysica Acta, 378 (1975) 251--259 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98202
SYNTHESIS OF a- AND fi-GLOBIN DIRECTED BY MESSENGER R I B O N U C L E O P R O T E I N F R O M RABBIT RETICULOCYTES
VIVIAN ERNST and HENRY R.V. ARNSTEIN
Department of Biochemistry, University of London King's College, Strand, London WC2R 2LS (U.K.) (Received August 14th, 1974)
Summary The translation of globin messenger ribonucleoprotein (mRNP) obtained from high salt-washed rabbit reticulocyte ribosomes by treatment with EDTA was investigated using a cell-free system from mouse Krebs II ascites t u m o u r cells. The messenger activity of the m R N P and the m R N A derived from it by mild deproteinization was compared in the presence and absence of reticulocyte initiation factors. Both forms gave identical products over a wide range of messenger concentration and there was no qualitative or quantitative difference in their efficiency as messengers. It is concluded that the proteins associated with polysomal m R N A do n o t alter the specificity of translation of a- and fi-globin messengers or the requirement for initiation factors.
Introduction Eukaryotic messenger RNA may be obtained in the form of characteristic ribonucleoprotein complexes (mRNP) by treatment of polysomes with EDTA [1]. In the case of globin messenger, the m R N P released by EDTA from rabbit reticulocyte polysomes has been shown to bind to the small ribosome subunit [2,3], to be incorporated into polysomes in a cell-free system from reticulocytes under conditions of protein synthesis [3] and to stimulate the incorporation of [ 14 C] leucine into protein -by cell-free extracts from Krebs ascites cells [4]. The functional significance of the association of specific proteins with messenger RNA is still unclear. It has been shown, however, that the proteins of rabbit globin m R N P differ physically and functionally from initiation factors [4], although one of them may be involved in some way in the binding of m R N A to the small ribosomal subunit [2,5]. Abbreviations: m R N P , messenger ribonucleoproSein.
252 The translation of the messenger RNAs for the a- and ~-globin chains is controlled independently. In vivo differences in the initiation frequency of aand fi-globin synthesis have been observed in rabbit reticulocytes [6] and in cell-free extracts from Krebs II ascites cells the ratio of ~/~ globin synthesis varies markedly with both the concentration of globin messenger RNA and initiation factors [7]. In these experiments with globin messenger, as in nearly all other examples of messenger-directed cell-free protein synthesis reported in the literature, free m R N A rather than mRNP was used. In view of the possibility that the proteins associated with natural messenger in the ribonucleoprotein complexes might affect the translation of mRNA it was of interest to compare the activity of globin m R N A and mRNP in directing the synthesis of a- and ~-globin chains in a heterologous cell-free system. Experimental
Materials ATP, GTP, creatine phosphate and creatine phosphokinase were purchased from Boehringer Mannheim GmbH (Mannheim, G.F.R.). Dithiothreitol was obtained from Koch-Light Laboratories Ltd. (Colnbrook, Berks., U.K.) and the radioactively labelled amino acids were from the Radiochemical Centre {Amersham, Bucks., U.K.). Grade I purified sucrose and N-laurylsarcosine were obtained from Sigma Chemicals Co. (St. Louis, Missouri, U.S.A.).
Isolation of reticulocyte ribosomes White New Zealand rabbits (2 kg body wt) were made anaemic by four consecutive daily injections of phenylhydrazine (0.6 ml of a neutralised solution containing 2.5% (w/v) phenylhydrazine and 1 mM mercaptoethanol). On the sixth day reticulocytes were obtained as previously described [8] and lysed with an equal volume of ice-cold distilled water. Ribosomes were isolated by centrifugation of the post-mitochondrial supernatant at 100 000 × g for 3 h. The ribosomes were purified by sucrose and salt washings according to the methods described by Blobel [9,10] with the following modifications. Ribosomes were resuspended at a concentration of 50--75 A26o n~n units/ml in 50 mM Tris--HC1 (pH 7.5 at 20°C), 25 mM KC1 and 5 mM MgC12. Aliquots (2 ml) were layered onto a sucrose bilayer consisting of 2 ml of 1.35 M sucrose and 2 ml 2 M sucrose in the same buffer. The ribosomes were pelleted by centrifugation in the 10 × 10 aluminium rotor of the MSE 50 ultracentrifuge at 162 000 X g for 16.5 h. The ribosomal pellets were resuspended in ice-cold distilled water at a concentration of 100--150 A260 nm units/ml and buffer was added to give a final concentration of 500 mM KC1, 50 mM Tris--HC1, pH 7.5, 5 mM MgC12 and 2 mM dithiothreitol. Aliquots (2 ml) of this ribosomal suspension were layered over 2 ml of 1 M sucrose in the same buffer. A pellet of salt-washed ribosomes was obtained after centrifugation at 260 000 X g for 1 h in the 10 X 10 titanium angle rotor of the MSE 65 ultracentrifuge.
Preparation of globin mRNP and m R N A The salt-washed ribosomal pellets obtained above were resuspended in 10 mM Tris--HC1, pH 7.6, and made 33 mM with EDTA to release globin mRNP
253 2.0
2.0
(a) rnRNP E c o
(b)
mRNA
1.5
8 ,~ 1.c
1.0
o 0.5
%
' ; 10
.
8
.
.
.
.
7 6 5 4 Fraction No.
.
.
3
2
1
Top Fraction No.
Fig. 1. P u r i f i c a t i o n of r n R N A a n d m R N P . A s o l u t i o n o f m R N P c o n t a i n i n g 0.1 r a g R N A in 0 . 2 m l o f w a t e r w i t h or w i t h o u t 1% N - l a u r y l s a r c o s i n e w a s l a y e r e d o n t o a 5.5 m l 8 - - 4 0 % s u c r o s e g r a d i e n t in 50 m M Tris'--HC1 ( p H 7 . 5 ) - - 2 5 m M KC1. A f t e r c e n t r i f u g a t i o n at 2 5 5 0 0 0 X g a y f o r 7 h at 4 ° C t h e g r a d i e n t w a s c o l l e c t e d u s i n g an I S C O d e n s i t y g r a d i e n t f r a c t i o n a t o r e q u i p p e d w i t h a 0 . 5 - c m f l o w cell to m o n i t o r t h e u l t r a v i o l e t a b s o r b a n c e . (a) U n t r e a t e d m R N P . (b) m R N P a f t e r d e p r o t e t h i z i n g w i t h 1% N - l a u r y l s a r c o s i n e .
