Messenger ribonucleoprotein-directed globin synthesis in an embryonic brain cell-free system

278

Biochimica et Biophysica Acta, 340 (1974) 2 7 8 - - 2 8 4 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m -- Printed in The Netherlands

BBA 97947

MESSENGER R I B O N U C L E O P R O T E I N - D I R E C T E D GLOBIN SYNTHESIS IN AN EMBRYONIC BRAIN CELL-FREE SYSTEM

DON H E N D R I C K , W A L T E R S C H W A R Z , S A B I N E P I T Z E L and H E I N Z T I E D E M A N N Institut fiir Molekularbiologie und Biochemie der Freien Universiti~t Berlin, 1 Berlin 33, Arnimallee 22 (Germany) (Received O c t o b e r l l t h , 1973)

Summary Globin messenger ribonucleoprotein (mRNP) from rabbit reticulocyte polysomes was translated in cell-free systems from ascites cells and chick embryo brain. Messenger ribonucleoprotein stimulated the incorporation of [3 H] leucine by the systems at a higher rate (30--40%) than did globin mRNA. Analysis of tryptic peptides of in vitro synthesized products showed that globin messenger ribonucleoprotein directed the synthesis of complete chains of rabbit hemoglobin in the chick e m b r y o brain cell-free system.

Introduction m R N A of eucaryotes occurs as a complex of RNA and protein when released from polysomes by EDTA or salt treatment [1,2]. This is also true for rabbit hemoglobin (Hb) m R N A which sediments as a messenger ribonucleoprotein (mRNP) complex [3] at a b o u t 14 s and consists of a b o u t 40% RNA and 60% protein [3,4]. Free ribonucleoprotein particles similar to m R N P from polysomes are found in the nucleus and cytoplasm [4--6] of eucaryotic cells. Recently it was shown that two proteins are c o m m o n to and b o u n d to several species of m R N A [7,8], suggesting that m R N P does not result from the nonspecific association of proteins with RNA. To investigate the possible role of the protein moiety of H b - - m R N P in translational control, we prepared cell-free protein-synthesizing systems from ascites cells and compared the degree of stimulation of [3 H] leucine incorporation in this system by rabbit H b - - m R N P and H b - - m R N A . Since the translation of heterologous messengers may be less restricted in t u m o r cells, the translatability of H b - - m R N P was also tested in extracts of e m b r y o brain. Brain was

Abbreviation: mRNP, messenger

ribonucleoprotein.

279 used since it is of ectodermal origin and separates very early in embryonic development from erythropoietic tissue which is of mesodermal origin. Materials and Methods H b - - m R N P was obtained by zonal sucrose centrifugation of EDTA-treated polysomes from rabbit reticulocytes [9]. m R N A was prepared from mRNP by extraction with phenol--chloroform--isoamyl alcohol [10] and purification on sucrose gradients [9]. Cell-free systems were prepared from chick embryo brain (13 days old, free of meninges and blood) and Ehrlich ascites cells from mice by preincubating post-mitochondrial supernatants of the cells with an energy source and amino acids for 30 min at 37°C before desalting on Sephadex G-25 [11]. Void volumes from the column were fractionated, stored in liquid N2, and are referred to as S-30. The system for assaying cell-free protein synthesis has been described [11]. Routine assays (50 pl) contained 10 pCi/ml [3H]leucine (1.9 Ci/mmole). For analysis of the products of the cell-free system [3 H] leucine was increased to 20 pCi/ml (38 Ci/mmole). Globin labeled with [14 C] leucine was prepared by incubating rabbit reticulocytes with [14 C] leucine [12] and subsequently precipitating the globin with acetone [13]. To analyze the in vitro synthesized products, ,4 C-labeled globin was added and cell-free incubations were chromatographed over Sephadex G-100. The globin-containing fractions were pooled and re-chromatographed over a CMcellulose column to separate the a and ~ chains. The ~ chains were oxidized with performic acid [14] and digested with trypsin [15]. The radioactive tryptic peptides were analyzed automatically with an ion-exchange column [16]. Results

