39

Biochimica et Biophysica Acta, 418 ( 1 9 7 6 ) 39--51 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in The N e t h e r l a n d s

BBA 9 8 4 7 3

CALF LENS MESSENGER RIBONUCLEOPROTEIN COMPLEXES CHARACTERIZATION AND COMPARISON OF TEMPLATE ACTIVITY WITH CORRESPONDING mRNAs*

J.H. CHEN, G.C. L A V E R S * * and A. S P E C T O R

Department of Ophthalmology, College of Physicians and Surgeons, Columbia University, New York, N.Y. 10032 (U.S.A.) (Received J u n e 4th, 1975)

Summary Lens messenger ribonucleoprotein complexes have been isolated from calf lens polysomes by sucrose gradient centrifugation after puromycin-induced dissociation. A 10 S mRNA was released from a 13 S messenger ribonucleoprotein complex and a 14 S mRNA from a 19 S messenger ribonucleoprotein complex. Two major protein components with molecular weights of approx. 64 000 and 40 000 were isolated from each of the messenger ribonucleoprotein complexes after RNAase digestion. Buoyant density determinations suggest that the messenger ribonucleoprotein complexes contain approximately one mol of each major protein species per mol mRNA. In contrast to lens mRNA, lens messenger ribonucleoproteins are poor templates for transcription with avian myeloblastosis virus reverse transcriptase. Similar results were also obtained with globin messenger ribonucleoprotein containing either two major protein species (or deficient in the lower molecular weight protein species). Polynucleotide phosphorylase eliminates the reverse transcription template activity of the lens mRNA. This effect is blocked in the messenger ribonucleoprotein. Such observations suggest that at least one of the protein components associated with lens messenger ribonucleoprotein may be located in the 3'-terminal region. Only a small variation in translation activity was observed between the messenger ribonucleoproteins and their respective mRNAs.

* A preliminary report o f this work was presented at the First International Congress of E y e Research, June 2--7, 1974, in Capri, Italy. ** Present address: D e p a r t m e n t of B i o c h e m i s t r y , College of Dentistry, N e w York University, N e w York, N.Y., U.S.A.

40 Introduction In 1971 it was shown that mammalian messenger ribonucleoproteins released by EDTA [1,2] contain two major protein species. In 1972, Blobel [3], utilizing a puromycin high salt concentration procedure, has isolated a globin messenger ribunucleoprotein particle which contained 78 000- and 52 000dalton proteins. Globin messenger ribonucleoprotein particles partially deficient in the 78 000- or completely deficient in the 52 000-dalton protein species have also been prepared (personal communication, Gilnter Blobel, Rockefeller University). These proteins are probably associated to untranslated regions of the m R N A with the 78 000-dalton species being located in the vicinity of poly(A) segments. Such proteins are usually released from the messenger ribonucleoprotein complexes by ribonuclease digestion or sodium dodecyl sulphate treatment of the messenger ribonucleoproteins [4]. The relationship of these proteins to their corresponding m R N A s in terms of binding sites and molecular binding ratios, remains obscure [5,6]. Furthermore, the physiological role of such protein c o m p o n e n t s has not been clearly defined [7]. Recent studies have suggested that they may be involved in m R N A transport [8,9], post-transcriptional modification (processing) and pre-translational or translational control via some u n k n o w n mechanisms such as protein phosphorylation [10]. Other experiments indicate that such proteins may have a direct effect u p o n m R N A translation [11] and initiation complex formation [12,13]. It has also been proposed that the specific interaction of these proteins with m R N A may be associated with the secondary structures of m R N A [5], protection of m R N A s against nuclease attack and thus m R N A stability [14]. The lens is an ideal system for the isolation of specific messenger ribonucleoprotein complexes. It is a highly specialized avascular tissue, containing a single layer of epithelial cells which transform into a population of anucleated fibers. The lens synthesizes primarily a small group of structural proteins. Approximately one half of the total protein synthesized in calf lens is a single species, alpha-crystallin [15]. The lens thus offers a unique system for the study of translation and transcription mechanisms. Previous investigations have demonstrated that alpha-crystallin m R N A s can be readily isolated b y zonal centrifugation or affinity chromatography [16--18]. A 10 S m R N A has been shown to code for the alpha-crystallin B chain and a 14 S m R N A for the A chain [19]. Both translation and reverse transcription experiments have been conducted successfully with such m R N A material [ 1 7 , 1 8 , 2 0 ] . Poly(A) segments of approx. 50 nucleotide residues in length have been detected at the 3'-termini of the lens mRNAs. Such sequences are believed to be necessary for reverse transcription [21]. In this paper, we report the isolation and characterization of lens messenger ribonucleoprotein complexes and the m R N A s associated with them. Two major protein species were b o u n d to each messenger ribonucleoprotein. The ability of the messenger ribonucleoprotein complexes to act as templates in translation and transcription processes was also examined.