[2]. The 14 S m R N P was separated from the ribosomal subunits by centrifugation in a 10--40% sucrose gradient in 10 mM Tris--HC1, pH 7.6, in the Spinco B XIV zonal rotor [ 1 1 ] . The m R N P was precipitated from the appropriate gradient fractions by making the solution 0.3% with respect to NaC1 and adding 2.5 vol. of ethanol. The precipitate was kept at --20°C overnight, washed twice with 70% ethanol and dried in vacuo. A portion of this mRNP was treated with 1% N-lauryl sarcosine and the m R N A separated from the proteins by sucrose density gradient centrifugation (Fig. lb). A parallel sucrose gradient in the absence of detergent was performed to purify m R N P (Fig. la). Protein-free m R N A and m R N P were recovered from the pooled gradient fractions as described above. The precipitated m R N A and m R N P were dried and resuspended in ice-cold distilled water at a concentration of 0.2--1.0 mg RNA per m! and stored frozen at --70°C in small aliquots. The RNA concentration was calculated from a nomogram (California Corporation for Biochemical Research, 3025 Nedford Street, Los Angeles, California, U.S.A.) which corresponds closely to alternative estimations based on the absorbance at 260 nm [12].
Cell-free incubations Krebs II ascites cells were obtained from Dr A.E. Smith (Imperial Cancer Research Fund Laboratories, Lincolns Inn Fields, London, W.C.2) and maintained by intraperitoneal injection of 0.2 ml ascitic fluid every seven days in male Schofield strain mice, 20--25 g b o d y wt. Preincubated $30 was obtained from these cells as previously described [13]. The incubation mixtures (50 pl) contained 15 #l preincubated ascites $30, 20 mM Tris--HC1 (pH 7.5), 3 mM MgC12, 85 mM KC1, 1 mM ATP, 0.2 mM GTP, 1 mM dithiothreitol, 10 mM creatine phosphate, 0.2 mg/ml creatine phosphokinase, 0.12 pmole/ml of each of the protein amino acids except the labelled precursor. The radioactivity of the labelled amino acids used is given in the legends to figures and tables. Where indicated reticulocyte initiation factors, prepared by extracting rabbit reticulocyte ribosomes with 0.5 M KC1 as described by Miller and Schweet [14] were also added.
254
The reaction mixtures (50 pl) were incubated for 30 min at 37°C. Then 0.3 M NaOH (0.5 ml) was added and the incubation continued for 15 min before precipitating the protein with 10% (w/v) trichloroacetic acid. The precipitate was filtered on to Whatman glass fibre discs and samples were counted in a Packard 574 liquid scintillation spectrometer using 2,5-diphenyloxazole (PPO) and 1,4-bis-(4-methyl-5-phenyloxazole-2-yl)benzene (dimethyl POPOP) in toluene, giving an efficiency of 80% for ~4C and 15--20% for ~H.
Product analysis When globin messenger RNA is added to the pre-incubated ascites cell-free system, the main product of protein synthesis is globin, but some endogenous protein synthesis also occurs and in the presence of reticulocyte initiation factors there is stimulation of both globin and endogenous protein synthesis [15]. All results have therefore been corrected for endogenous protein synthesis, as well as for the synthesis of globin due to mRNA present in reticulocyte initiation factors [15] by subtracting the incorporation of labelled amino acids in appropriate control incubation mixtures in the absence of globin messenger with or w i t h o u t initiation factors. When required, the radioactivity of protein samples was converted into moles of amino acid incorporated, assuming no dilution of isotope, and the a//3 globin ratio was obtained from the molar ratio of valine and isoleucine [7 ]. Where indicated, c~- and fi-globin were separated by chromatography on c a r b o x y m e t h y l cellulose [16] using a gradient of formic acid (0:6--1.5 M)--pyridine (0.06--0.15 M) as previously described [7]. The radioactivity in each chain was determined directly after precipitation of the protein in the appropriate gradient fractions by adding an equal volume of 20% trichloroacetic acid. Results
Characterization of globin m R N P To be able to compare rigorously the efficiency of translation of mRNA with mRNP it was necessary to deproteinize the mRNP using as mild a procedure as possible in order to avoid any inactivation of messenger or preferential isolation of either a- or ~-globin mRNA. Phenol and sodium dodecylsulphate extraction at neutral pH results in a considerable loss of the poly(A) containing m R N A from the aqueous phase and cleavage of mRNA [17]. To eliminate such effects mild detergent treatment, as described in the methods, was used as a means of obtaining m R N A from mRNP. The protein moiety of the mRNP was analysed on polyacrylamide gels (Fig. 2a). Two main protein bands were observed with molecular weights of 49 000 -+ 1000 and 72 000 +- 1000 using the following standards: phosphorylase (91 000), alkaline phosphatase (80 000), bovine serum albumin (68 000), pyruvate kinase (57 000), ovalbumin (43 000), creatine kinase (40 000). When mRNP was poorly separated from ribosomal subunits by overloading of the sucrose gradient other proteins were also present although in much lower amounts (unpublished observations). A maximum loading of the gradient of 2000 A 2 6 0 nm units of ribosomes was observed to give an mRNP preparation with little or no contamination with ribosomal proteins. The integ-
255
Fig. 2. A n a l y s i s of p u r i f i e d m R N A a n d m R N P b y p o l y a c r y l a m i d e - g e l e l e c t r o p h o r c s i s . (a) T h e m R N P f r o m the g r a d i e n t s h o w n in Fig. l a (20 t~g) in s a m p l e b u f f e r ( 2 0 pl) w a s i n c u b a t e d at 37C'C for 15 m i n , a d d e d to a 8% a c r y l a m i d e gel w i t h 3% a c r y l a m i d e s p a c e r and e l e c t r o p h o r e s e d a c c o r d i n g to t h e m e t h o d of L a e m m l i [ 1 8 ] . T h e gel w a s s t a i n e d for p r o t e i n w i t h 0.2% C o o m a s s i e brilliant blue in 30% m e t h a n o l - - l O % acetic acid. (b) T h e m R N A (10 pg) f r o m the g r a d i e n t s h o w n in Fig. l b was a d d e d to a 4% a c r y l a m i d e gel and e l e c t r o p h o r e s e d a c c o r d i n g to L o e n i n g [ 1 9 , 2 0 ] . T h e gel was s t a i n e d f o r R N A w i t h 0.2% t o l u i d i n e blue in 0.4 M s o d i u m a c e t a t e b u f f e r ( p H 4.7).
rity of the RNA samples obtained after the second sucrose gradient centrifugations was analysed on 4% RNA gels [19,20]. A single RNA band (Fig. 2b) with no visible degradation of the RNA was observed in both cases. The m R N A contained no detectable protein contaminants when protein gels loaded with 40 pg were examined. The RNA coincided with 9-S m R N A kindly donated as marker by P. Hamlyn (Biophysics Department, King's College, Drury Lane, London, W.C.2). The m R N A had a molecular weight of approximately 220 000 using RNA standards (rabbit reticulocyte 18 S, 5 S and 4 S, Escherichia coli 23 S and 16 S) as described by Gould and Hamlyn [11].
