Isolation and translation of non-crystallin messenger RNA from calf lens

447

Biochimica et Biophysica Acta, 454 (1976) 447--456 © Elsevier/North-Holland Biomedical Press

BBA 98785

ISOLATION AND T R A N S L A T I O N OF NON-CRYSTALLIN MESSENGER RNA FROM CALF LENS

A.J.M. VERMORKEN, J.M.C.H. HILDERINK, W.J.M. VAN DE VEN and H. BLOEMENDAL

Department of Biochemistry, University of Nijmegen, Geert Grooteplein Noord 21, Nijmegen (The Netherlands) (Received May 5th, 1976)

Summary Affinity chromatography of lens polyribosomal RNA on oligo(dT)-cellulose yields three fractions. As a rule the second fraction has been neglected in other studies reported in the literature. According to our investigations this fraction in particular contains the messengers for the non-crystallin lens proteins.

Introduction Previous studies from our laboratory [1,2] demonstrated that messenger R N A (mRNA), coding for crystallins, the water-soluble lens proteins which consist of subunits whose molecular weights range from approximately 18 000 to 32 000, can be isolated from sodium dodecyl sulphate-treated polyribosomes by sucrose gradient centrifugation. The majority of the lens membrane specific proteins appeared to be located in a region of higher molecular weight than the crystallins [3,4]. In recent studies, Kibbelaar and Bloemendal [5] characterized the water-insoluble proteins from the lens cortex. Also in this work a number of polypeptides of molecular weights higher than that of the crystallins were detected. One may ask whether or not these proteins are synthesized in the lens cortex, or whether they originate from the lens epithelium and remain in the cell during the process of differentiation into the lens fibers. In a recent paper [6] we described that also non-crystaUin proteins are synthesized in the lens cortex. Both in the lens cell-free system and in a heterologous system after addition of lens polyribosomes, non-crystallin protein could be detected. We have stressed previously that the isolation and characterization of the non-crystallin m R N A s is relevant for the study and understanding of membrane synthesis and assembly [6]. Moreover, the translation of these mRNAs in heter-

448 ologous systems provides a valuable tool for the study of possible immunological interrelationships between the crystallins and the water-insoluble proteins. In this paper we describe the isolation and the translation of a poly(A)-containing RNA fraction that codes for the total set of lens proteins. Materials and Methods L-[3SS]Methionine (spec. act. 200 Ci/mmol) was purchased from the Radiochemical Centre (Amersham, U.K.). For the synthesis of oligo(dT)-cellulose Whatman cellulose p o w d e r CC41 obtained from Reeve Angel; thymidine 5'm o n o p h o s p h a t e from Sigma; and N1Nl-dicyclohexylcarbodiimide from Aldrich and pronase free of nucleases from Calbiochem were used. Preparation of oligo(dT)-cellulose. Oligo(dT)-cellulose was prepared according to Gilham [7], using the N1N~-dicyclohexylcarbodiimide reaction for the polymerization of thymidine 5'-monophosphate on cellulose. One gram (dry weight) oligo(dT)-cellulose prepared in this way retained and was saturated by a b o u t 175 pg of calf lens m R N A under the conditions mentioned below. Preparation of calf lens mRNA. Calf lens polyribosomes were isolated by procedures described previously [8,9]. A b o u t 5 mg of polyribosomes per 1000 lenses were obtained. Sucrose gradient centrifugation revealed that the major part of the polyribosome preparations from cortex cells had an absorbance profile corresponding with an o p t i m u m of eight ribosomes per polysome. The polyribosomes were treated with pronase which was added to a final concentration of 1 mg/ml: RNA was extracted with phenol/chloroform as described by Perry et al. [10]. Calf lens m R N A was prepared from the polyribosomes or from polyribosomal RNA by oligo(dT)-cellulose chromatography using a modification of the Aviv and Leder technique [11]. Three mg of calf lens polyribosomes or, alternatively, 5 mg of polyribosomal RNA dissolved in application buffer containing 0.5 M NaC1/0.01 M Tris • HC1, pH 7.5/0.5% sodium dodecyl sulphate/1.0 mM EDTA (buffer A) were applied to a 2-ml oligo(dT)-cellulose column previously washed with the same buffer. The non-absorbed material was eluted by continued washing with the buffer and the material retained by the column was eluted in two steps with buffers of reduced ionic strength. The first elution buffer (buffer B) contained 0.1 M NaC1/0.01 M Tris • HC1, pH 7.5; the second one (buffer C) 0.01 M Tris • HC1, pH 7.5. The material eluted in this way was precipitated by the addition of 0.1 vol. of 2 M sodium acetate (pH 5.0) and 2 vol. of cold ethanol. Synthesis of lens protein in vitro. Rabbit reticulocytes were prepared as described by Evans and Lingrel [12] and lysed by addition of water. A 30 000 × g supernatant fraction of these lysed cells was used as cell-free system and incubations were performed at 30°C for 1 h. The reaction mixture contained per ml: 0.5 ml of reticulocyte cell-free extract, 1 pmol ATP, 0.2 pmol GTP, 5.0 pmol 2-mercaptoethanol, 10 pmol creatine phosphate, 50/ag creatine phosphokinase, 50 pmol Tris • HC1, pH 7.4, 100 pmol KC1, 3 pmol magnesium acetate and 0.1 pmol of 19 amino acids. 40 pCi [3SS]methionine was added as only labeled amino acid. Unless indicaLed otherwise m R N A was added in a concentration of 40 pg per ml. Analyses were performed by sodium dodecyl sulphate-polyacrylamide gel