41 Materials and Methods Oligo(dT)-cellulose, oligo(dT)l 2 - , s and all other polynucleotides were obtained from Collaborative Research, Inc., Mass. d[ 3H] TTP (spec. act. 1800 cpm • pmol -~) was purchased from New England Nuclear while dATP, dCTP, dGTP, and 3'-deoxyadenosine were obtained from Sigma Chemical Company. Polynucleotide phosphorylase (EC 2.7.7.8} was purchased from Worthington Biochemicals, Freehold, N.J. The avian myeloblastosis virus RNA-directed DNA polymerase was either a generous gift of Dr. D. Kacian of the Institute of Cancer Research, Columbia University or obtained from Boehringer Mannheim Biochemicals. All globin messenger ribonucleoprotein particles from rabbit reticulocytes were graciously provided by Drs. G. Blobel and C. Freienstein of Rockefeller University, New York. Polysome preparations and puromycin-high salt procedure for the isolation o f messenger ribonucleoproteins. Calf lenses excised from the eyes of approximately 3-month old animals were homogenized in a buffer system containing 50 mM Tris (pH 7.5) 25 mM KC1, 8 mM MgCl:, 8 mM mercaptoethanol (buffer A) and 340 mM sucrose. The homogenate was then centrifuged at 18 000 X g for 20 min at 4°C. Triton X-100 was added to the supernatant to a final concentration of 0.5% and incubated for 20 min at 4°C. The polysome fractions in the supernatant were isolated by sedimentation through a discontinuous sucrose density gradient in medium A containing 3 ml of 1.3 M sucrose over 2 ml of 2 M sucrose at 150 000 X g for 150 min. The detailed preparation of the polysomal fraction and the extraction of polysomal m R N A were described previously [18]. Lens messenger ribonucleoprotein particles were prepared by puromycin/ high salt treatment [3]. The purified polysomal pellet was first washed in high-salt buffer containing 50 mM triethanolamine . HC1, (pH 7.5), 5 mM MgC12, 500 mM KC1, and 2 mM dithiothreitol (buffer B), and layered on a discontinuous gradient of 2 ml 10% sucrose over 3 ml of 30% sucrose both in buffer B. Centrifugation was carried o u t at 50 000 rev./min in a Beckman 50 rotor for 1.5 h at 4°C. This purification step was repeated two or three times. The resultant salt-washed polysome pellet was adjusted to a final concentration of 1.2 mM neutralized puromycin in buffer B and the preparation was incubated at 37°C for 15 min. Subsequent analysis of the dissociated polysomal fraction was carried o u t on sucrose gradients. Characterization of messenger ribonucleoproteins, m R N A s and proteins by sucrose gradients and electrophoreses. The size and homogeneity o f messenger ribonucleoproteins and m R N A s was determined in a linear 15%--30% sucrose gradient in 0.05 M Tris • HC1 pH 7.5. Standard reference markers of yeast t R N A and 16 S and 23 S Escherichia coli m R N A s were run in a parallel gradient. RNA samples were electrophoresed in 3% polyacrylamide gels; ultraviolet-absorbing material of the gel was extracted by soaking 3 times in fresh running buffer, and prerun at 3 mA/gel for 20 min. RNA samples were applied in 50% sucrose containing 0.1% bromophenol blue on t o p o f the gel. Then electrophoreses together with reference RNA markers in parallel gels were performed for 180 min at 7 mA/gel at 5°C. Protein gel electrophoreses were run

