The status of small nuclear RNA in the ribonucleoprotein fibrils containing heterogeneous nuclear RNA

109

Biochimica et Biophysica A cta, 6 5 2 ( 1 9 8 1 ) 1 0 9 - - 1 2 0 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 99805

THE STATUS O F SMALL N U C L E A R R N A IN THE R I B O N U C L E O P R O T E I N F I B R I L S CONTAINING H E T E R O G E N E O U S N U C L E A R R N A HELENE GALLINARO and MONIQUE JACOB *

Laboratoire de Gdndtique Moldculaire des Eucaryotes (CNRS) et Unitd 184 de I'INSERM, 67085 Strasbourg (France) (Received June 27th, 1980)

Key words: small nuclear R N A ; h n R N A ; Salt treatment; Ribonucleoprotein particle; Proteinase K; (Rat brain)

Summary hnRNP are made of two classes of unit, monoparticles and heterogeneous complexes. The monoparticles are much more easily dissociated by salt than the heterogeneous complexes. We made use of this differential salt sensitivity to determine the localization of snRNA in hnRNP. 1, A b o u t 50% of the snRNA were released by NaC1 under the conditions of dissociation of monoparticles. U1 R N A which was enriched in monoparticles was preferentially released. 2, When the proteins resistant to salt dissociation were digested with proteinase K, an additional small proportion of snRNA was released, in particular a species designated as 5 Sa RNA. Therefore, 5 Sa R N A seems to be preferentially associated with the proteins of heterogeneous complexes. 3, 40% of the snRNA remained associated with the h n R N A in the absence of any detectable protein. U1 and U2 R N A were the major RNAs in this fraction. The same R N A pattern was obtained for phenol-extracted RNA. The results indicate that all snRNA species are associated with the proteins of monoparticles, with those of heterogeneous complexes and with hnRNA. The existence of these pools of snRNA m a y reflect different functional states.

Introduction

Several observations suggest that small nuclear R N A (snRNA) m a y play a role in the processing of premessenger RNA. First, snRNA are associated with * TO whom correspondence should be addressed. Abbreviations: snRNA, small nuclear RNA(s); hnRNP, ribonucleoprotein(s) containing h e t e r o g e n e o u s nuclear RNA ; nRNP. ribonucleoprotein(s) containing nuclear RNA.

II0

phenol-extracted poly(A) ÷ nuclear and cytoplasmic RNA, probably by hydrogen bonding [1]. Second, snRNA m a y associate with h n R N A under conditions of formation of hydrogen bonds [2]. Third, snRNA are constituents of the hnRNP which are the nuclear ribonucleoproteins assumed to be site of premessenger RNA processing [ 3 - 6 ] . The hypothesis that snRNA may participate in splicing by association with specific sites of the premessenger RNA molecule has been formulated [7,8], but n o t experimentally verified. A variety of snRNA species has been described and this suggests a certain diversity of function. On the other hand, it seems unlikely that these snRNA f o u n d associated with premessenger RNA at a given time remain continuously so. Rather, we may assume t h a t each snRNA species may be taken out from one or several pools according to the requirements of processing. The metabolic stability of snRNA as compared to h n R N A [9,10] suggests the possibility of re-utilization. In fact, for continuous processing, snRNA should be readily available and we wondered if one of the functions of hnRNP was n o t to prepare snRNA for ready use. This hypothesis might imply that a fraction of the snRNA of hnRNP is n o t directly associated with hnRNA, hnRNP are made of two classes of units which may be separated from each other by a mild ribonuclease treatment [11]. Each class of unit ~as a typical protein composition and snRNA might be associated with the proteins of one or the other class of unit. In this report, we show that snRNA may be b o u n d to proteins of the two classes of unit and, in addition, m a y be directly associated with hnRNA. Materials and Methods