Cell-free protein synthesis directed by m R N P A comparison of cell-free protein synthesis in the presence of increasing amounts of either m R N A or mRNP shows that both preparations are equally active, in relation to their RNA content, in stimulating the incorporation of [ 14 C] leucine over the range 10--40 pg of RNA per ml of incubation mixture (Fig. 3). In agreement with earlier results [7], globin m R N A had a differential effect on the incorporation of valine and isoleucine into protein (Fig. 4a), which is particularly marked in the presence of initiation factors. Thus, with initiation factors present the incorporation of valine increases approximately linearly in response to mRNA up to a concentration of 30 pg/ml and then levels off, whereas the incorporation of isoleucine reaches a m a x i m u m at 20 pg/ml (Fig. 4a). In a parallel experiment with mRNP very similar results were obtained and the same differential effect on the incorporation of valine and isoleucine was observed {Fig. 4b).
256 1600 mRNA
~'~ 1400 w .~ 1200
/"
boool '~ 80C "F. 60C o
f
N
~ 4oc 2OC
I
I
I
I
10 20 30 40 Messenger concentration (/~g RNA ml 4)
Fig. 3. U t i l i z a t i o n of m R N A a n d m R N P for cell-free p r o t e i n synthesis. T h e s t a n d a r d cell-free m i x t u r e s ( 5 0 /~l) c o n t a i n i n g 0 . 1 5 pCi or L-[ 14C] l e u c i n e ( 3 4 2 C i / m o l e ) as t h e r a d i o a c t i v e a m i n o acid w e r e i n c u b a t e d at 3 7 ° C for 30 rain w i t h i n c r e a s i n g a m o u n t s of e i t h e r m R N A or m R N P . o - - i , m R N A ; o . . . . . a, mRNP. (a) mRNA
(b) mRNP
-
O-
U
6--
_
/ //+
Val
Val ,o---<)
o-~-o
5
/ / + Factors
Factors
c !
2
/I
. -O"-F~c;r~ ~
v
I
I
10
I
--Ile
I
20 30 40 10 20 30 Messenger concentration (#g R N A / m l )
40
Fig. 4. T h e e f f e c t of globin m R N A a n d m R N P o n t h e i n c o r p o r a t i o n of valine a n d i s o l e u c i n e i n t o p r o t e i n . I n c u b a t i o n m i x t u r e s (50 /~1) c o n t a i n i n g 0 . 0 8 /~Ci of L - [ 1 4 C ] v a l i n e ( 2 6 5 C i / m o l e ) a n d 1.5 /zCi of L - [ 3 H ] isoleucine (10 C i / m m o l e ) w e r e i n c u b a t e d for 30 rain at 3 7 ° C . Where i n d i c a t e d i n i t i a t i o n f a c t o r s in 0.2 M KC1 (15 M1) a n d i n c r e a s i n g a m o u n t s of e i t h e r m R N A (a) or m R N P ( b ) w e r e a d d e d . O p e n s y m b o l s ( o a): R a d i o a c t i v i t y due to [ 14C] valine; c l o s e d s y m b o l s (0, i ) : R a d i o a c t i v i t y d u e to [3 H ] i s o l e u c i n e . , o: w i t h i n i t i a t i o n f a c t o r s ; a m: w i t h o u t i n i t i a t i o n factors. 2,5 --
(a) m R N A
•~ 2.0 --
~t5 £
(b) m R N A
2.5
2.0
r-
7.5Factors
+ Factors
~1.0
1.C
._o 0.~
0.5 - actors
I
lO
I
1
-
I
20 30 40 Messenger concentration
,I
1,
10 20 ( #g R N A / m ; )
I
30
I
40
Fig, 5. S y n t h e s i s of o~- a n d ~-giobin d i r e c t e d b y giobin m R N A or m R N P . T h e results given in Fig. 4 w e r e u s e d to c a l c u l a t e [ 7 ] the r a t i o of ~x//3 globin s y n t h e s i z e d at d i f f e r e n t c o n c e n t r a t i o n s of m R N A (a) or m R N P ( b ) e i t h e r in the p r e s e n c e ( e ) or a b s e n c e (o) of i n i t i a t i o n factors.
257 TABLE
I
CELL-FREE
SYNTHESIS
O F ce- A N D ~ - G L O B I N
DIRECTED
BY mRNA
OR mRNP
Incubation mixtures (0.1 ml) containing 24 pCi of L-[35S] methionine (195 Ci/mmole) were incubated at 3 7 ° C f o r 3 0 m i n . W h e r e i n d i c a t e d , i n i t i a t i o n f a c t o r s i n 0 . 2 M KC1 ( 3 0 #1) w e r e a d d e d . A t t h e e n d o f t h e incubation period, postribosomal supernatant (0.3 ml) was added to provide carrier haemoglobin (approx. 1 5 r a g ) a n d t h e p r o d u c t w a s p r e c i p i t a t e d w i t h 1 % I4Cl in a c e t o n e a t - 2 0 ° C . T h e p r e c i p i t a t e w a s d r i e d , d i s s o l v e d i n d i l u t e c o l u m n b u f f e r (1 m l ) a n d c h r o m a t o g r a p h e d on carboxymethyl cellulose for determinat i o n o f t h e r a d i o a c t i v i t y i n c o r p o r a t e d i n t o t h e s e p a r a t e d c~- a n d ~ - g l o b i n c h a i n s [ 7 ] . Messenger
mRNA mRNP mRNA m RNP mRNA mRNP mRNA mRNP
RNA concentration (pg/ml)
Initiation factors
10 10 40 40 10 10 40 40
+ + + +
Radioactivity
in p r o t e i n
(cpm)
(~-globin
~-globin
5570 5970 6 380 6 400 36700 37 400 37 6 0 0 38 800
4930 5 120 15200 16 000 15400 16 9 0 0 60700 63600
Ratio of ~/~ globin
1.13 1.16 0.42 0.40 2.38 2.21 0.62 0.61
When the results given in Fig. 4 are used to calculate the ratio of a- and ~-globin synthesized in response to mRNA and mRNP it can be seen (Fig. 5) that in both cases there is a decrease in the a/f~ ratio as the messenger concentration is increased from 10 to 40 pg/ml. Both with and without initiation factors this ratio is greater than one at 10 pg of RNA per ml and approximately 0.4--0.6 at 40 pg of RNA per ml. In another experiment, the incorporation of labelled methionine into aand ~-globin was determined after separating the chains by chromatography with c a r b o x y m e t h y l cellulose. The results (Table I) confirm that the cell-free system synthesizes similar amounts of globin in response to messenger whether added as free m R N A or as mRNP. In both cases, an increase in messenger concentration results in a relatively greater stimulation of the synthesis of ~-globin compared with that of a-globin. Thus, the activities of m R N A and mRNP in this cell-free system are both qualitatively and quantitatively identical. Discussion