42 on 5% polyacrylamide gel in 0.01 M phosphate buffer, pH 7.4, containing 0.1% sodium docedyl sulphate. After completion of electrophoresis, gels were cut into 0.75-mm slices and incubated overnight at 60°C in either I ml of NCS solubilizer (Amersham-Searle) or in 1 ml H ~O 2. The digested gel samples were then counted in a Beckman liquid scintillation counter after addition of 10 ml Aquasol (New England Nuclear). In some cases the RNA sample gels were also scanned at 260 nm in a Gilford spectrophotometer equipped with an automatic scanning device. Preparation of ' 2 Si_labelled m R N A and messenger ribonucleoprotein. Iodination of mRNA was carried out in a closed 15 ml siliconized vial containing 3 mCi of carrier-free t 2 s I in a total volume of 340 pl. After addition of T1C1 (150 pl of 5 mM in 0.1 M sodium acetate, pH 4.5) to the RNA sample (1 pg/pl) the reaction was carried out for 20 min at 20°C. One volume (340 pl) of 10 mM Na2SO3 in 100 mM Tris, pH 9.5, was added and further incubation was performed at 60°C for 30 min to decompose the unstable intermediates [221. Two volumes (680 pl) of buffer containing 10 mM Tris, pH 7.5, 1 mM EDTA, 100 mM NaC1 and 0.1% sodium dodecyl sulphate was added and the RNA was precipitated with 2 volumes of ethanol redissotved in the same buffer and bound to a CF-11 cellulose (Whatman) column. The RNA was then washed with 10--20 column volumes of the buffer described above to remove the unreacted label and finally eluted with H20. Any remaining free label associated with the RNA was removed with a Sephadex G-75 column (0.8 × 14 cm) equilibrated with 1.5 × SSC (0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0) containing 0.01% sodium azide and run at 24°C. The procedure used for labelling the protein moiety of the messenger ribonucleoprotein was carried out at neutral pH and low temperature according to the modified method of Greenwood and Hunter [23]. After addition of 25 pl of 0.5 M phosphate buffer, pH 7.5, to a rubber-capped vial, containing 15 pl of 12 s I solution (1 mCi), 50 pg mRNP, 100 pg of freshly prepared chloramineT (sodium p-toluenesulfonchloramide) was introduced. After 10 min at 0°C, the reaction was continued for 5 min at 20°C. Addition of 100 pl sodium metabisulphite (0.1 ml of 2.4 mg/ml solution) was followed by 200 pg K1 (10 mg/ml) in the phosphate buffer. The reaction mixture was then transferred to a Sephadex column to separate ~2 s I-labelled messenger ribonucleoprotein from the reaction mixture. The ~2 s I-labelled messenger ribonucleoprotein was pelleted at 150 000 × g and then precipitated with ethanol. ~251 was counted in a Beckman liquid scintillation counter. Bray's solution [24] was used as the scintillation fluid. Preparation of wheat germ cell-free translation system. Fresh commercial untoasted wheat germ (NiBlack Foods Inc., Rochester, N.Y.) was routinely used as the starting material. Preincubation of the extracts and assay of protein synthesis were performed as described by Roberts and Patterson [25]. Reactions were incubated at 24°C for 90 min. RNA-directed DNA polymerase and polynucleotide phosphorylase assays. Avian myeloblastosis virus RNA-directed DNA polymerase assays were carried out as previously described [13]. The polynucleotide phosphorylase reaction mixture contained 10 pg lens mRNA, 30 pg of polynucleotide phosphorylase, 100 mM Tris, pH 7.7, 10 mM MgC12, and 15 mM KC1 in a final volume of 100

43 pl. The reaction mixture was incubated at 5°C for 15 min and the reactions were terminated b y adding 2 volumes of sodium dodecyl sulphate (10%). The poly(A)-free m R N A preparation was extracted with phenol/chloroform (1 : 1, v/v) and precipitated with 2.5 volumes of ethanol {100%). Results