Labeling o f RNA. Rats were injected intracisternally with 25 pCi of [3H]uridine for 4 h. Under such conditions, no radioactivity was detected in brain snRNA after electrophoresis and fluorography, whereas h n R N A was heavily labeled. Therefore, the radioactivity may be considered as t h a t of hnRNA. Preparation o f a nuclear extract. A nuclear extract was prepared by ultrasonication of purified brain nuclei as previously described [11]. The nuclear extract contains hnRNP plus nucleosol and is n o t significantly contaminated by chromatin or cytoplasm [12]. Salt treatment. The salt Concentration of a nuclear extract was adjusted to a chosen concentration at 0--4°C and the extract was layered on a sucrose gradient. Conditions were adjusted so that 30 min elapsed between the addition of NaC1 and the start of centrifugation. The conditions of centrifugation are given in the next or in the legends of figures. Proteinase K treatment. The salt concentration of a nuclear extract was adjusted to 0.4 M. Proteinase K was added to the chosen concentration. Incubation was for 30 min at 0--4 ° C, 30 min being calculated as in the case of salt treatment. Preparation o f RNA. Pooled fractions of gradients (untreated hnRNP or salttreated hnRNP) were precipitated overnight with 2vol. ethanol a t - - 2 0 ° C . After centrifugation, the pellets were extracted with phenol at pH 8.3 in the presence of 0.5% sodium dodecyl sulfate as previously described [6]. In certain experiments, the RNA was directly extracted from the nuclear extract by the

111 same method. The proteinase K treated samples were not phenol-extracted. Electrophoresis of RNA. For the simultaneous analysis of hnRNA and snRNA, linear 2.2--15% polyacrylamide slab gels (11 cm long} were used after denaturation of the RNA by heating [6]. For the analysis of snRNA alone, 12% polyacrylamide slab gels (28 cm long) containing 8 M urea were used. The samples were denatured by heating at 65°C in the presence of 8 M urea and 1% sodium dodecyl sulfate. In both methods, staining was with Methylene Blue and destaining with a methanol/water mixture (1 : 1, v/v). The nomenclature was that established by Ro-Choi and Busch [13] in aqueous gels. For calibration of urea gels, the major bands were eluted from aqueous gels and run in urea gels. A splitting of certain bands was observed in particular in the 5 S and 4.5 S regions. Electrophoresis of proteins. Ethanol-precipitated sucrose gradient fractions were dissolved in 10 mM Tris-HC1, pH 7.5/1 mM dithiothreitol/6 M urea. After overnight incubation in the cold, they were treated with 1% sodium dodecyl sulfate at 60°C for 10 min immediately before electrophoresis on 9% polyacrylamide slab gels. Proteins were stained with Coomassie Brillant Blue R. Results

Prerequisites. For the study of the mode of association of snRNA with hnRNP, certain conditions must be fulfilled: 1, hnRNP must be as close to the native state as possible, since losses of material and rearrangements may occur upon partial hydrolysis [14]; 2, hnRNP should not be contaminated by cellular constituents which may introduce RNA from other sources; 3, non-specific adsorption (of snRNA in our particular case) should be avoided. All these conditions were controlled in the past for the standard preparation of rat brain hnRNP used here [6,12,15] and only certain controls were performed in the course of the present experiments. For the first requirement, we showed that hnRNP were heterogeneous in size and sedimented up to 200 S (Fig. 1). They contained a complex pattern of proteins from 23 000--200 000 daltons. This indicated very moderate ribonuclease hydrolysis since even a mild digestion transforms the hnRNP into 30--50 S material enriched in 28 000--38 000 dalton proteins [11,14]. This does not exclude the occurence of some nicks in the hnRNA which are unavoidable during subcellular fractionation. Concerning the second requirement, RNA analysis demonstrated the very low level of cytoplasmic ribosomal RNA (28 S, 18 S, 5.8 S, 5 S) and transfer RNA. The absence of nucleoli was controlled by the absence of U3 RNA specifically located in the nucleolus [6]. No histories were detected b y protein analysis, indicating that chromatin was not a major contaminant. Finally, previous work [6] showed that the non-specific adsorption of snRNA from the nucleosol did not exceed 2% of the snRNA truly present in hnRNP under the experimental conditions of preparation described under Materials and Methods. Rationale. Besides-protein composition, the two classes of unit of hnRNP have different characteristics [11,12]. In particular, the monoparticles (30-50 S) are very sensitive to salt dissociation in contrast to the heterogeneous complexes {50--200 S). We made use of this differential sensitivity to dis-

