Polyadenylic acid in RNA extracted by thermal phenol fractionation from chick embryo brain and liver

262

Biochimiea et Biophysica Acta, 340 (1974) 262--268

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 97956 POLYADENYLIC ACID IN RNA EXTRACTED BY THERMAL PHENOL FRACTIONATION FROM CHICK EMBRYO BRAIN AND LIVER

KARI HEMMINKI Department o f Medical Chemistry, University o f Helsinhi, Siltavuorenpenger 10 A, SF-O01 70 Helsinki 17 (Finland)

(Received September 17th, 1973) (Revised manuscript received November 16th, 1973)

Summary Cytoplasmic, nucleolar and heterogeneous nuclear RNA were extracted by thermal phenol fractionation from chick embryo brain and liver. The RNA fractions were passed through an oligodeoxythymidylate cellulose column. 60% of the labelled cytoplasmic RNA of brain and 30% of the liver were bound to the column. The gel electrophoretic separation of the fractions indicated that the bulk of the u n b o u n d cytoplasmic RNA was ribosomal and transfer RNA. Thus most of the messenger RNA in chick embryo brain and liver contain a polyadenylic acid segment. The percentage of oligodeoxythymidylate binding in heterogeneous nuclear RNA was around 30% in brain and liver.

Introduction The thermal phenol fractionation of RNA, introduced by Georgiev and Mantieva [1] provides a useful technique permitting the isolation of three major RNA classes, including cytoplasmic, nucleolar and heterogeneous nuclear RNA, from a single sample. The fractionation is especially valuable in studies of RNA synthesis in tissues, where actinomycin D is ineffective in inhibiting the synthesis of ribosomal RNA. The thermal phenol fractionation specifically separates ribosomal precursor RNA and ribosomal RNA from the heterogeneous nuclear RNA [2,3] permitting studies on the synthesis of this rapidly labelled species and on the processing of premessenger RNA [4]. In this study the thermal phenol fractionation of RNA is applied to embryonic tissue, which is used in our laboratory in attempts to find developmental changes in the synthesis of RNA. The RNA fractions are characterized by their binding to oligodeoxythymidylate (oligo(dT)) cellulose. The bound and u n b o u n d RNA is separated on agarose--acrylamide gels and the distribution of the labelled RNA species is determined. The reliability of the thermal

263 phenol fractionation in separating various RNA classes is confirmed and differences between the RNA bound and u n b o u n d to oligo(dT) columns are compared. Materials and Methods Chick embryos, at 9 to 13 days of incubation, were labelled by injecting 30 pCi of [3H]uridine (24 Ci/mmole) into the air space of the eggs. The incubation was continued at 37°C for 10 h. The brains and livers were rapidly removed and rinsed with ice-cold 0.14 M NaC1. The tissue was homogenized with non-radioactive carrier brain and liver tissue in 15 ml of ice-cold 0.14 M NaC1 using 12 strokes of a !posely fitting Teflon--glass homogenizer. 20 ml of freshly distilled phenol (pH 6.2), saturated with water and containing 0.1% 8-hydroxyquinoline were added and the thermal fractionation of RNA was performed as described by Markov and Arion [3]. Some minor modifications were introduced. After extracting the 45°C RNA the interphase was extracted once at 53°C. The interphase obtained was washed from phenol with 0.14 M NaC1 using a Pasteur pipette to collect phenol. The washed interphase was suspended in 0.14 M NaC1 and 10 mM MgC12. Deoxyribonuclease (Worthington, ribonuclease free) was added at 50 pg/ml and the samples were incubated at room temperature for 25 min. Sodium dodecylsulphate, final concentration of 1%, and phenol were added and heterogeneous nuclear RNA was extracted at 75°C for 15 min. Deproteinization was performed by adding sodium dodecylsulphate to the water phase to 1% followed by an equal vol. of phenol. Phenol extraction was carried out once (twice with the 4°C RNA) at 4°C for 15 min. Deproteinization was continued once with phenol--chloroform (1:1, v/v) and once with chloroform. The spectral ratios, A260 to A280 of the deproteinized samples ranged from 1.8 to 2.1. RNA was precipitated with 3 vol. of ethanol and 0.1 M sodium acetate at --20°C overnight. Three RNA fractions were obtained: 4°C RNA containing cytoplasmic, 45°C RNA containing nucleolar, and 75°C RNA containing heterogeneous nuclear RNA species [1--3]. The RNA precipitates were dissolved in the binding buffer containing 400 mM NaC1, 1 mM EDTA, 0.1% sodium dodecylsulphate and 10 mM Tris buffer, pH 7.6. The oligo(dT) cellulose fractionation of RNA was performed as originally described by Edmonds and Caramela [5]. About 0.3 ml of oligo(dT) cellulose (Collaborative Research, Waltham, Mass.} was loaded in 1.5-ml syringes, and the columns were equilibrated with the binding buffer. The samples, containing no more than 3--4 A260 units of RNA were passed three times through the columns. The u n b o u n d fractions were collected and the columns were washed with 5 0.9-ml batches of the binding buffer. The elution of the bound RNA was performed with 5 batches of the elution buffer containing all other constituents of the binding buffer except for NaC1. Aliquots of each fraction were precipitated with 6% trichloroacetic acid and carrier RNA at --20°C for 30 min. RNA was collected on glass fibre filters (Whatman}, dried and counted with 0.3 ml of Soluene 100 and 5 ml of toluol scintillation fluid. The bound and u n b o u n d RNA obtained from the oligo(dT} columns were pooled and precipitated. The pellets were dissolved in 50--100 pl of 20 mM