Isolation of lens messenger ribonucleoprotein complexes Lens polysomes were isolated by discontinuous sucrose gradients as described in Material and Methods. It has been shown that a high salt concentration of 0.5 M KC1 can eliminate a considerable amount of the probably nonspecific protein bound to polysomal RNA [3]. The lens polysomes were, therefore, washed two or three times with 0.5 M KC1. The washed polysomes were then incubated at 37°C for 15 min with puromycin and the puromycin-dissociated material was centrifuged on a 15--30% linear sucrose gradient. From Fig. 1 it can be seen that in addition to the ribosomal subunits some particles greater than 23 S were also pelleted down to the b o t t o m of the gradient while free puromycin and small oligonucleotides remained in the top fractions. The two major peaks, designated by A and B, were pooled and subjected to further purification on similar sucrose gradients. Figs 1A and 1B represent typical sedimentation patterns of the rerun A and B peaks. These puromycin-dissociated components have sedimentation values corresponding to 19 S and 13 S. The peak regions delineated by the bars were isolated for further experiments.

I

,6s

h5 o_

x E= 1,0 0 0.5

i

,o

0

i

3~

,~ 20 2's FRAcT.O~ NU.'~R fl

1,0

' 23S

16S

4$ |

-I,,o == i0,5 (19S) 0

I

I0

(~3S) i

20

I

,-7,/

30 0 IO FRAGTION NUMBER

20

| 30

Fig. 1. 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 profiles o f lens 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 c o m p l e x e s . Initial f r a c t i o n a t i o n o f p u r o m y c i n - d i s s o c i a t e d lens p o l y s o m e s in a l i n e a r 1 5 - - 3 0 % s u c r o s e g r a d i e n t is s h o w n in t h e t o p p a n e l . H a t c h e d areas, A a n d B, i n d i c a t e t h e f r a c t i o n s p o o l e d for f u r t h e r p u r i f i c a t i o n o n s u c r o s e g r a d i e n t s . Panels A a n d B r e p r e s e n t t y p i c a l s e d i m e n t a t i o n p a t t e r n of the A and B P e a k s o f t h e t o p p a n e l . A r r o w s i n d i c a t e s t a n d a r d m a r k e r s o f 4 S y e a s t t R N A a n d 16 S a n d 23 S E. c o l i r R N A s . Bars d e l i n e a t e the f r a c t i o n s p o o l e d for s u b s e q u e n t e x p e r i m e n t s .

44

A

19S ,"n-RNP

B

13S m - R N P

1,7

1,5 16 i 1,4 1.3 1.2 ~

x =E 2 o. ¢.)

I0

30 0 I0 FRACTION NUMBER

20

20

30

Fig. 2. CsCI e q u i l i b r i u m d e n s i t y c e n t r i f u g a t i o n o f l e n 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 c o m p l e x e s . L e n 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 s w e r e f i x e d in 4% f o r m a l d e h y d e for 24 h at 5°C. C e n t r i f u g a t i o n w a s c a r r i e d o u t at 2 0 ° C in a S p i n c o 56 r o t o r at 38 0 0 0 r e v . / m i n f o r 70 h. (A), 19 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 : (B), 13 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 .

Buoyant density of lens messenger ribonucleoprotein complexes The densities of the two messenger ribonucleoprotein particles were measured by CsC1 b u o y a n t density equilibrium centrifugation. A major peak with a density of approx. 1.59 gm/ml was obtained for b o t h messenger ribonucleoprotein complexes (Fig. 2). It should also be noted that a small fraction of the messenger ribonucleoprotein was sedimented at lower densities of 1.4--1.5 gm/ml. Based on the relationship reported b y Perry [26] the protein contents for b o t h major messenger ribonucleoproteins were calculated to be a b o u t 35-40%. This figure is consistent with a model that would contain approximately equal molar quantities of m R N A and protein. Characterization o f m R N A components derived from messenger ribonucleoprotein complexes The m R N A c o m p o n e n t s were released from the purified messenger ribonucleoprotein complexes by extraction with phenol/chloroform ( 1 : 1, v/v). Fig. 3 illustrates the fractionation profile of the released m R N A c o m p o n e n t s on sucrose gradients. It is evident that 10 S m R N A (Fig. 3A) was isolated from the 13 S messenger ribonucleoprotein and a 14 S m R N A (Fig. 3B) from the 19 S messenger ribonucleoprotein complex. The m R N A fractions from the gradients were also analyzed b y electrophoresis on polyacrylamide gel. Fig. 3C and 3D represent the 260 nm densitometric tracings o f the gels. Again two major m R N A peaks of 10 S and 14 S were observed. The 10 S m R N A species is relatively homogenous while the 14 S m R N A appeared to be somewhat heterogenous.