112

tinguish between the snRNA bound to monoparticle proteins and those bound to the proteins of the heterogeneous complexes (Fig. 1). Small nRNA may be associated with hnRNP in four different ways: I, bound to monoparticles proteins; II, bound to the proteins of heterogeneous complexes; III, bound to the hnRNA in monoparticles; IV, bound to the hnRNA in heterogeneous complexes. A salt t r e a t m e n t under the conditions of monoparticle dissociation, followed by a protease treatment, would release the snRNA of class I in the first step and those of class II in the second. As hnRNA-snRNA hybrids are stable at high salt concentration, the snRNA of classes III and IV would remain associated with the naked hnRNA. The distinction between classes III and IV would be possible, provided that the units are first separated. However, this would require a ribonuclease t r e a t m e n t (see the structure hnRNP in Fig. 1).

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Fig. 1. Schematic representation of the possible status of small nuclear R N A in h n R N P , h n R N P are m a d e of t w o classes of unit, 30--50 S monoparticles (open circles) and 30--200 S heterogeneous complexes (between dashed lines) joined together by h n R N A (full lines). A statistical representation is s h o w n and is compatible with previous data [11,12,14,20]. s n R N A (black dots) m a y be associated with proteins (I and If) or with h n R N A (Ill and IV) as detailed in the text. N a C l provokes the dissociation of m o n o particles with concomitant release of monoparticle proteins and of the associated s n R N A (1). T h e saltresistant R N P (st R N P ) m a y be further deproteinized b y proteinase K and this releases the s n R N A of class II. T h e s n R N A of classes Ill and I V remain associated with the h n R N A . This schematic representation does not imply that all classes of s n R N A are simultaneously present in a individual h n R N P fibril. F i g . 2. S e d i m e n t a t i o n of a nuclear extract before and after NaC1 treatment. The NaC1 concentration of a nuclear extract was adjusted to 1 M. The extract was then centrifuged on a 10--40% linear sucrose gradient made in 10 mM triethanolamine-HCl ( P H 7 . 4 ) / 1 m M M g C 1 2 / 1 0 0 m M KC1. C e n t r i f u g a t i o n w a s f o r 16 h at 24 000 rev./min (70 000 X g) in a SW 25-2 rotor. An untreated nuclear extract was centrifuged under the same conditions. 2-ml fractions were collected and acid-insoluble radioactivity was determined on aliquots. Fractions were pooled as indicated. Ribosomal subunits obtained by EDTA dissociation o f p o l y s o m e s s e r v e d as m a r k e r s o f s e d i m e n t a t i o n (e e ) , a, c o n t r o l : ( v . . . . . . v), b, NaCl-treated nuclear extract.

113 As s n R N A are associated with h n R N A on no more than 25 nucleotides [2], ribonuclease may simultaneously hydrolyse the unpaired tails of snRNA which would not be recognizable any longer. A partial hydrolysis of snRNA under conditions allowing the separation of the units was indeed observed (unpublished observations). Previous work showed that hnRNP contained snRNA resistant to salt dissociation (II, III, IV) [16,17] or snRNA bound to proteins (I, II) and to h n R N A (III, IV) [2,3]. However, distinction between classes I and II was not realised and no quantitative or semi-quantitative data concerning the respective importance of the snRNA pools was published. Release of small nRNA from hnRNP by NaCl. A nuclear extract (labeled for 4 h with [3H]uridine) was prepared and its NaC1 concentration adjusted to 1 M. A fraction of the extract was not treated and served as control. Both fractions were sedimented on a 10--40% sucrose gradient containing 100 mM KC1 (Fig. 2). The sedimentation profile of the control showed the expected large hetero-