449 electrophoresis according to Laemmli [ 13] with the modification that a slab gel instead of gel rods was used. The gel was 12 cm long and contained 13% acrylamide, 0.4% methylene-bisacrylamide and 0.1% sodium dodecyl sulphate. In this method a stacking gel was applied. Staining and destaining was performed as described by Weber and Osborn [14]. Gels were processed for autoradiography. For the detection of the labeled proteins the procedure of Bonnet and Laskey [15] was used in combination with the drying procedure described by Berns and Bloemendal [9]. Gradient centrifugation. RNA was solubilized in buffer (50 mM Tris • HC1 pH 7.8, 1 mM EDTA) and 1% sodium dodecyl sulphate, layered on 15--35.5% (w/v) isokinetic sucrose gradients in the same buffer, and centrifuged at 4°C in a SB 283 rotor at 41 000 rev./min for 11 h. After centrifugation, absorbance was monitored at 260 nm using a Gilford spectrophotometer, equipped with a 2 mm flow cell of IEC. Results and Discussion Krystosek et al. [16] recently evaluated a method for the isolation of mRNA. In this procedure polyribosomes are applied directly onto an oligo(dT)cellulose column without deproteinization by phenol extraction. Application of this technique in our investigations revealed that the messenger population isolated in this way codes almost exclusively for the synthesis of proteins in the 18 000--32 000 mol. wt. range [6]. Our experiments further indicated that different potassium and magnesium concentrations are required for Optimal translation in the reticulocyte lysate of the various messengers on lens polyribosomes [6]. These results led us to investigate in a similar way the translation of isolated m R N A preparations under different ionic conditions. It appeared, however, that under the various conditions studied no high molecular weight products were formed. Since we noticed that the translatability of the lens

A26o/ml

A260/ml

A A

30

i

30

20

20

10

10

/

B

b

Jt

/

c

\

r,

,

2

4

6

8

10

12

14

16 18 ml b u f f e r

8

16

24

32

40

48

56 64 fraction

Fig. 1. E l u t i o n p r o f i l e s o f R N A fractionated o n o l i g o ( d T ) - c e l l u l o s e c o l u m n s . (a) Present w o r k ; ( b ) O b t a i n e d b y Aviv a n d L e d e r ( w i t h p e r m i s s i o n ) .

450 m R N A coding for the non-crystallin lens proteins disappeared after affinity chromatography on oligo(dT)-cellulose the latter purification step, was studied in more detail. By using the modified procedure of Aviv and Leder [11] described in the methods section, we obtained three fractions. Aviv and Leder restricted the assay for translation capacity to the material present in the peak fractions A and C {compare Fig. l b with Fig. la). In material from peak A no m R N A was detectable, while it was in material from peak C. If the high molecular weight m R N A is n o t very tightly b o u n d to the column,