Several reports in the literature [2,10,21,22] show that globin m R N A which is released upon dissociation of ribosomes with EDTA in the presence of low salt [2,20,21] or by puromycin and high salt [10] is bound to two main distinct proteins. The molecular weights of 72 000 and 49 000 determined by us for the two proteins agree closely with the values (78 000 and 52 000 respectively) obtained by Blobel [10] for rabbit globin mRNP and with those (73 000 and 49 000) of Morel et al. [21,22] for duck globin mRNP proteins. In agreement with Blobel's findings [10] we observed that mRNP released from ribosomes that had not been subjected to a salt wash prior to dissociation with EDTA contained other proteins in variable quantities, notably one of molecular weight 115 000 and several others with molecular weights less than
258 40 000. Since these components were almost absent from mRNP prepared from salt-washed ribosomes, they are considered to be ribosomal proteins which are non-specifically adsorbed on to mRNP during isolation. For this reason we have used only the purified mRNP obtained from salt-washed ribosomes for the cell-free experiments. Previous work has shown that the incorporation of leucine into protein by a cell-free mouse liver S 30 system is increased in response to rabbit globin mRNP [23] and rabbit globin mRNP also stimulates protein synthesis by similar cell-free preparations from rat liver [24]. A comparison of the activities of the globin mRNA and mRNP in these cell-free systems showed that the free m R N A was somewhat more active, but no product analysis was made in the experiments with messenger ribonucleoprotein [23,24]. In a more recent report, rabbit globin mRNP was found to be 10--20% more active than the m R N A in stimulating the incorporation of leucine into protein by cell-free systems from chick-embryo brain or Ehrlich ascites cells [25]. The results of our experiments , which were done with more highly purified preparations of mRNP, show that the ascites cell-free system translates globin messenger equally efficiently whether it is added as free m R N A or as the purified ribonucleoprotein complex released from polysomes by EDTA treatment. In both cases the product is mainly globin, as determined by chromatography of the chains on c a r b o x y m e t h y l cellulose. The same decrease in the a/~ globin ratio with increasing messenger concentrations as previously found with globin m R N A [7] has now been observed with mRNP. At any given messenger concentration the addition of crude initiation factors, obtained by extracting polyribosomes with 0.5 M KC1, not only stimulates protein synthesis but also increases the proportion of a-globin synthesized [7], indicating a differential requirement of the a- and ~-globin mRNAs for initiation factors. Any effect of the mRNP protein on initiation would therefore be expected to result in changes of the a/~ globin ratio as compared with the corresponding ratio for mRNA. Since the observed effects were, however, quantitatively identical with both m R N A and mRNP it is unlikely that the proteins alter the specific recognition of the mRNA by ribosomes and initiation factors. These conclusions are consistent with recent results obtained by others [4] showing that in the ascites cell-free system the incorporation of [ L4C]leucine into protein has similar kinetics whether directed by globin m R N A of globin mRNP. Blobel [26] has shown that the larger of the two proteins in globin mRNP is associated with the poly(A) region at the 3' end. Several recent reports [27--29] indicate that messenger RNAs from which poly(A) has been removed are translated in cell-free systems with equal efficiency as the native m R N A [27--29]. These results, together with the experiments reported here, suggest that the protein attached to the poly(A) region is not involved in the translation process. The location of the other protein in mRNP is still unclear and there is no convincing evidence that it participates in protein synthesis. The main function of both proteins may therefore be concerned with nuclear processing, transport or maturation of mRNA.
259
Acknowledgments We should like to thank P. Hamlyn for gifts of globin messenger RNA and for valuable advice in the use of zonal rotor centrifugation. We also thank the Science Research Council for the award of a postgraduate studentship to V.E. and the Medical Research Council for an equipment grant to the Department. References 1 W i l l i a m s o n , R. ( 1 9 7 3 ) F E B S L e t t . 3 7 , 1 - - 6 2 L e b l e u , B., M a r b a i x , G., H u e z , G., T e m m e r m a n , J., B u r n y , A. a n d C h a n t r e n n e , H., ( 1 9 7 1 ) E u r . J. Biochem. 19,264--269 3 P r a g n e l l , I.B., M a r b a i x , G., A r n s t e i n , H . R . V . a n d L e b l e u , B. ( 1 9 7 1 ) F E B S L e t t . 1 4 , 2 8 9 - - 2 9 2 4 N u d e l , U., L e b l e u , B., Z e h a v i - W i U n e r , T. a n d Revel, M. ( 1 9 7 3 ) E u r . J. B i o c h e m . 3 3 , 3 1 4 - - 3 2 2 5 C a s h i o n , L.M. a n d S t a n l e y , J r , W.M. ( 1 9 7 4 ) P r o c . N a t l . A c a d . Sci. U.S. 7 1 , 4 3 6 - - 4 4 0 6 L o d i s h , H . F . ( 1 9 7 1 ) J . Biol. C h e m . 2 4 6 , 7 1 3 1 - - 7 1 3 8 7 Hall, N.D. a n d A r n s t e i n , H . R . V . ( 1 9 7 3 ) F E B S L e t t . 3 5 , 4 5 - - 5 0 8 A r n s t e i n , H . R . V . , C o x , R . A . a n d H u n t , J . A . ( 1 9 6 4 ) B i o c h e m . J. 9 2 , 6 4 8 - - 6 6 1 9 Blobel, G. a n d S a b a t i n i , D. ( 1 9 7 1 ) P r o c . N a t l . A c a d . Sci. U.S. 6 8 , 3 9 0 - - 3 9 4 10 B l o b e l , G. ( 1 9 7 2 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 4 7 , 8 8 - - 9 5 11 G o u l d , H . J . a n d H a m l y n , P. ( 1 9 7 3 ) F E B S L e t t . 