Fig. 1 shows a zonal gradient profile for the large-scale preparation of mRNP released from reticulocyte polysomes. Fixation of the m R N P with formaldehyde [17] and centrifugation to equilibrium in CsC1 showed that the fixed m R N P migrated into the gradient as a single major peak banding at a density of 1.47 g/cm 3 . This agrees with results previously published for this material [3]. A comparison of the ultraviolet absorption spectra showed a 260 nm/280 nm ratio of 2.00 for m R N A and 1.77 for mRNP. As shown in Fig. 2 concentrations of mRNP up to a b o u t 0.15 A260 nm unit/ml caused the amino acid-incorporating activity of the e m b r y o brain system to increase in an almost linear manner. Control experiments showed that the K ÷ and Mg 2÷ optima for exogenous m R N A and m R N P in the ascites and brain cell-free systems did not differ and that the brain system (with H b - - m R N P ) synthesized protein at a linear rate for the first 30 min. mRNP consistently stimulated the e m b r y o brain system somewhat more than did the mRNA. Since protein in m R N P makes a small contribution to the absorbance at 260 nm, stimulation by m R N P is, on an RNA basis, somewhat larger than shown in Fig. 2. Attempts to concentrate mRNP by re-centrifugation, dialysis, or lyophilization yielded less active mRNP. Therefore, a saturation of the cell-

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Fig. 1. Z o n a l s e d i m e n t a t i o n of E D T A - t r e a t e d r a b b i t r e t i c u l o c y t e p o l y s o m a l R N A . 3 0 0 m g of p o l y s o m e s in 10 m l o f b u f f e r ( 0 . 2 5 M sucrose, 10 m M Tris--HC1, 1 5 m M KCI, 3 0 m M E D T A , p H 7.4) w e r e p u m p e d o n t o a g r a d i e n t of 7 . 5 - - 3 1 % sucrose in o v e r l a y e r b u f f e r ( 1 0 m M Tris--HC1, 15 m M KC1, 3 m M E D T A , p H 7.4) in t h e B e c k m a n Ti 14 z o n a l r o t o r a n d c e n t r i f u g e d for 23 h at 3 5 0 0 0 r e v . / m i n a n d 0°C. A z o n a l g r a d i e n t r u n c o n s i s t e d of 4 5 0 m l of s u c r o s e g r a d i e n t , 10 m l of p o l y s o m e s , a n d 1 4 0 m l of o v e r l a y e r b u f f e r . D u r i n g c o l l e c t i o n of t h e f r a c t i o n s (12 m l e a c h ) f r o m t h e z o n a l c e n t r i f u g a t i o n r u n , t h e a b s o r b a n e e at 2 6 0 n m was r e c o r d e d a u t o m a t i c a l l y . F o r use in cell-free p r o t e i n s y n t h e s i s , t h e m a t e r i a l in t h e h a t c h e d p o r t i o n of t h e m R N P r e g i o n w a s d i a l y z e d for 5 h a g a i n s t 5 0 0 vol. of 10 m M Tris--HC1, 35 m M KC1, 1.7 m M MgC12, p H 7.5 w i t h t h r e e c h a n g e s .

free system with mRNP could not be achieved. In contrast to an earlier report [18] the highest concentration of mRNP used (0.4 A2 6o n m unit/ml) showed no inhibition of amino acid incorporation. Higher concentrations of m R N A up to 1 A 2 6 0 n m unit/ml did not further stimulate the system. In Table I the stimulation of the rate of [3 H] leucine incorporation by mRNA and mRNP into protein is compared in systems from ascites cells and chick embryo brain. There was a 4--5-fold increase in the amount of [3 H]leucine incorporated in the ascites system and a 2--3-fold increase in the system from chick embryo brain.