F i g . 2. T h e b e n t a r r o w o n t h i s f i g u r e a n d F i g s . 3, 4 a n d 6 is i n d i c a t i n g t h e t o p o f t h e gel, t h e l o w e r o n e is indicating the globin front. Autoradiography of the translation products of lens mRNA isolated by affinity chromatography on oligo(dT)-cellulo~e (without deproteinization), mRNA from peak B was added to 2 5 $zl o f i n c u b a t i o n m i x t u r e ; a = 0 . 5 p g ; a' = 1 . 5 pg. m R N A f r o m p e a k C w a s a d d e d t o 2 5 p l o f i n c u b a t i o n m i x t u r e ; b = 0 . 5 p g ; b ' = 1 . 5 # g . c : A u t o r a d i o g r a p h o f p r o d u c t s s y n t h e s i z e d i n t h e l e n s c e l l - f r e e system.

451

this material might be lost in the second, neglected peak. Fig. 2 shows that the translation of high molecular weight m R N A is indeed better with material isolated from the second peak (a,a') than from the third one (b,b'), (see straight arrows). However, the translation is not so effective as in the cell-free system (c), (compare the intensity of the high molecular weight bands with that of the crystallins in both systems). Since the m R N A preparations were isolated in a very gentle way, without phenol extraction, destruction of the messengers was not very likely. On the other hand by applying the method of Krystosek et al. [16], it could n o t be excluded that in the preparations protein was still present which might have inhibited to some extent the translation. In order to eliminate the effect of possibly contaminating proteins and to be able to compare our method with that of Aviv and Leder, in all further experiments, polysomal RNA, isolated by pronase treatment and phenol-chloroform extraction was applied to the column. Fig. l a presents our elution profile in comparison with that of Aviv and Leder ( l b ) . The profile depicted in l a shows that elution of peak A did n o t lead to zero extinction and that a clear-cut fraction B could be obtained. The

~

b c

de

f

g

Fig. 3. (A + B) A u t o r a d i o g r a p h o f the translation p r o d u c t s f o r m e d after a d d i t i o n o f R N A e l u t e d w i t h b u f f e r B a n d b u f f e r C, r e s p e c t i v e l y . ( a f t e r d e p r o t e i n i z a t i o n ) (a) b l a n k i n c u b a t i o n . T h e a d d e d R N A c o n c e n t r a t i o n s are: Co) 0 . 1 2 5 ~g, (e) 0 . 2 5 ~g, (d) 0 . 5 / ~ g , (e) 1 /~g, (f) 2 ~g, (g) 4 ~g, p e r 2 5 ~1 i n c u b a t i o n m i x t u r e . ( T h e n u m b e r s o n t h e b a n d s i n d i c a t e t h e m o l e c u l a r w e i g h t s o f t h e c o r r e s p o n d i n g p o l y p e p t i d e s in 1000 daltons.)

452 material of peak A isolated in this way, was totally devoid of m R N A as could be demonstrated by translation experiments. Therefore we concluded that the column was not overloaded. Deproteinization of the messengers improves the translation of high molecular weight mRNA, both from fraction B and C, as can be seen in Fig. 3A and 3B. In Fig. 3A which represents proteins synthesized under direction of material eluted with buffer B, the effect is most pronounced. Here the a m o u n t of high molecular weight proteins is much greater in comparison to the a m o u n t of crystallins formed, than with the buffer C preparation. Even in the very high molecular weight range {above 68 000) specific lens protein bands can be observed, which are not present in the translation products obtained after addition of RNA from fraction C. Gozes et al. [17] studying the translation in vitro of m R N A coding for tubulin and actin also reported that messengers coding for proteins of higher molecular weight were found in the " B " fraction after stepwise elution of their messenger preparation from an oligo(dT)-cellulose column. In Fig. 3A and 3B also the influence of the addition of increasing amounts of both preparations is visualized. Whereas, with the u IJ

68

Fig. 4. A u t o r a d i o g r a p h of the translation products of fractions from a sucrose gradient onto which total polysomal RNA was applied. The sedimentation coefficients of some of the fractions are indicated.