264 Tris buffer containing 2 mM EDTA, 0.2% sodium dodecylsulphate and sucrose. RNA was separated on agarose--acrylamide composite gels as described by Peacock and Dingman [6]. The concentration of agarose was 0.25% and that of acrylamide 2%. The buffer contained 20 mM Tris, pH 7.8, 2 mM EDTA and 0.2% sodium dodecylsulphate. The gels were first pre-run for 30 min followed by a 60 min run with the samples at 2.5 mA/tube. The gels were frozen and sliced in 2-mm cylinders to be counted in 300 pl of Soluene 100 and 5 ml of toluol scintillation fluid. Results Chick embryos were labelled with [s H] uridine for various time periods and the thermal fractionation of brain and liver RNA was performed essentially as described by Markov and Arion [3]. Three fractions were recovered including the 4°C RNA containing cytoplasmic RNA, the 45°C RNA containing nucleolar RNA and the 75°C RNA containing heterogeneous nuclear RNA [1--3] (Fig. 1). At all time points the specific activity of heterogeneous nuclear RNA was much higher than t h a t of nucleolar or cytoplasmic RNA in brain as well as in liver. The labelling of nuclear RNAs started to level off after 9 h, while that of cytoplasmic RNA still actively increased. The slow appearance of labelled RNA in cytoplasm and the late levelling off of nuclear incorporation are indicative of a delayed absorption of uridine from the air space of the egg. In later experiments 10 h was chosen for the incorporation time to ensure sufficiently high labelling of each RNA fraction. It is, however, unclear, how justified this selection is because of the kinetic complexity of the RNA metabolism in various subcellular fractions and in different tissues. RNA containing polyadenylic acid (poly(A)) sequences is known to bind to oligo(dT) by base pairing [5]. RNA containing poly(A) was isolated from each thermal RNA fraction using oligo(dT) columns (Fig. 2). The u n b o u n d

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RNA was collected largely in the two first fractions eluted with the binding buffer, while the bound RNA was recovered with three batches of the elution buffer containing a low ionic concentration. The radioactivity of the thermal phenol RNA fractions selected by their binding to oligo(dT) cellulose was determined to evaluate the amount of poly(A)-containing R N A (Table I). The highest proportion of poly(A)-containing RNA was detected in the cytoplasmic RNA fraction of brain, 61% of the total radioactivity. This suggests a relatively active synthesis of messenger R N A as compared to ribosomal RNA. The liver cytoplasmic fraction appeared to contain only 32% poly(A)-attached RNA. The amount of poly(A)-containing molecules was fairly similar in the heterogeneous nuclear RNAs of brain and liver, about 30%. The smallest binding to oligo(dT) cellulose was observed in nucleolar RNA, about 20%. The gel electrophoretic analysis of this fraction, shown later, indicated that in this fraction the bound R N A contained no species identified as ribosomal precursor RNA. Thus the bound 45°C RNA may be TABLE I B I N D I N G OF B R A I N A N D L I V E R R N A E X T R A C T E D TO O L I G O D E O X Y T H Y M I D Y L A T E CELLULOSE

BY T H E R M A L P H E N O L F R A C T I O N A T I O N

Means -+ S.E. o f three e x p e r i m e n t s . Fraction

Brain

Liver

Radioactivity (cpm/embryo)

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Unbound

Bound

Bound fraction (% o f t o t a l )