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114

geneity (Fig. 2a). hnRNP above 50 S were found in fractions A, B, C and 30--50 S hnRNP (or monoparticles) in D. Fraction E (10--25 S) contained small nRNP, large proteins or protein aggregates, and fraction D (0--10 S) free snRNA and proteins [6]. After NaC1 treatment, the monoparticles were dissociated as shown by the shift of a large amount of the proteins from fraction A--D to fraction F (Fig. 6, I, II, III). Previous studies on the action of NaC1 on hnRNP demonstrated that the most easily dissociated proteins were in the 2 8 0 0 0 - - 3 8 0 0 0 dalton range [12] which were subsequently shown to belong to monoparticles [11]. More than 90% of these proteins were released under the conditions of the experiments against 60--70% of the other proteins. The remaining salt-resistant RNP contained a set of proteins of typical composition (Fig. 6, II). In spite of the removal of a large quantity of proteins, their sedimentation coefficient was not considerably modified (Fig. 2b). This is due to the persistance of large size hnRNA and probably to differences of shape between salt-resistant RNP and hnRNP. Fractions A to F were collected in the experiments shown in Figs. 2a and 2b. RNA was extracted and analyzed in polyacrylamide gradient gels allowing

TABLE I D I S T R I B U T I O N O F s n R N A A N D h n R N A IN D I F F E R E N T F R A C T I O N S O F A N U C L E A R E X T R A C T T h e p r o p o r t i o n s o f h n R N A and s n R N A w e r e d e t e r m i n e d b y p l a n i m e t r y a f t e r r e c o r d i n g o f s t a i n e d gels. T h e a c c u m u l a t i o n o f s n R N A at 0 - - 1 0 S m a k e s their release b y salt or p r o t e i n a s e K t r e a t m e n t particularly o b v i o u s ( u n d e r l i n e d values). T h e f r a e t i o n size is t h a t o f R N P e x c e p t for the t w o last e x p e r i m e n t s o f panel I I I w h e r e it c o r r e s p o n d s t o d e p r o t e i n i z e d R N A .

I

II

Treatment of nuclear extract

NaC1 concentration in g r a d i e n t (M)

Fraction size (S)

hnRNA (%)

snRNA (%)

None

0.1

1 M NaCl

0.1

0.4 M NaCl

0.4

>30 10--25 0--10 )30 10--25 0--10 ~>30 10--25 0--10

77 15 8 79 12 9 60 26 14

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None

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24 25 51 18 11 71 19 16 65

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0 . 4 M NaC1/10 p g / m l p r o t e i n a s e K

0.4

0.4 MNaC1]100 p g / m l p r o t e i n a s e K

0.4

115 the simultaneous study of hnRNA and snRNA (Fig. 3). The hnRNA was shifted from A--C (panel I) to B--D (panel II). A specific distribution of snRNA was previously described in untreated hnRNP [6] and was confirmed here (panel I). In particular, U1 RNA was enriched in the regions where 30--50 S monoparticles and free small nRNP sediment (panel I, D, E). This distribution was markedly changed after NaC1 treatment (panel II). A fraction of the major snRNA (U2, U1, 5 Sa and 4.5 S) was released from hnRNP (A, B, C, D, panels I and II) and recovered in E and especially F. The release of 5 Sa RNA was less marked than that of U2, U1 and 4.5 S. U1 RNA was no m o r e enriched in D. The RNA designated as Ux was present only in the large hnRNP (A to C) and apparently was not released by NaC1. The distributions of hnRNA and snRNA in the different size fractions under various conditions of salt dissociation are shown in Table I. Under the conditions described above (1 M NaC1 treatment, centrifugation on sucrose gradients containing 0.1 M salt), approximately half of the snRNA was released from hnRNP (>30 S, fractions A to D) and the proportion of free snRNA (0--10 S) increased several-fold (Table I, panel I). When 0.4 M NaC1 replaced 1 M NaC1, the dissociation of snRNA was less marked (Table I, panel II). However, when the duration of action of 0.4 M NaC1 was extended to 16 h by centrifugation on gradients containing 0.4 M NaC1, the dissociation of snRNA was similar to that obtained with 1 M NaC1 during a shorter period (Table I, panels I and II). In parallel, protein dissociation was less marked in the presence of