45

A

23S

16S

4S

(-)

_ 23S

16S

C

(+)

4S

5 x O

~o

B

o

IO

i 0

i I i I0 20 30 FRACTION NO.

[.i I 0 2 (Origin)

I I I 4 6 8 DISTANCE (cm)

j IO

Fig. 3. S u c r o s e g r a d i e n t a n d p o l y a c r y l a m i d c gel electrophoresis o f m R N A s r e l e a s e d f r o m 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 c o m p l e x e s . D e t a i l s o f m R N A release a n d gel electrophoresis are described in the M a t e r i a l a n d M e t h o d s s e c t i o n ; s u c r o s e g r a d i e n t s (A a n d B) a n d e l e c t r o p h o r e s i $ (C a n d D) o f 1 0 S a n d 1 4 S m R N A s e x t r a c t e d f r o m their i 3 S a n d 1 9 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 c o m p l e x e s , respectively. Bars indicate f r a c t i o n s p o o l e d f o r s u b s e q u e n t assays.

Characterization of protein 'components To study further the messenger ribonucleoprotein particles, they were iodinated with 12 s I under conditions which would preferentially label the protein moiety [23]. Analyses of the iodinated messenger ribonucleoprotein by sucrose gradient centrifugation indicate that the sedimentation rate of the messenger ribonucleoprotein was not effected by iodination. The messenger ribonucleoprotein particles were digested by RNAase A, T~ and T2 and the released protein components were precipitated with 12% cold trichloroacetic acid, washed with 75% alcohol and dissolved in a phosphate buffer containing sodium dodecyl sulphate. Fig. 4 indicates the electrophoretic pattern of the nuclease-released protein components from the messenger ribonucleoprotein complexes. The method of Shapiro et al. [27] was utilized. Both 19 S and 13 S messenger ribonucleoproteins contained two major protein species. Minor components were also noted, particularly in the high molecular weight region. Such high molecular weight fractions were also observed by Gander et al. [28] with messenger ribonucleoprotein released from reticulocyte polysomes by EDTA treatment. Fig. 4C illustrates the electrophoretic profile of a pronase-digested sample, indicating the protein nature of the released macromolecules. The molecular weight of these proteins was determined by sodium dodecyl sulphate gel electrophoresis and comparison with standard reference protein markers. As shown in Fig. 4D, peaks I and III each have a molecular weight of 64 000 while peak II from the 19 S messenger ribonucleoprotein and IV from the 13 S messenger ribonucleoprotein have molecular weights of 42 000 and 40 000 respectively. Some high molecular weight species in the range of 90 000--130 000 were also observed. Their relationship to the messenger ribonucleoprotein complexes remains to be clarified.

Translation and transcription template activity It is apparent that two major protein components can be isolated from

46

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Peak I ~ ~4~'l-- MW = 6 4 0 0 0

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Peak :]E

.m..t-.~%'~- MW = 4 2 O 0 0 -

(-)

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Myoglobin --='~

I I I (+) PRONASE DIGESTED

I 0

4 J W 3 J O

5

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F i g . 4. E l e c t r o p h o r e t i c patterns of p r o t e i n c o m p o n e n t s released by r i b o n u c l e a s e digestions from the lens 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 c o m p l e x e s . E l e c t r o p h o r e s e s were run according to the m e t h o d of Shapiro et al. [ 2 7 ] . T h e released Protein samples were precipitated w i t h 12% trichloroacetic acid and w a s h e d w i t h a l c o h o l , dried and dissolved in b u f f e r c o n t a i n i n g 0.1% s o d i u m d o d e c y l sulphate. Proteins released f r o m R N A a s e - t r e a t e d 1 9 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 ) and 1 3 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 (B) and pronase-digested 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 s (C). Molecular weight d e t e r m i n a t i o n o f p r o t e i n c o m p o n e n t s released f r o m lens 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 c o m p l e x e s (D). T h e ribonuclease-released p r o t e i n c o m p o n e n t s were run o n ,5% p o l y a c r y l a m i d e gels w i t h proteins o f k n o w n m o l e c u l a r weight: r i b o n u c l e a s e A , 1 . 3 5 • 1 0 4 ; m y o g l o b i n , 1 . 8 6 • 1 0 4 ; o v a l a l b u m i n , 4 . 5 5 • 1 0 4 ; b o v i n e serum albumin, 1 . 2 5 • 1 0 S ; "},-globulin, 1 . 6 4 • 1 0 5 .