453

preparation C increasing amounts eventually inhibit translation, this does not occur with material from fraction B, at least not in the concentration range tested. Fig. 4 shows the translation products of fractions from a sucrose gradient onto which total polysomal RNA was applied. A sedimentation coefficient of 18--20 S can be derived from these results for the high molecular weight lens mRNA. Separation of this material from ribosomal 18 S RNA can therefore not be achieved by gradient centrifugation. One may ask whether the proteins synthesized after addition of RNA from the 18--20 S range are specific lens proteins or proteins of the reticulocyte lysate that have been more actively synthesized after the addition of 18 S rRNA. Such an effect of 18 S rRNA has recently been described by Kabat [18]. In our experiments this is unlikely because the formation of the 68 000 dalton product of the reticulocyte lysate was not stimulated by any of the fractions. To obtain a better insight in the difference between the buffer B and the buffer C preparation, both fractions were analyzed on sucrose gradients. In Fig. 5b and 5c the extinction profiles are shown. It can be seen that in both cases an 18 S and a 28 S RNA component are still present. The amount of free messenger RNA which sediments in a peak of 10--14 S, is much higher in the buffer C fraction than in the buffer B fraction. In the figures d, e and f it is visualized absorbonce at 260 nm

28S

100

a

C

j

0.75 28S 0.50 025-

O-

f 28S

075285

O~

0

top

bottom

top

bottom

top

bottom

Fig. 5. Sedimentation profiles of gradients of different R N A preparations. (a) polysomal R N A eluted with buffer A, (b) polysomal R N A eluted with buffer B; (c) polysomal R N A eluted with buffer C; (d) R N A from fraction B eluted with buffer A; (e) R N A from fraction B eluted with buffer B~ (f) R N A from fraction B eluted with buffer C.

454

Fig. 6 A . See o p p o s i t e page for l e g e n d .

what happens when the buf f e r B preparation is re-chromatographed on the oligo(dT)-cellulose column. The material is split up in a fraction which is eluted with buffer A and anot her one which is eluted with buffer C. This finding suggests that the original buf f er B fraction consists of a poly(A)-containing compon e n t presumably b o u n d to ribosomal RNA. This is consistent with the fact that we were n o t able to obtain zero extinction after peak A emerged and the column was washed with the application buf fer (compare Fig. l a). A certain a m o u n t of the ribosomal RNA seems to be b o u n d very tightly to the messengers but in such a way that the poly A piece is not involved in this binding. This part emerges with fraction C. A n o t h e r part of the ribosomal RNA can be rem o v e d by re-chromatography. This latter RNA seems to weaken the poly(A) oligo(dT)-cellulose interaction. It is unlikely that the size of the poly(A) tracks is responsible for the differential elution o f messengers f r o m the oligo(dT)-cellulose column, since u p o n r e c h r o m a t o g r a p h y no messenger is eluted in the B fraction and buffer C had to be applied to release the m RN A from the column (see Fig. 5). The m R N A fractions also contain a peak sedimenting at 21 S. One may ask whether or n o t this peak represents m R N A bound to 18 S ribosomal RNA. Such a c o m p l e x f o r m a t i o n has been proposed by Kabat [18] for the globin

455

Fig. 6. A u t o r a d i o g r a p h s o f t h e t r a n s l a t i o n p r o d u c t s o f f r a c t i o n s f r o m a s u c r o s e g r a d i e n t o n t o w h i c h t o t a l p o l y s o m a l R N A w a s a p p l i e d . ( A ) F r a c t i o n s f r o m t h e 8 S - - 2 0 S r e g i o n ; (B) F r a c t i o n s f r o m t h e 2 0 S - - 2 S S r e g i o n . It c a n be s e e n t h a t in b o t h r e g i o n s t h e s a m e m R N A s are p r e s e n t .

messenger. The fact that the peak is retained to some extent by the oligo{dT)cellulose column suggests that this might be the case. However, a 21 S peak is also present in the buffer A fraction where no m R N A activity is detectable. Translation of the individual fractions from a sucrose gradient onto which polysomal RNA had been applied shows that complex formation may have indeed occurred, since the products coded for by the 10--14 S fractions are also formed by fractions under the 21 S peak {compare Fig. 6A with 6B). Since some mRNA activity is observed all over the gradient, aggregation and binding of messenger to 28 S seems also to occur. In conclusion our results are in favor of the assumption that fraction B consists of a complex of ribosomal RNA and mRNA while fraction C contains mainly free mRNA. It seems that especially the high molecular weight mRNAs are the candidates for complex formation. Acknowledgements This work was supported in part by the Netherlands Foundation for Chemical Research (S.O.N.) and by financial aid from the Netherlands organization