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Fig. 4. S e d i m e n t a t i o n o f a n u c l e a r e x t r a c t a f t e r N a C I t r e a t m e n t i n t h e p r e s e n c e o r a b s e n c e o f p r o t e i n a s e K. T h e NaC1 c o n c e n t r a t i o n o f a n u c l e a r e x t r a c t w a s a d j u s t e d t o 0 . 4 M NaC1. A f r a c t i o n o f t h e e x t r a c t w a s t r e a t e d f o r 3 0 r a i n at 0 - - 4 ° C i n t h e p r e s e n c e o f 1 0 0 # g / m 1 o f p r o t e i n a s e K. A n o t h e r f r a c t i o n w a s incubated in the absence of the enzyme. Both samples were centrifuged on a 10--25% linear sucrose g r a d i e n t m a d e in 10 m M t r i e t h a n o l a m i n e - H C 1 ( p H 7 . 4 ) / 1 m M M g C I 2 as i n d i c a t e d . C y t o p l a s m i c R N A s e r v e d as m a r k e r s o f s e d i m e n t a t i o n . S a m p l e s i n c u b a t e d i n t h e p r e s e n c e (A . . . . . . A) o r i n t h e a b s e n c e (e e ) o f p r o t e i n a s e K.

116

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Fig. 5. P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s of R N A . F r a c t i o n s w e r e p o o l e d as i n d i c a t e d in Fig. 4. N u c l e a r e x t r a c t s w e r e i n c u b a t e d in t h e a b s e n c e of p r o t e i n a s e K ( p a n e l I) or in its p r e s e n c e ( p a n e l II). T h e e l e c t r o p h o r e t i c c o n d i t i o n s w e r e t h e s a m e as in Fig. 3.

0.4 M NaC1 when the time of contact was short and was similar to that observed in the presence of 1 M NaC1 when the time of contact was extended (not shown). In contrast, the distribution of h n R N A did not change significantly in any of these experimental conditions. We conclude t h a t approximately half of the RNAs are associated with hnRNP through proteins sensitive to salt dissociation. The released snRNA are likely to belong to monoparticles (Fig. 1, class I). This seems particularly obvious for U1 RNA which was enriched in the 30--50 S monoparticle region of control (Fig. 3, panel I, D) and was no more so after dissociation and release of the monoparticle proteins. Small nRNA might be released as small nRNP. As most released snRNA were detected at 0--10 S, indicating that they were free or associated with only a small quantity of proteins, this would suggest t h a t the putative small nRNP might also be dissociated by salt. Additional release o f small nRNA by proteinase K. A protease treatment was used in order to eliminate the proteins resistant to salt dissociation. Frac-

117

tions of a nuclear extract were treated with 10 and 100 #g/ml of proteinase K in the presence of 0.4 M NaC1, and another fraction w i t h o u t addition of proteinase K served as control. They were centrifuged on sucrose gradients containing 0.4 M salt. As R N A rather than RNP was to be analyzed, 10--25% instead of 10--40% sucrose gradients were used. A marked shift of sedimentation coefficient was observed (Fig. 4). The R N A obtained after proteinase K treatment was heterogeneous and sedimented from approximately 4 S to 40 S. The large size might partially be due to aggregation (denaturing gradients had to be avoided as hydrogen bonding m a y be the w a y of association of snRNA and hnRNA). All the residual proteins were hydrolyzed (Fig. 6, panel IV). Small polypeptides were detected primarily in fraction E and were less abundant after 100 pg/ml than after 10 pg/ml of proteinase K. The proteinase treatment provoked an additional small release of most of the snRNA as compared to salt treatment (Table I, panel III). But the most salient feature was the preferential release of 5 Sa R N A which accumulated in fraction E (Fig. 5). Therefore, it seems likely that a large fraction of 5 Sa R N A is asso-