each of the lens messenger ribonucleoprotein complexes. Since such proteins were not removed by 0.5 M KC1 and appear to have a 1 : 1 stoichiometric relationship with their mRNAs, it was of interest to determine their effect on in vitro translation and transcription systems. Globin messenger ribonucleoprotein was included in the study. Table I indicates the result of [ 3H] leucine incorporation directed by the lens and globin messenger ribonucleoprotein complexes and their released corresponding m R N A s in a wheat germ cell-free translation system. It can be seen that with either the lens 10 S or 14 S m R N A , approximately the same level of incorporation was obtained as that found with their counterpart messenger ribonucleoprotein complexes. Similar results were also observed when globin messenger ribonucleoprotein and its corresponding m R N A were employed as templates. These results indicate that the messenger ribonucleoproteins do not affect the translation system significantly. The estimated efficiency of translation was determined by calculating the incorporation of amino acid into protein per mol of either m R N A or messenger ribonucleoprotein complex. The 10 S and 14 S m R N A s have been shown to code for the B chain and A chain of alpha crystallin, respectively. Thus it is possible to determine the rounds of translation associated with the observed results. Such data clearly shows that not only the total incorporation but the

47 TABLE I I N C O R P O R A T I O N O F [ 3 H ] L E U C I N E D I R E C T E D BY L E N S A N D G L O B I N M E S S E N G E R NUCLEOPROTEINS AND THEIR RELEASED mRNAs

RIBO-

I n c o r p o r a t i o n was c a r r i e d o u t in a 1 0 0 / J l r e a c t i o n m i x t u r e a t 2 4 ° C for 9 0 m i n as p r e v i o u s l y d e s c r i b e d [ 2 5 ] . I n all assays, e q u i v a l e n t m o l a r q u a n t i t i e s o f 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 or t h e i r r e l e a s e d m R N A w e r e e m p l o y e d . E s t i m a t e d t r a n l a t i o n a l efficiencies w e r e b a s e d on t h e f o l l o w i n g m o l e c u l a r p a r a m e t e r s : m R N A m o l e c u l a r w e i g h t s : globin m R N A [ 2 9 ] , 2.2 • 105 ; lens 10 S m R N A [ 3 0 ] , 2.5 ' 105 ; lens 14 S m R N A [ 3 0 ] , 5.1 • 1 0 5 ; specific a c t i v i t y of [ 3 H ] leucine: 7.5 • 107 c p m / ~ m o l ; tool of l e u c i n e in B2 subu n i t o f a i p h a - c r y s t a i l i n [ 3 1 ] , 15, in A 2 , 14 a n d in r a b b i t globin c h a i n s [ 3 2 ] , 1 7 . 5 . Template

A m i n o acid i n c o r p o r a t i o n

Estimated efficiency ( m o l leucine/mol R N A )

cpm

pmot

A. L e n s 13 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 I0 S mRNA

27 4 8 0 32 9 0 0

366 439

45,8 54.9

B. L e n s 19 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 14 S m R N A

18 6 2 0 19 4 5 0

248 259

63.3 66.4

41 4 0 0 39 5 4 0

552 527

60.7 58.0

34 2 0 0

456

50.1

C. G l o b i n Messenger ribonucleoprotein mRNA Messenger r i b o n u c l e o p r o t e i n ( d e f i c i e n t in 78 0 0 0 - d a l t o n p r o t e i n )

initiation and elongation efficiencies are the same for both the m R N A and its messenger ribonucleoprotein complex. Transcriptional activity was determined with the avian myeloblastoma virus RNA-directed D N A polymerase system. Table II illustrates the results of a T A B L E II AVIAN MYELOBLASTOSIS VIRUS RNA-DIRECTED DNA POLYMERASE ACTIVITY VARIOUS MESSENGER RIBONUCLEOPROTEINS AND THEIR CORRESPONDING mRNAs