3 0 , 3 0 1 - - 3 0 4 12 B r u n s , G.P., F i s c h e r , S. a n d L o w y , B . A . ( 1 9 6 5 ) B i o c h i m . B i o p h y s . A c t a 9 5 , 2 8 0 - - 2 9 0 13 M a t h e w s , M.B. a n d K o r n e r , A. ( 1 9 7 0 ) E u r . J. B i o c h e m . 1 7 , 3 2 8 - - 3 4 3 1 4 Miller, R . L . a n d S c h w e e t , R.S. ( 1 9 6 8 ) A r c h . B i o c h e m . B i o p h y s . 1 2 5 , 6 3 2 - - 6 4 6 15 M a t h e w s , M.B., P r a g n e l l , I.B., O s b o r n , M. a n d A r n s t e i n , H . R . V . ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2 8 7 , 113--123 1 6 D i n t z i s , H.M. ( 1 9 6 1 ) P r o c . N a t l . A c a d . Sci. U.S. 4 7 , 2 4 7 - - 2 6 1 17 P e r r y , R . P . , La T o r r e , T., K e l l e y , D.E. a n d G r e e n b e r g , J . R . ( 1 9 7 2 ) B i o c h i m . B i o p h y s . A c t a 2 6 2 , 220--226 18 L a e m m l i , U . K . ( 1 9 7 0 ) N a t u r e 2 2 7 , 6 8 0 - - - 6 8 5 19 L o e n i n g , U.E. ( 1 9 6 8 ) J. Mol. Biol. 3 8 , 3 5 5 - - 3 6 5 2 0 L o e n i n g , U.E. ( 1 9 6 7 ) B i o c h e m . J. 1 0 2 , 2 5 1 - - 2 5 7 21" M o r e l , C., K a y i b a n d a , B. a n d S c h e r r e r , K. ( 1 9 7 1 ) F E B S L e t t . 1 8 , 8 4 - - 8 8 2 2 M o r e l , C., G a n d e r , E.S., H e r z b e r g , M., D u b o c h e t , J. a n d S c h e r r e r , K. ( 1 9 7 3 ) E u r . J. B i o c h i m . 3 6 , 455--464 2 3 S a m p s o n , J., M a t h e w s , M.B., O s b o r n , M. a n d B o r g h e t t i , A . F . ( 1 9 7 2 ) B i o c h e m i s t r y 1 1 , 3 6 3 6 - - 3 6 4 0 2 4 S a m p s o n , J. a n d B o r g h e t t i , A . F . ( 1 9 7 2 ) N a t . N e w Biol. 2 3 8 , 2 0 0 - - 2 0 2 2 5 H e n d r i c k , D., S c h w a r z , W., Pitzel, S. a n d T i e d e m a n n , H. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 4 0 , 278--284 2 6 B l o b e l , G. ( 1 9 7 3 ) P r o c . N a t l . A c a d . Sci, U.S. 7 0 , 9 2 4 - - 9 2 8 2 7 w i l l i a m s o n , R., C r o s s l e y , J. a n d H u m p h r i e s , S. ( 1 9 7 4 ) B i o c h e m i s t r y 1 3 , 7 0 3 - - 7 0 7 28 B a r d , E., M a r c u s , A. a n d P e r r y , R . P . ( 1 9 7 4 ) Cell 1, 1 0 1 - - 1 0 6 2 9 H u m p h r i e s , S., D o e l , M. a n d W i l l i a m s o n , R . ( 1 9 7 4 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 5 8 , 9 2 7 - - 9 3 1
Biochimica et Biophysica Acta, 378 (1975) 251--259 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98202
SYNTHESIS OF a- AND fi-GLOBIN DIRECTED BY MESSENGER R I B O N U C L E O P R O T E I N F R O M RABBIT RETICULOCYTES
VIVIAN ERNST and HENRY R.V. ARNSTEIN
Department of Biochemistry, University of London King's College, Strand, London WC2R 2LS (U.K.) (Received August 14th, 1974)
Summary The translation of globin messenger ribonucleoprotein (mRNP) obtained from high salt-washed rabbit reticulocyte ribosomes by treatment with EDTA was investigated using a cell-free system from mouse Krebs II ascites t u m o u r cells. The messenger activity of the m R N P and the m R N A derived from it by mild deproteinization was compared in the presence and absence of reticulocyte initiation factors. Both forms gave identical products over a wide range of messenger concentration and there was no qualitative or quantitative difference in their efficiency as messengers. It is concluded that the proteins associated with polysomal m R N A do n o t alter the specificity of translation of a- and fi-globin messengers or the requirement for initiation factors.
Introduction Eukaryotic messenger RNA may be obtained in the form of characteristic ribonucleoprotein complexes (mRNP) by treatment of polysomes with EDTA [1]. In the case of globin messenger, the m R N P released by EDTA from rabbit reticulocyte polysomes has been shown to bind to the small ribosome subunit [2,3], to be incorporated into polysomes in a cell-free system from reticulocytes under conditions of protein synthesis [3] and to stimulate the incorporation of [ 14 C] leucine into protein -by cell-free extracts from Krebs ascites cells [4]. The functional significance of the association of specific proteins with messenger RNA is still unclear. It has been shown, however, that the proteins of rabbit globin m R N P differ physically and functionally from initiation factors [4], although one of them may be involved in some way in the binding of m R N A to the small ribosomal subunit [2,5]. Abbreviations: m R N P , messenger ribonucleoproSein.
252 The translation of the messenger RNAs for the a- and ~-globin chains is controlled independently. In vivo differences in the initiation frequency of aand fi-globin synthesis have been observed in rabbit reticulocytes [6] and in cell-free extracts from Krebs II ascites cells the ratio of ~/~ globin synthesis varies markedly with both the concentration of globin messenger RNA and initiation factors [7]. In these experiments with globin messenger, as in nearly all other examples of messenger-directed cell-free protein synthesis reported in the literature, free m R N A rather than mRNP was used. In view of the possibility that the proteins associated with natural messenger in the ribonucleoprotein complexes might affect the translation of mRNA it was of interest to compare the activity of globin m R N A and mRNP in directing the synthesis of a- and ~-globin chains in a heterologous cell-free system. Experimental
Materials ATP, GTP, creatine phosphate and creatine phosphokinase were purchased from Boehringer Mannheim GmbH (Mannheim, G.F.R.). Dithiothreitol was obtained from Koch-Light Laboratories Ltd. (Colnbrook, Berks., U.K.) and the radioactively labelled amino acids were from the Radiochemical Centre {Amersham, Bucks., U.K.). Grade I purified sucrose and N-laurylsarcosine were obtained from Sigma Chemicals Co. (St. Louis, Missouri, U.S.A.).