456

for Pure Research (Z.W.O.). The authors also wish to thank Professor E.L. Benedetti (Institut de Biologie Moldculaire, Paris) for valuable and stimulating discussions. References 1 Berns, A.J.M., Strous, G.J.A.M. a n d B l o e m e n d a l , H. ( 1 9 7 2 ) Nat. New. Biol. 2 3 6 , 7--9 2 B l o e m e n d a l , H., Berns, A., Strous, G., Mathews, M. a n d Lane, C.D. ( 1 9 7 2 ) R N A V i r u s e s / R i b o s o m e s , P r o c e d i n g s of the E i g h t h Meeting o f the F e d e r a t i o n o f E u r o p e a n B i o c h e m i c a l Societies, A m s t e r d a m , Vol. 27, pp. 2 3 7 - - 2 5 5 , Elsevier, A m s t e r d a m 3 B l o e m e n d a l , H., Zweers, A., V e r m o r k e n , F., Dunia. I. a n d B e n e d e t t i , E.L. ( 1 9 7 2 ) Cell Diff. 1, 9 1 - - 1 0 6 4 Dunia, I., Sen G h o s h , C., B e n e d e t t i , E.L., Zweers, A. a n d B l o e m e n d a l , H. ( 1 9 7 4 ) FEBS Lett. 45, 139--144 5 K i b b e l a a r , M. a n d B l o e m e n d a l , H. ( 1 9 7 5 ) E x p . Eye. Res. 21, 2 5 - - 3 6 6 V e r m o r k e n , A.J.M., Hilderink, J.M.H.C., van de Ven, W.J.M. a n d B l o e m e n d a l , H. ( 1 9 7 5 ) Biochim. Biophys. Acta, 4 1 4 , 1 6 7 - - 1 7 2 7 Gilham, P. ( 1 9 6 4 ) J. Am. Chem. Soc. 86, 4 9 8 2 - - 4 9 8 5 8 B l o e m e n d a l , H., S c h o e n m a k e r s , J . J . G . , Zweers, A., Matze, R. a n d B e n e d e t t i , E.L. ( 1 9 6 6 ) Biochim. B i o p h y s . A c t a 123, 2 1 7 - - 2 2 0 9 Berns, A.J.M. a n d B l o e m e n d a l , H. ( 1 9 7 4 ) M e t h o d s in E n z y m o l o g y , Vol. XXX, pp. 6 7 5 - - 6 9 4 , A c a d e m ic Press, N e w Y o r k 10 Perry, R.P., La T o r t e , J., Kelly, D.E. a n d G r e e n b e r g , J . R . ( 1 9 7 2 ) Biochim. Biophys. A c t a 2 6 2 , 2 2 0 - 226 11 Aviv, A. a n d L e d e r , P. ( 1 9 7 2 ) Proc. Natl. Acad. Sci. U.S. 69, 1 4 0 8 - - 1 4 1 2 12 Evans, M.J. a n d Lingrel, J.B. ( 1 9 6 9 ) B i o c h e m i s t r y 8, 8 2 9 - - 8 3 1 13 L a e m m l i , U.K. ( 1 9 7 0 ) N a t u r e 227, 6 8 0 - - 6 8 5 14 Weber, K. a n d O s b o r n , M. ( 1 9 6 9 ) J. Biol. Chem. 2 4 4 , 4 4 0 6 - - 4 4 1 2 15 B o n n e t , W.M. a n d L a s k e y , R . A . ( 1 9 7 4 ) Eur. J. B i o c h e m . 4 6 , 8 3 - - 8 8 16 K r y s t o s e k , A., L a w r e n c e C a w t h o n , M. a n d K a b a t , D. ( 1 9 7 5 ) J. Biol. C h e m . 2 5 0 , 6 0 7 7 - - 6 0 8 4 17 Gozes, I., S c h m i t t , H. a n d L i t t a u e r , U.L. ( 1 9 7 5 ) Proc. Natl. A c a d . Sci. U.S. 72, 7 0 1 - - 7 0 5 18 K a b a t , D. ( 1 9 7 5 ) J. Biol. Chem. 250, 6 0 8 5 - - 6 0 9 2