WITH

A v i a n m y e l o b l a s t o s i s virus R N A - d i r e c t e d D N A p o l y m e r a s e assays w e r e p e r f o r m e d as p r e v i o u s l y d e s c r i b e d [ 1 7 ] . I n all assays, e q u i v a l e n t m o l a r q u a n t i t i e s of m e s s e n g e r f i b o n u c l e o p r o t e i n o f t h e i r r e l e a s e d m R N A were employed. Template

d[3H]TMP incorporation (cpm)

Estimated percent inhibition

A. L e n s 10 S m R N A 13 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

21 3 7 5 6 314

--70

B. L e n s 14 S m R N A 19 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

17 9 2 2 3 840

--78

20 6 4 9 6 374

--69

24 791

+12

i 0 100

--51

C. G l o b i n mRNA Messenger r i b o n u c l e o p r o t e i n Messenger ribonucleoprotein ( d e f i c i e n t in 78 0 0 0 - d a l t o n p r o t e i n ) Messenger ribonucleoprotein* (lacking 52 0 0 0 - d a l t o n p r o t e i n )

* This p r e p a r a t i o n o b t a i n e d f r o m G. Blobel, R o c k e f e l l e r U n i v e r s i t y , c o n t a i n s n o 52 0 0 0 - d a l t o n p r o t e i n a n d r e d u c e d a m o u n t s o f t h e 78 0 0 0 - d a l t o n p r o t e i n .

48 typical experiment with lens and globin m R N A s and their corresponding messenger ribonucleoproteins. Template activity observed with the released m R N A s was inhibited to 70% and 78% by the 13 S and 19 S lens messenger ribonucleoprotein particles respectively. With globin messenger ribonucleoprotein, a 69% inhibition was observed. No inhibitory effect was observed with globin messenger ribonucleoprotein containing predominantly the 52 000dalton protein component. Approximately 50% of the template activity was abolished with a messenger ribonucleoprotein containing no 52 000-dalton protein and reduced amounts of the 78 000-dalton species, thus suggesting the presence of some protein-free mRNA. The inhibition caused by the 78 000dalton protein associated with the globin m R N A is consistent with the finding that this protein is attached to the poly(A) region of the macromolecule. The inhibition of template activity observed with the lens messenger ribonucleoprotein suggests that a protein on or near the 3'-terminal poly(A) region blocks in vitro reverse transcription.

Polynucleotide phosphorylase activity Since polynucleotide phosphorylase attacks polynucleotides at their 3'terminal end, it was of interest to examine the effect of such enzyme digestion on the transcription activity of lens m R N A s and messenger ribonucleoproteins. Table III shows that phosphorylase attack on the 14 S m R N A causes little change in translational activity b u t almost completely abolishes template activity for reverse transcription. However, with the messenger ribonucleoprotein, there is considerable protection from polynucleotide phosphorylase action since 85% of the original template activity is still found with the m R N A iso-

TABLE In E F F E C T OF T R A N S L A T I O N AND T R A N S C R I P T I O N T E M P L A T E A C T I V I T I E S A F T E R M O D I F I C A T I O N OF m R N A s A N D 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 S C o n d i t i o n s o f assay f o r a m i n o acid i n c o r p o r a t i o n a n d R N A - d i r e c t e d D N A p o l y m e r a s e a c t i v i t y w e r e d e s c r i b e d p r e v i o u s l y [ 1 7 , 2 5 ] . T h e details of t h e p o l y n u c l e o t i d e p h o s p h o r y l a s e assays are o u t l i n e d i n t h e Material and Methods section. After p o l y n u e l e o t i d e phosphorylase digestion the preparations were p h e n o l - e x t r a c t e d a n d t h e m R N A i s o l a t e d for assay. C o n t r o l s w e r e t r e a t e d in a s i m i l a r m a n n e r . Template

A. Lens 14 S m R N A

19 S m e s s e n g e r ribonucleoprotein

B. L e n s 10 S m R N A

13 S messenger ribonucleoprotein

Modification

Translation template activity (%)

Transcription template activity (%)