6100± 330 300 ± 75 180 ± 40 10300 ± 2900 660± 150 195 ± 40

9600 92 72 4780 141 100

61 23 29 32 17 34

±2950 ± 28 ± 2 ± 1270 ± 34 ± 27

266 a nucleoplasmic contamination or a high molecular weight aggregate as it does not move far into the gel. The gel electrophoretic separation of the bound and u n b o u n d RNA of the thermal phenol fractions was performed to test the reliability of thermal phenol fractionation as well as to compare the poly(A)-containing RNAs to those lacking poly(A). Fig. 3 shows the patterns of radioactivity produced with the liver RNA fractions. Cytoplasmic RNA separation is shown in Fig. 3A. The fraction lacking poly(A) consisted largely of ribosomal 28-S and 18-S RNAs and transfer RNA. The poly(A)-containing fraction, consisting presumably of messenger RNA, was heterogeneously distributed along the gel. The bulk of newly synthesized messenger RNA appeared to range in size from about 50 to 10 S. No detectable peaks of ribosomal or transfer RNA were present. Judged by the gel patterns oligo(dT) selection appeared effective in separating cytoplasmic RNA in two different fractions. Yet the obtained gels do n o t exclude a minor contamination of the 4°C RNA with heterogeneous nuclear RNA (compare Figs 3A and 3C). Incorporation kinetics (Fig. 1) also confirm that this possible contamination should be small. The 45°C RNA (Fig. 3B) contained one major peak, which could be identified as the 45-S ribosomal precursor [7]. This fraction did not bind to the oligo(dT) column and was clearly absent from other thermal fractions

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267 indicating the specificity of the extraction technique. The distribution of heterogeneous nuclear RNA is shown in Fig. 3C. The radioactivity is evenly distributed along the gel in the bound and u n b o u n d fraction. The largest of these RNA species had molecular weights of over 100 S, which were higher than the molecular weights of messenger RNA (Fig. 3A). The gels containing brain R N A (not shown) were apparently not different from those shown in Fig. 2 containing liver RNA. It appears that thermal fractionation is a valid technique to extract RNA from embryonic chick brain as well as from liver. Discussion

Messenger R N A and heterogeneous nuclear RNA contain a segment of poly(A) of a b o u t 150 to 200 nucleotides long [8]. It has been reported that 50 to 80% of messenger RNA in cultured cells contain a poly(A) sequence [9,10]. By contrast, only 10 to 40% of heterogeneous nuclear RNA appears to contain poly(A) sequences [11]. These are minimal estimates, since the degradation of RNA tends to decrease the figures. Recent evidence indicates that heterogeneous nuclear RNA is a precursor of messenger RNA [4,11,12], and it has been suggested that the poly(A) segment may be involved in the processing of messenger RNA. The half-life of heterogeneous nuclear RNA is very short, whereas that of messenger RNA is reported to be several hours [13,14]. The thermal phenol fractionation of RNA [1--4] was applied in this study to prepare cytoplasmic, nucleolar and heterogeneous nuclear RNA from chick e m b r y o brain and liver. The RNA classes appeared fairly pure by gel electrophoretic analysis. The only RNA fraction, which was obviously contaminated to a small extent, was the 45°C RNA. This contained oligo(dT)-attached material, which did not appear to be ribosomal precursor RNA. The proportion of poly(A)-containing R N A in the thermal fractions was determined by oligo(dT) selection. Cytoplasmic heterogeneous RNA was bound to a large extent to oligo(dT) cellulose indicating that the bulk of embryonic brain and liver messenger RNA contain a poly(A) segment. A b o u t 30% of heterogeneous nuclear R N A contained poly(A) which was comparable to cultured cells [ 10,11]. Differentiation appears to be accompanied with the appearance of new R N A transcripts in simple eukaryotic cells [15] as well as in mammalian cells [16,17]. It is, however, possible that some control of differentiation may be exerted after transcription, for instance at the level of processing premessenger RNA or translating messenger RNA. This may be indicated by developmental changes in the amounts of poly(A)-containing RNA species [18,19]. In the present study, where growing and differentiating chick embryo [20,21] was used, brain tissue appeared to produce a higher relative proportion of messenger RNA than liver when compared to cytoplasmic RNA lacking poly(A) or to heterogeneous nuclear RNA. Thus brain tissue may be more efficient than liver in processing messenger RNA. Acknowledgements The author is thankful to the teachers of the FEBS Course on Eukaryotic