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Fig. 2. Effect of m R N P and m R N A concentrations on protein synthesis in a cell-free system from chick e m b r y o brain. Each 50-I~I incubation contained 0.4 A 2 6 0 n m unit of e m b r y o brain S-30. K + and M g 2+ were present at 63 m M and 3.0 raM, respectively. After a 45-rain incubation at 37°C, the hot trichloroacetic acid-insoluble radioactivity was determined. --, with rabbit globin m R N A ; - - , with rabbit globin mRNP.

281 TABLE I E F F E C T OF E X O G E N O U S M E S S E N G E R R I B O N U C L E O P R O T E I N A N D M E S S E N G E R R I B O N U C L E I C A C I D ON [ ~ H ] L E U C I N E I N C O R P O R A T I O N BY H E T E R O L O G O U S C E L L - F R E E P R O T E I N - S Y N T H E SIZING SYSTEMS E a c h 50-/21 i n c u b a t i o n c o n t a i n e d 0 . 4 A 2 6 0 n m u n i t o f c y t o p l a s m i c e x t r a c t f r o m c h i c k e m b r y o b r a i n or E h r l i c h ascites cells a n d w h e r e i n d i c a t e d 0.02 A 2 6 0 rim u n i t o f r a b b i t globin m e s s e n g e r r i b o n u c l e o p r o t e i n o r m e s s e n g e r r i b o n u c l e i c acid. Tris--HC1, K +, a n d Mg 2+ w e r e p r e s e n t in all s y s t e m s at 20 m M , 63 m M a n d 3.0 m M , r e s p e c t i v e l y . T h e final p H of t h e i n c u b a t i o n s was 7.5. A f t e r i n c u b a t i o n f o r 4 5 m i n at 3 7 ° C , the h o t t r i c h l o r o a c e t i c acid-insoluble r a d i o a c t i v i t y w a s d e t e r m i n e d . S o u r c e o f cell-free s y s t e m ( c p m i n c o r p o r a t e d / a s s a y )

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C h i c k E m b r y o Brain

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8 300 26 3 0 0 22000

When the [3 H] leucine-labeled products of the embryo brain system (with or without Hb--mRNP) were analyzed (Fig. 3) it was found that the protein synthesized in vitro by the brain system with Hb--mRNP consisted almost entirely of material co-chromatographing with rabbit a- and ~-globin chains. Without Hb--mRNP there were no proteins synthesized which co-chromatographed with a- or ~-globin chains (not shown).

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Trypsinization of the a chains synthesized in the brain system with mRNP and ion-exchange chromatography of the peptides showed good agreement between the elution profile of [3H]leucine peptides sythesized in vitro and peptides labeled with [, 4C]leucin e from carrier globin. All 13 peaks contain ,4 C-labeled peptides and 3 H-labeled peptides (Fig. 4). 12 peaks are fairly congruent. The incongruency of the peaks at Fractions 127 and 128 could be due to residual endogenous protein synthesis by the S-30. Trypsinization of the chain of rabbit hemoglobin should yield nine leucine-containing peptides. The four extra leucine-containing peptides {Fig. 4) could be due to incomplete digestion by trypsin or contamination of trypsin with other proteolytic enzymes. 58