None Polynucleotide phosphorylase digestion

i00

i00

88

11

None Polynucleotide phosphorylase digestion

96

20

73

85

None Polynucleotide phosphorylase digestion

100

100

66

8

84

30

None

49 4ated by phenol extraction after such treatment. In a similar manner, the 10 S lens mRNA loses almost all template activity after polynucleotide Ph0sphorylase digestiom It was also observed again that the proteins associated with this mRNA prevented template activity. In the case of both lens mRNA and messenger ribonucleoprotein preparations a moderate decrease in translation activity was observed after polynucleotide phosphorylase digestion. This effect may be due to a nonspecific degradation of the mRNA during the enzyme incubation. However, the observed losses in translation template activity are small in comparison to the almost total loss of transcription template activity. Discussion

In the present communication, we have shown that two major protein species of approx. 40 000 and 60 000 daltons can be isolated from each of two lens messenger ribonucleoprotein particles. These proteins are similar in molecular weight to the major protein species associated with a number of other eukaryotic mRNAs [1,3,10]. Strong dissociating conditions such as high ionic strength or EDTA treatment have failed to remove such protein components from the mRNA. These findings suggest that such tightly bound proteins may be common to all eukaryotic mRNAs. The buoyant densities of these complexes as determined by CsC1 equilibrium centrifugation are consistent with a model in which there is one mol of each of the two major protein components per mol of mRNA. Similar results have been reported by Infante and Nemer [33] who observed buoyant densities from approx. 1.50 to 1.75 gm/cm 3. Perry and Kelley [ 34] have also demonstrated that after treating L cell messenger ribonucleoprotein particles with 0.55 M LiC1 the remaining messenger ribonucleoprotein complexes have buoyant densities of approx. 1.62 gm/ml. Utilizing high concentrations of KC1 as a messenger ribonucleoprotein wash, Olsnes [35] has obtained similar buoyant densities. However, the densities found in these investigations differ from earlier observations [36,37] on messenger ribonucleoproteins released by EDTA treatment. The discrepancy may be partially due either to the incomplete removal of extraneous proteins or to the binding of nonspecific proteins during the isolation procedure [38]. It has been previously demonstrated that poly(A) segments are present at the 3'-terminal region of lens mRNA as well as other eukaryotic mRNAs [39,40]. Work with globin [41] and mouse sarcoma mRNAs [42] have clearly indicated a protein binding to such poly(A) regions. The present investigation also suggests that at least one protein is located at or near the 3'-poly(A) region of the lens mRNA. The observed stoichiometry as well as the apparent binding specificity of at least one of the mRNA associated proteins suggests they may have a biological role. This view is reinforced by the observation that the protein located at or near the 3'-terminal region inhibits the mRNA template activity for reverse transcription and protects the mRNA molecules against 3'-exonuclease attack. It has previously been reported that the mRNAs for the A and B chains of alpha crystallin have sedimentation values of 14 S and 10 S respectively [19]. From the 13 S and 19 S messenger ribonucleoprotein particles isolated in this study, 10 S and 14 S mRNA species have also been obtained. Since the same

50 lens polysomal material was used for the isolation of messenger ribonucleoproteins or alpha crystallin mRNAs, it is probable that the 10 S and 14 S mRNA species released from the messenger ribonucleoprotein particles are the alpha crystallin messengers. It is of interest to note that the translational template activity of the messenger ribonucleoproteins observed in the wheat germ cell-free system is similar to that of their corresponding mRNA molecules as shown by total incorporation and rounds of translation. This data is also consistent with the report of Freienstein and Blobel [43] who found a similar translation efficiency between globin mRNA and messenger ribonucleoprotein in a mammalian reconstituted translation system. Other investigators [44,45] have also demonstrated that mRNA-associated proteins have essentially no effect upon translation.

Acknowledgements The authors are indebted to Drs. G. Blobel and C. Freienstein of Rockefeller University for their constructive discussions and for kindly providing the globin messenger ribonucleoprotein particles and to Dr. D. Kacian of the Institute of Cancer Research, Columbia University for generously supplying avian myeloblastosis virus RNA-directed DNA polymerase. We are also indebted to Ms. Ta-Wen Chen for her fine technical assistance. This investigation was supported by grants from the National Eye Institute, National Institutes of Health. G.C.L. was a Postdoctoral Fellow of the National Eye Institute, N.I.H.

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