Isolation of reticulocyte ribosomes White New Zealand rabbits (2 kg body wt) were made anaemic by four consecutive daily injections of phenylhydrazine (0.6 ml of a neutralised solution containing 2.5% (w/v) phenylhydrazine and 1 mM mercaptoethanol). On the sixth day reticulocytes were obtained as previously described [8] and lysed with an equal volume of ice-cold distilled water. Ribosomes were isolated by centrifugation of the post-mitochondrial supernatant at 100 000 × g for 3 h. The ribosomes were purified by sucrose and salt washings according to the methods described by Blobel [9,10] with the following modifications. Ribosomes were resuspended at a concentration of 50--75 A26o n~n units/ml in 50 mM Tris--HC1 (pH 7.5 at 20°C), 25 mM KC1 and 5 mM MgC12. Aliquots (2 ml) were layered onto a sucrose bilayer consisting of 2 ml of 1.35 M sucrose and 2 ml 2 M sucrose in the same buffer. The ribosomes were pelleted by centrifugation in the 10 × 10 aluminium rotor of the MSE 50 ultracentrifuge at 162 000 X g for 16.5 h. The ribosomal pellets were resuspended in ice-cold distilled water at a concentration of 100--150 A260 nm units/ml and buffer was added to give a final concentration of 500 mM KC1, 50 mM Tris--HC1, pH 7.5, 5 mM MgC12 and 2 mM dithiothreitol. Aliquots (2 ml) of this ribosomal suspension were layered over 2 ml of 1 M sucrose in the same buffer. A pellet of salt-washed ribosomes was obtained after centrifugation at 260 000 X g for 1 h in the 10 X 10 titanium angle rotor of the MSE 65 ultracentrifuge.
Preparation of globin mRNP and m R N A The salt-washed ribosomal pellets obtained above were resuspended in 10 mM Tris--HC1, pH 7.6, and made 33 mM with EDTA to release globin mRNP
253 2.0
2.0
(a) rnRNP E c o
(b)
mRNA
1.5
8 ,~ 1.c
1.0
o 0.5
%
' ; 10
.
8
.
.
.
.
7 6 5 4 Fraction No.
.
.
3
2
1
Top Fraction No.
Fig. 1. P u r i f i c a t i o n of r n R N A a n d m R N P . A s o l u t i o n o f m R N P c o n t a i n i n g 0.1 r a g R N A in 0 . 2 m l o f w a t e r w i t h or w i t h o u t 1% N - l a u r y l s a r c o s i n e w a s l a y e r e d o n t o a 5.5 m l 8 - - 4 0 % s u c r o s e g r a d i e n t in 50 m M Tris'--HC1 ( p H 7 . 5 ) - - 2 5 m M KC1. A f t e r c e n t r i f u g a t i o n at 2 5 5 0 0 0 X g a y f o r 7 h at 4 ° C t h e g r a d i e n t w a s c o l l e c t e d u s i n g an I S C O d e n s i t y g r a d i e n t f r a c t i o n a t o r e q u i p p e d w i t h a 0 . 5 - c m f l o w cell to m o n i t o r t h e u l t r a v i o l e t a b s o r b a n c e . (a) U n t r e a t e d m R N P . (b) m R N P a f t e r d e p r o t e t h i z i n g w i t h 1% N - l a u r y l s a r c o s i n e .
[2]. The 14 S m R N P was separated from the ribosomal subunits by centrifugation in a 10--40% sucrose gradient in 10 mM Tris--HC1, pH 7.6, in the Spinco B XIV zonal rotor [ 1 1 ] . The m R N P was precipitated from the appropriate gradient fractions by making the solution 0.3% with respect to NaC1 and adding 2.5 vol. of ethanol. The precipitate was kept at --20°C overnight, washed twice with 70% ethanol and dried in vacuo. A portion of this mRNP was treated with 1% N-lauryl sarcosine and the m R N A separated from the proteins by sucrose density gradient centrifugation (Fig. lb). A parallel sucrose gradient in the absence of detergent was performed to purify m R N P (Fig. la). Protein-free m R N A and m R N P were recovered from the pooled gradient fractions as described above. The precipitated m R N A and m R N P were dried and resuspended in ice-cold distilled water at a concentration of 0.2--1.0 mg RNA per m! and stored frozen at --70°C in small aliquots. The RNA concentration was calculated from a nomogram (California Corporation for Biochemical Research, 3025 Nedford Street, Los Angeles, California, U.S.A.) which corresponds closely to alternative estimations based on the absorbance at 260 nm [12].
Cell-free incubations Krebs II ascites cells were obtained from Dr A.E. Smith (Imperial Cancer Research Fund Laboratories, Lincolns Inn Fields, London, W.C.2) and maintained by intraperitoneal injection of 0.2 ml ascitic fluid every seven days in male Schofield strain mice, 20--25 g b o d y wt. Preincubated $30 was obtained from these cells as previously described [13]. The incubation mixtures (50 pl) contained 15 #l preincubated ascites $30, 20 mM Tris--HC1 (pH 7.5), 3 mM MgC12, 85 mM KC1, 1 mM ATP, 0.2 mM GTP, 1 mM dithiothreitol, 10 mM creatine phosphate, 0.2 mg/ml creatine phosphokinase, 0.12 pmole/ml of each of the protein amino acids except the labelled precursor. The radioactivity of the labelled amino acids used is given in the legends to figures and tables. Where indicated reticulocyte initiation factors, prepared by extracting rabbit reticulocyte ribosomes with 0.5 M KC1 as described by Miller and Schweet [14] were also added.
254
The reaction mixtures (50 pl) were incubated for 30 min at 37°C. Then 0.3 M NaOH (0.5 ml) was added and the incubation continued for 15 min before precipitating the protein with 10% (w/v) trichloroacetic acid. The precipitate was filtered on to Whatman glass fibre discs and samples were counted in a Packard 574 liquid scintillation spectrometer using 2,5-diphenyloxazole (PPO) and 1,4-bis-(4-methyl-5-phenyloxazole-2-yl)benzene (dimethyl POPOP) in toluene, giving an efficiency of 80% for ~4C and 15--20% for ~H.