268

RNA in Sofia for methodological advice. The study was supported by the Sigrid Juselius Foundation and the National Research Council for Medical Sciences, Finland. References 1 G e o r g i e v , G.P. a n d M a n t i e v a , V . L . ( 1 9 6 2 ) B i o c h i m . B i o p h y s . A c t a 6 1 , 1 5 3 - - 1 5 5 2 G e o r g i e v , G.P. ( 1 9 6 7 ) P r o g r e s s in N u c l e i c A c i d R e s e a r c h a n d M o l e c u I a r B i o l o g y ( D a v i d s o n , J . N . a n d C o h n , W.E., eds), V o l . 6, p p . 2 5 9 - - 3 5 1 , A c a d e m i c Press, N e w Y o r k a n d L o n d o n 3 M a r k o v , G . G . a n d A r i o n , V.J. ( 1 9 7 3 ) E u r . J. B i o c h e m . 35, 1 8 6 - - 2 0 0 4 G e o r g i e v , G.P., R y s k o v , A . P . , C o u t e l l e , C., M a n t i e v a , V . L . a n d A v a k y a n , R . R . ( 1 9 7 2 ) B i o c h i m . Biophys. Acta 259, 259--283 5 E d m o n d s , M. a n d C a r a m e l a , M.G. ( 1 9 6 9 ) J. Biol. C h e m . 2 4 4 , 1 3 1 4 - - 1 3 2 4 6 P e a c o c k , A.C. a n d D i n g m a n , C.W. ( 1 9 6 8 ) B i o c h e m i s t r y 7, 6 6 8 - - 6 7 4 7 P e n m a n , S., F a n , H., P e r l m a n , S., R o s b a s h , M., W e i n b e r g , R. a n d Z y l b e r , E. ( 1 9 7 0 ) C o l d S p r i n g H a r b o r S y m p . Q u a n t . Biol. 35, 5 6 1 - - 5 7 5 8 K a t e s , J. ( 1 9 7 0 ) C o l d S p r i n g H a r b o r S y m p . Q u a n t . Biol. 35, 7 4 3 - - 7 5 2 9 A d e s n i k , M., S a l d i t t , M., T h o m a s , W. a n d D a r n e l l , J . E . ( 1 9 7 2 ) J. Mol. Biol. 2 1 - - 3 0 1 0 S u l l i v a n , N. a n d R o b e r t s , W.K. ( 1 9 7 3 ) B i o c h e m i s t r y 12, 2 3 9 5 - - 2 4 0 3 11 J e l i n e k , W., A d e s n i k , M., S a l d i t t , M., S h e i n e s s , D., Wall, R., M o l l o y , G., P h i l i p s o n , L. a n d D a r n e l l , J.E. ( 1 9 7 3 ) J. Mol. Biol. 7 5 , 5 1 5 - - 5 3 2 1 2 W i l l i a m s o n , R., D r e w i e n k i e w i c z , C.E. a n d P a u l , J. ( 1 9 7 3 ) N a t . N e w Biol. 2 4 1 , 6 6 - - 6 8 13 B r a n d h o r s t , B . R . a n d H u m p h r e y s , T. ( 1 9 7 2 ) J. Cell Biol. 53, 4 7 4 - - 4 8 2 1 4 S i n g e r , R . H . a n d P e n m a n , S. ( 1 9 7 3 ) J. Mol. Biol. 78, 3 2 1 - - 3 3 4 15 F i r t e l , R . A . , B a x t e r , L. a n d L o d i s h , H . F . ( 1 9 7 3 ) J. Mol. Biol. 79, 3 1 5 - - 3 2 7 16 H a h n , W.E. a n d L a i r d , C.D. ( 1 9 7 1 ) S c i e n c e 1 7 3 , 1 5 8 - - 1 6 1 17 C h u r c h , R . B . a n d B r o w n , I.R. ( 1 9 7 2 ) in N u c l e i c A c i d H y b r i d i z a t i o n in t h e S t u d y of Cell D i f f e r e n t i a t i o n ( U r s p r u n g , H., e d . ) , p p . 1 1 - - 2 4 , S p r i n g e r - V e r l a g , Berlin 18 S c h u l t z , G., Manes, C. a n d H a h n , W.E. ( 1 9 7 3 ) Devel. Biol. 30, 4 1 8 - - 4 2 6 19 B a n k s , S.P. a n d J o h n s o n , T.C. ( 1 9 7 3 ) S c i e n c e 1 8 1 , 1 0 6 4 - - 1 0 6 5 2 0 J u d e s , C., S e n s e n b r e n n e r , M., J a c o b , M. a n d M a n d e l , P. ( 1 9 7 3 ) B r a i n Res. 51, 2 4 1 - - 2 5 1 21 J u d e s , S. a n d J a c o b , M. ( 1 9 7 3 ) B r a i n Res. 51, 2 5 3 - - 2 6 7