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Fig. 4. C h r o m a t o g r a p h i c e l u t i o n p r o f i l e o f l e u c i n e - c o n t a i n i n g p e p t i d e s o b t a i n e d b y t r y p t i c d i g e s t i o n o f t h e c~ s u b u n i t o f r a b b i t h e m o g l o b i n s y n t h e s i z e d i n v i t r o . T h e f r a c t i o n s c o n t a i n i n g t h e ~ c h a i n s f r o m t h e C M - 5 2 c h r o m a t o g r a p h i c s e p a r a t i o n were p o o l e d a n d d i a l y z e d a g a i n s t w a t e r , l y o p h i l i z e d , r e - d i s s o l v e d in cold formic acid methanol (4:1 v/v) and oxidized with performic acid for 2 h at --10°C. After repeated lyophilization the protein was digested with trypsin. Analysis of the tryptic peptides was performed automatically [16] using a column of Aminex A (Bio-Rad Laboratories, Richmond, Cal.) 0.45 cm × 40 c m in c o n n e c t i o n w i t h a 1 6 0 - m l linear g r a d i e n t f r o m 0.2 M p y r i d i n e , 4.5 M acetic acid to 2.0 M p y r i d i n e , 25 M acetic acid. Adsorbed peptides were eluted at a buffer pumping rate of 7 ml/h. Counting was c a r r i e d o u t w i t h a B e c k m a n L S - 2 3 0 L i q u i d S c i n t i l l a t i o n s y s t e m e q u i p p e d w i t h a d i s c o n t i n u o u s f l o w cell. C o r r e c t i o n w a s m a d e f o r s p i l l o v e r o f 14 C i n t o t h e 3 H c h a n n e l . T h e p e p t i d e s l a b e l e d w i t h [3 H ] l e u e i n e are derived from the a chain synthesized in vitro with rabbit globin mRNP in the chick embryo brain cell-free s y s t e m . T h e p e p t i d e s l a b e l e d w i t h [ ] 4 C ] l e u c i n e are d e r i v e d f r o m t h e c~ c h a i n s y n t h e s i z e d e n d o g e n o u s l y i n whole rabbit reticulocytes.

283 The labeling of all leucine-containing tryptic peptides together with the fact that the amino- and carboxyl-terminal tryptic peptides of the ~ chain of rabbit hemoglobin contain leucine indicates that the brain system with H b - - m R N P synthesized complete ~ subunits. Discussion

Earlier reports showed that rabbit H b - - m R N P stimulates protein synthesis in extracts from t u m o r cells [ 1 9 ] , rat liver [ 2 0 ] , and mouse liver [18], though no product analysis was made. R o d e n t liver is erythropoietic in embryos and this could perhaps be preserved as a potentiality in the adult. The present work was undertaken to determine whether in an embryonic brain cell-free system the protein moiety of H b - - m R N P exerts stringent translational control which might determine which messengers can be translated in heterologous systems. A crude extract of brain was used to avoid the possibility of losing naturally occurring factors which might interact with the mRNP. Analysis of the in vitro synthesized products revealed that complete globin chains are synthesized in the embryo brain system when H b - - m R N P was added. That H b - - m R N A is translated in a partially purified brain derived system to which brain initiation factors were added is already known [21]. Obviously the protein moiety of the globin m R N P does not exert a stringent tissue specific translational control. The existence of protein factors which specifically stimulate the synthesis of the ~- or ~-globin chain has been reported [ 2 2 , 2 3 ] . But this does not imply that a stringent tissue:specific control of translation is exerted by such factors. One could argue that translational control mechanisms normally operative in cells are impaired in cell-free systems. But stringent translational control may not be required, since free H b - - m R N P cannot be detected in chick embryo brain within the limits of sensitivity of the H b - - m R N A assay in the ascites system (Hendrick, D., unpublished) (< 0.6% of free H b - - m R N A or < 0.03% of the total amount of H b - - m R N A found in erythroblasts is present in brain). This suggests pre-translational control for the synthesis of globin chains and probably for other tissue-specific proteins. Several roles for the protein moiety of the mRNP have been suggested [1,4,24], including a role in binding of m R N A to the 40-S ribosomal subunit [25] or in initiation [2]. On the other hand, protein-free m R N A is faithfully translated in vivo [26] and in vitro [27] in heterologous systems. The fact that mRNP stimulates globin synthesis to a somewhat higher extent than m R N A may suggest that proteins present in mRNP may also be present in limiting amounts in the cell-free systems. This question needs further experimentation. Acknowledgement These investigations were supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich Embryonale Entwicklung und Differenzierung).

284

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

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