Product analysis When globin messenger RNA is added to the pre-incubated ascites cell-free system, the main product of protein synthesis is globin, but some endogenous protein synthesis also occurs and in the presence of reticulocyte initiation factors there is stimulation of both globin and endogenous protein synthesis [15]. All results have therefore been corrected for endogenous protein synthesis, as well as for the synthesis of globin due to mRNA present in reticulocyte initiation factors [15] by subtracting the incorporation of labelled amino acids in appropriate control incubation mixtures in the absence of globin messenger with or w i t h o u t initiation factors. When required, the radioactivity of protein samples was converted into moles of amino acid incorporated, assuming no dilution of isotope, and the a//3 globin ratio was obtained from the molar ratio of valine and isoleucine [7 ]. Where indicated, c~- and fi-globin were separated by chromatography on c a r b o x y m e t h y l cellulose [16] using a gradient of formic acid (0:6--1.5 M)--pyridine (0.06--0.15 M) as previously described [7]. The radioactivity in each chain was determined directly after precipitation of the protein in the appropriate gradient fractions by adding an equal volume of 20% trichloroacetic acid. Results
Characterization of globin m R N P To be able to compare rigorously the efficiency of translation of mRNA with mRNP it was necessary to deproteinize the mRNP using as mild a procedure as possible in order to avoid any inactivation of messenger or preferential isolation of either a- or ~-globin mRNA. Phenol and sodium dodecylsulphate extraction at neutral pH results in a considerable loss of the poly(A) containing m R N A from the aqueous phase and cleavage of mRNA [17]. To eliminate such effects mild detergent treatment, as described in the methods, was used as a means of obtaining m R N A from mRNP. The protein moiety of the mRNP was analysed on polyacrylamide gels (Fig. 2a). Two main protein bands were observed with molecular weights of 49 000 -+ 1000 and 72 000 +- 1000 using the following standards: phosphorylase (91 000), alkaline phosphatase (80 000), bovine serum albumin (68 000), pyruvate kinase (57 000), ovalbumin (43 000), creatine kinase (40 000). When mRNP was poorly separated from ribosomal subunits by overloading of the sucrose gradient other proteins were also present although in much lower amounts (unpublished observations). A maximum loading of the gradient of 2000 A 2 6 0 nm units of ribosomes was observed to give an mRNP preparation with little or no contamination with ribosomal proteins. The integ-
255
Fig. 2. A n a l y s i s of p u r i f i e d m R N A a n d m R N P b y p o l y a c r y l a m i d e - g e l e l e c t r o p h o r c s i s . (a) T h e m R N P f r o m the g r a d i e n t s h o w n in Fig. l a (20 t~g) in s a m p l e b u f f e r ( 2 0 pl) w a s i n c u b a t e d at 37C'C for 15 m i n , a d d e d to a 8% a c r y l a m i d e gel w i t h 3% a c r y l a m i d e s p a c e r and e l e c t r o p h o r e s e d a c c o r d i n g to t h e m e t h o d of L a e m m l i [ 1 8 ] . T h e gel w a s s t a i n e d for p r o t e i n w i t h 0.2% C o o m a s s i e brilliant blue in 30% m e t h a n o l - - l O % acetic acid. (b) T h e m R N A (10 pg) f r o m the g r a d i e n t s h o w n in Fig. l b was a d d e d to a 4% a c r y l a m i d e gel and e l e c t r o p h o r e s e d a c c o r d i n g to L o e n i n g [ 1 9 , 2 0 ] . T h e gel was s t a i n e d f o r R N A w i t h 0.2% t o l u i d i n e blue in 0.4 M s o d i u m a c e t a t e b u f f e r ( p H 4.7).
rity of the RNA samples obtained after the second sucrose gradient centrifugations was analysed on 4% RNA gels [19,20]. A single RNA band (Fig. 2b) with no visible degradation of the RNA was observed in both cases. The m R N A contained no detectable protein contaminants when protein gels loaded with 40 pg were examined. The RNA coincided with 9-S m R N A kindly donated as marker by P. Hamlyn (Biophysics Department, King's College, Drury Lane, London, W.C.2). The m R N A had a molecular weight of approximately 220 000 using RNA standards (rabbit reticulocyte 18 S, 5 S and 4 S, Escherichia coli 23 S and 16 S) as described by Gould and Hamlyn [11].
Cell-free protein synthesis directed by m R N P A comparison of cell-free protein synthesis in the presence of increasing amounts of either m R N A or mRNP shows that both preparations are equally active, in relation to their RNA content, in stimulating the incorporation of [ 14 C] leucine over the range 10--40 pg of RNA per ml of incubation mixture (Fig. 3). In agreement with earlier results [7], globin m R N A had a differential effect on the incorporation of valine and isoleucine into protein (Fig. 4a), which is particularly marked in the presence of initiation factors. Thus, with initiation factors present the incorporation of valine increases approximately linearly in response to mRNA up to a concentration of 30 pg/ml and then levels off, whereas the incorporation of isoleucine reaches a m a x i m u m at 20 pg/ml (Fig. 4a). In a parallel experiment with mRNP very similar results were obtained and the same differential effect on the incorporation of valine and isoleucine was observed {Fig. 4b).
256 1600 mRNA
~'~ 1400 w .~ 1200
/"
boool '~ 80C "F. 60C o
f
N
~ 4oc 2OC
I
I
I
I
10 20 30 40 Messenger concentration (/~g RNA ml 4)
Fig. 3. U t i l i z a t i o n of m R N A a n d m R N P for cell-free p r o t e i n synthesis. T h e s t a n d a r d cell-free m i x t u r e s ( 5 0 /~l) c o n t a i n i n g 0 . 1 5 pCi or L-[ 14C] l e u c i n e ( 3 4 2 C i / m o l e ) as t h e r a d i o a c t i v e a m i n o acid w e r e i n c u b a t e d at 3 7 ° C for 30 rain w i t h i n c r e a s i n g a m o u n t s of e i t h e r m R N A or m R N P . o - - i , m R N A ; o . . . . . a, mRNP. (a) mRNA
(b) mRNP
-
O-
U
6--
_
/ //+
Val
Val ,o---<)
o-~-o
5
/ / + Factors
Factors
c !
2
/I
. -O"-F~c;r~ ~
v
I
I
10
I
--Ile
I
20 30 40 10 20 30 Messenger concentration (#g R N A / m l )
40
Fig. 4. T h e e f f e c t of globin m R N A a n d m R N P o n t h e i n c o r p o r a t i o n of valine a n d i s o l e u c i n e i n t o p r o t e i n . I n c u b a t i o n m i x t u r e s (50 /~1) c o n t a i n i n g 0 . 0 8 /~Ci of L - [ 1 4 C ] v a l i n e ( 2 6 5 C i / m o l e ) a n d 1.5 /zCi of L - [ 3 H ] isoleucine (10 C i / m m o l e ) w e r e i n c u b a t e d for 30 rain at 3 7 ° C . Where i n d i c a t e d i n i t i a t i o n f a c t o r s in 0.2 M KC1 (15 M1) a n d i n c r e a s i n g a m o u n t s of e i t h e r m R N A (a) or m R N P ( b ) w e r e a d d e d . O p e n s y m b o l s ( o a): R a d i o a c t i v i t y due to [ 14C] valine; c l o s e d s y m b o l s (0, i ) : R a d i o a c t i v i t y d u e to [3 H ] i s o l e u c i n e . , o: w i t h i n i t i a t i o n f a c t o r s ; a m: w i t h o u t i n i t i a t i o n factors. 2,5 --
(a) m R N A
•~ 2.0 --
~t5 £
(b) m R N A
2.5
2.0
r-
7.5Factors
+ Factors
~1.0
1.C
._o 0.~
0.5 - actors
I
lO
I
1
-
I
20 30 40 Messenger concentration
,I
1,
10 20 ( #g R N A / m ; )
I
30
I
40
Fig, 5. S y n t h e s i s of o~- a n d ~-giobin d i r e c t e d b y giobin m R N A or m R N P . T h e results given in Fig. 4 w e r e u s e d to c a l c u l a t e [ 7 ] the r a t i o of ~x//3 globin s y n t h e s i z e d at d i f f e r e n t c o n c e n t r a t i o n s of m R N A (a) or m R N P ( b ) e i t h e r in the p r e s e n c e ( e ) or a b s e n c e (o) of i n i t i a t i o n factors.