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Fig. 6. P o l y a c r y l a m i d e gel e l e c t r o p h o r e s ~ s o f p r o t e i n s . A l i q u o t s f r o m f r a c t i o n s A t o E as i n d i c a t e d in Figs. 2 a n d 4 w e r e a n a l y z e d . Panel I: u n t r e a t e d r . u c l e a r e x t r a c t (Fig. 2a). 5% o f e a c h f r a c t i o n w a s anal y z e d . P a n e l II: t h e n u c l e a r e x t r a c t w a s t r e a t e d w i t h 1 M NaC1 (Fig. 2b). 10% of e a c h f r a c t i o n w a s a n a l y z e d . Panel I I I : in o r d e r to visualize t h e p r o t e i n b a n d s o f f r a c t i o n F f r o m p a n e l s I a n d II, a l i q u o t s c o r r e s p o n d i n g to 3% ( F I ) a n d 1 . 7 5 % ( F I I ) w e r e a n a l y z e d . P a n e l IV: n u c l e a r e x t r a c t t r e a t e d w i t h 1 0 0 /~g/ml o f p r o t e i n a s e K (Fig. 4). 15% of e a c h f r a c t i o n w a s a n a l y z e d . M i n d i c a t e s t h e m i g r a t i o n o f m a r k e r proteins. F r o m t o p to b o t t o m : p h o s p h o r y l a s e b ( 9 4 0 0 0 Mr), b o v i n e s e r u m a l b u m i n ( 6 7 0 0 0 Mr) , ovalb u m i n ( 4 3 0 0 0 Mr) , c a r b o n i c a n h y d r a s e ( 3 0 0 0 0 M r ) , c h y m o t r y p s i n o g e n ( 2 5 0 0 0 Mr).

118 A

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Fig. 7. D e n a t u r i n g p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s of R N A . R N A s w e r e a n a l y z e d o n 12% h o m o g e n e o u s gels in t h e p r e s e n c e o f 8 M u r e a . A n u n t r e a t e d n u c l e a r e x t r a c t was f r a c t i o n a t e d o n a 1 0 - - 4 0 % s u c r o s e g r a d i e n t a n d R N A w a s p r e p a r e d f r o m h n R N P a b o v e 50 S (A) a n d f r o m 3 0 - - 5 0 S m o n o p a r t i c l e s (D). A n o t h e r n u c l e a r e x t r a c t was t r e a t e d w i t h 1 0 0 ~tg/ml p r o t e i n a s e K a n d c e n t r i f u g e d o n a 1 0 - - 2 5 % s u c r o s e g r a d i e n t in t h e p r e s e n c e o f 0.4 M NaC1. T h e R N A w h i c h s e d i m e n t e d a b o v e 18 S (B) a n d at 1 0 - 18 S (E) w e r e a n a l y z e d , F i n a l l y , t h e R N A o f a n u c l e a r e x t r a c t w a s p r e p a r e d b y p h e n o l e x t r a c t i o n (in t h e p r e s e n c e of 0.4 M NaC1) a n d c e n t r i f u g e d o n a 1 0 - - 2 5 % s u c r o s e g r a d i e n t . T h e R N A a b o v e 18 S (C) a n d t h e 1 0 - - 1 8 S R N A (F) w e r e a n a l y z e d . T h e h n R N A of A , B, C o n o n e side o f D, G, F o n the o t h e r w e r e a p p r o x i m a t e l y of the s a m e size ( n o t s h o w n ) . T h e y r e m a i n o n t h e t o p o f t h e gels u n d e r t h e e l e c t r o p h o r e t i c c o n d i t i o n s u s e d h e r e . Gels w e r e c a l i b r a t e d w i t h a m i x t u r e of c y t o p l a s m i c s m a l l R N A f r o m H e L a cells i n f e c t e d w i t h a d e n o v i r u s . V a ! R N A is a v i r u s - c o d e d s m a l l R N A . Gels w e r e r e c o r d e d a f t e r s t a i n i n g w i t h a V e r n o n d e n s i t o m e t e r (Paris, F r a n c e ) .

ciated with a protein (or proteins) resistant to salt dissociation and is part of heterogeneous complexes. The hnRNA-bound small R N A . Approximately 40% of the snRNA sedim e n t e d with h n R N A after deproteinization. Were the same snRNA species present among h n R N A - b o u n d and protein-bound snRNA? In order to answer this question, we compared the snRNA b o u n d to large ( > 2 0 S) and middle (10--18 $) size h n R N A in untreated and in proteinase treated hnRNP. In addition phenol-extracted R N A which should be similar to RNA prepared b y proteinase K treatment was examined. Denaturing concentrated polyacrylamide gels were used for analysis in order to avoid divergences due to secondary structure and smears of h n R N A along the gels. Several snRNA species were present in large size hnRNP, the major ones being U2, U1 and a 5 S R N A species (Fig. 7A). After proteinase K treatment (Fig. 7B) or phenol extraction (Fig. 7C), an enrichment of UI and U2

119

RNA and a decrease of the proportions of the RNA of 5 S or less were observed. The 10--18 S RNA was examined independently because this size is approximately that of the hnRNA from 30--50 S monoparticles, one the classes of unit of hnRNP [11] in which U1 RNA clearly predominates (Ref. 6 and Figs. 3D panel I and 7D). After deproteinization (Figs. 7E and F), the relative proportion of U1 RNA considerably decreased (Figs. 7D and E, F, compare U, and U2 RNA, for instance), so that the electrophoretic profiles of snRNA bound to 10--18 S RNA or to larger hnRNA had the same general characteristics (Fig. 7B, C and Fig. 7E, F). The method of deproteinization, phenol extraction or proteinase treatment, did not significantly modify the distribution of the snRNA bound to hnRNA (compare Fig. 7B, E and C, F). Such behavior would be expected from hydrogen-bonded snRNA. From these experiments, we may deduce that the U-rich RNAs were enriched in the hnRNA-bound snRNA pool and the smaller snRNA in the protein-bound snRNA pool (40 and 60% of the total pool, respectively). However, at least the major species, and probably also the minor ones, were present in both pools. Discussion

The association of snRNA with hnRNA was first described in proteinase-Ktreated hnRNP [2] and in phenol-extracted poly(A) ÷ hnRNA [1]. Denaturation by melting, formamide or urea suggested that hydrogen bonding was the most likely way of association [1,2,18]. All the snRNA species, but with a relative predominance of Uz and U2, remained associated with hnRNA prepared from hnRNP by proteinase treatment [2]. This was also the case in our own experiments and, in addition, we showed that the same snRNA profiles could be obtained upon phenol extraction. In contrast, only an RNA 90--100 nucleotide long (the 4.5 S region) was isolated from phenol-extracted poly(A) ÷ hnRNA [1]. It is possible that 4.5 S RNA is the only snRNA which remains associated with hnRNA after polyadenylation. The major snRNA species were all found associated with hnRNA, with the salt-dissociated proteins and with the salt-resistant proteins. This was probably also the case of the minor snRNA species though this was more difficult to determine. The distribution between these three pools varied according to the snRNA species. Thus, most of Uz RNA was preferentially bound to the easily salt-dissociated monoparticle proteins and 5 Sa RNA to the salt-resistant proteins of heterogeneous complexes. The biological significance of such differences of distributions is not yet understood. Nevertheless, the fact that the same snRNA species were found in all pools suggests the possibility of exchanges within hnRNP. For instance, snRNA (free or as small nRNP) may first bind to hnRNP proteins and thus be readily available for association with hnRNA where they may play their (hypothetical) role in processing. Previous determination showed that there was about 1 molecule of snRNA per 2500 nucleotides of hnRNA in hnRNP [6]. Assuming that they are distributed randomly, this would indicate 1--2 molecules of snRNA per individual hnRNP

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fibril. A given snRNA species would be present only in one out of several fibrils. A possible interpretation of these data is that different snRNA species are required for different steps of pre-mRNA processing and may sequentially bind to hnRNP. After having performed their specific function, they might be re-utilized. However, there is n o t experimental evidence of random distribution of snRNA in hnRNP. All species might be present in a set of fibrils at a given time and none in the others. It is obvious that more data are required to understand the function of snRNA in hnRNP and we hope that our current investigations on the sites of h n R N A to which snRNA are associated will contribute to the solution of this problem. The first discovery of metabolically stable snRNA in hnRNP [19] led to the suggestion that these RNAs might be structural constituents of hnRNP (in the sense that ribosomal RNAs are structural constituents of ribosomal subunits). Our finding that U1 R N A was enriched in monoparticles and released together with their proteins made it an apparently good candidate for such a function. In fact, there is less than 1 U, R N A molecule per monoparticle [6]. Therefore, a structural role must be excluded and other functions found for U, RNA. Acknowledgement The excellent technical assistance of Mrs. L. Kister is acknowledged. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

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