257 TABLE
I
CELL-FREE
SYNTHESIS
O F ce- A N D ~ - G L O B I N
DIRECTED
BY mRNA
OR mRNP
Incubation mixtures (0.1 ml) containing 24 pCi of L-[35S] methionine (195 Ci/mmole) were incubated at 3 7 ° C f o r 3 0 m i n . W h e r e i n d i c a t e d , i n i t i a t i o n f a c t o r s i n 0 . 2 M KC1 ( 3 0 #1) w e r e a d d e d . A t t h e e n d o f t h e incubation period, postribosomal supernatant (0.3 ml) was added to provide carrier haemoglobin (approx. 1 5 r a g ) a n d t h e p r o d u c t w a s p r e c i p i t a t e d w i t h 1 % I4Cl in a c e t o n e a t - 2 0 ° C . T h e p r e c i p i t a t e w a s d r i e d , d i s s o l v e d i n d i l u t e c o l u m n b u f f e r (1 m l ) a n d c h r o m a t o g r a p h e d on carboxymethyl cellulose for determinat i o n o f t h e r a d i o a c t i v i t y i n c o r p o r a t e d i n t o t h e s e p a r a t e d c~- a n d ~ - g l o b i n c h a i n s [ 7 ] . Messenger
mRNA mRNP mRNA m RNP mRNA mRNP mRNA mRNP
RNA concentration (pg/ml)
Initiation factors
10 10 40 40 10 10 40 40
+ + + +
Radioactivity
in p r o t e i n
(cpm)
(~-globin
~-globin
5570 5970 6 380 6 400 36700 37 400 37 6 0 0 38 800
4930 5 120 15200 16 000 15400 16 9 0 0 60700 63600
Ratio of ~/~ globin
1.13 1.16 0.42 0.40 2.38 2.21 0.62 0.61
When the results given in Fig. 4 are used to calculate the ratio of a- and ~-globin synthesized in response to mRNA and mRNP it can be seen (Fig. 5) that in both cases there is a decrease in the a/f~ ratio as the messenger concentration is increased from 10 to 40 pg/ml. Both with and without initiation factors this ratio is greater than one at 10 pg of RNA per ml and approximately 0.4--0.6 at 40 pg of RNA per ml. In another experiment, the incorporation of labelled methionine into aand ~-globin was determined after separating the chains by chromatography with c a r b o x y m e t h y l cellulose. The results (Table I) confirm that the cell-free system synthesizes similar amounts of globin in response to messenger whether added as free m R N A or as mRNP. In both cases, an increase in messenger concentration results in a relatively greater stimulation of the synthesis of ~-globin compared with that of a-globin. Thus, the activities of m R N A and mRNP in this cell-free system are both qualitatively and quantitatively identical. Discussion
Several reports in the literature [2,10,21,22] show that globin m R N A which is released upon dissociation of ribosomes with EDTA in the presence of low salt [2,20,21] or by puromycin and high salt [10] is bound to two main distinct proteins. The molecular weights of 72 000 and 49 000 determined by us for the two proteins agree closely with the values (78 000 and 52 000 respectively) obtained by Blobel [10] for rabbit globin mRNP and with those (73 000 and 49 000) of Morel et al. [21,22] for duck globin mRNP proteins. In agreement with Blobel's findings [10] we observed that mRNP released from ribosomes that had not been subjected to a salt wash prior to dissociation with EDTA contained other proteins in variable quantities, notably one of molecular weight 115 000 and several others with molecular weights less than
258 40 000. Since these components were almost absent from mRNP prepared from salt-washed ribosomes, they are considered to be ribosomal proteins which are non-specifically adsorbed on to mRNP during isolation. For this reason we have used only the purified mRNP obtained from salt-washed ribosomes for the cell-free experiments. Previous work has shown that the incorporation of leucine into protein by a cell-free mouse liver S 30 system is increased in response to rabbit globin mRNP [23] and rabbit globin mRNP also stimulates protein synthesis by similar cell-free preparations from rat liver [24]. A comparison of the activities of the globin mRNA and mRNP in these cell-free systems showed that the free m R N A was somewhat more active, but no product analysis was made in the experiments with messenger ribonucleoprotein [23,24]. In a more recent report, rabbit globin mRNP was found to be 10--20% more active than the m R N A in stimulating the incorporation of leucine into protein by cell-free systems from chick-embryo brain or Ehrlich ascites cells [25]. The results of our experiments , which were done with more highly purified preparations of mRNP, show that the ascites cell-free system translates globin messenger equally efficiently whether it is added as free m R N A or as the purified ribonucleoprotein complex released from polysomes by EDTA treatment. In both cases the product is mainly globin, as determined by chromatography of the chains on c a r b o x y m e t h y l cellulose. The same decrease in the a/~ globin ratio with increasing messenger concentrations as previously found with globin m R N A [7] has now been observed with mRNP. At any given messenger concentration the addition of crude initiation factors, obtained by extracting polyribosomes with 0.5 M KC1, not only stimulates protein synthesis but also increases the proportion of a-globin synthesized [7], indicating a differential requirement of the a- and ~-globin mRNAs for initiation factors. Any effect of the mRNP protein on initiation would therefore be expected to result in changes of the a/~ globin ratio as compared with the corresponding ratio for mRNA. Since the observed effects were, however, quantitatively identical with both m R N A and mRNP it is unlikely that the proteins alter the specific recognition of the mRNA by ribosomes and initiation factors. These conclusions are consistent with recent results obtained by others [4] showing that in the ascites cell-free system the incorporation of [ L4C]leucine into protein has similar kinetics whether directed by globin m R N A of globin mRNP. Blobel [26] has shown that the larger of the two proteins in globin mRNP is associated with the poly(A) region at the 3' end. Several recent reports [27--29] indicate that messenger RNAs from which poly(A) has been removed are translated in cell-free systems with equal efficiency as the native m R N A [27--29]. These results, together with the experiments reported here, suggest that the protein attached to the poly(A) region is not involved in the translation process. The location of the other protein in mRNP is still unclear and there is no convincing evidence that it participates in protein synthesis. The main function of both proteins may therefore be concerned with nuclear processing, transport or maturation of mRNA.
259
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