BIOCHIMIE, 1975, 57, 227-233.
Nuclear and mitochondrial origin of rat liver double-stranded RNA. L o u i s e HAREL, (*) Guy Rxou ( * ' ) (*) l n s t i t u t d e R e c h e r c h e s s u r (*') Institut Gustave (*'*) D~partement de ViroIogie,
and L u c MONTAGNIER ( . . . . /). le C a p e e r - - B.P. 8 V i I l e j u i f . Roussg -- Villejuif. I n s t i t u t P a s t e u r - - P a r i s (XV~).
(4-12-1974).
S u m m a r y . - - We have stud.ted the intraeellular location of double-stranded RNA from rat liver. The m a j o r i t y of this dsRNA is associated with nuclear and mitochondrial fractions and the remaining portion is associated w i t h microsomes. Mitochondrial dsRNA hybridizes specifically with purified strands of mitochondrial DNA and seems therefore to arise from transcription of the latter. A fraction of nuclear dsRNA hybridizes with repetitive sequences of nuclear DNA. Because of self-hybridization of dsRNA strands, it was not possible to determine whether another fraction of dsRNA is homologous to nonrepetitivc sequences of DNA. Both mitochond'rial and nuclear types of dsRNA are not retained on oligo(dT) cellulose columns and therefore seem to lack long po]y A sequences. The mechanism of formation of dsRNA is discussed.
INTRODUCTION. W e h a v e p r e v i o u s l y r e p o r t e d t h e i s o l a t i o n of s m a l l a m o u n t s of d o u b l e - s t r a n d e d R N A in cells o r tissues n o t i n f e c t e d b y v i r u s e s [1]. T h e d s R N A e x t r a c t e d f r o m a d u l t r a t l i v e r w a s f o u n d to h y b r i dize to a l a r g e e x t e n t w i t h c e l l u l a r D N A of t h e h o m o l o g o u s tissue [2], a n d to be an i n d u c e r of i n t e r f e r o n [3]. T h e g e n e r a l o c c u r e n c e of d s R N A s e q u e n c e s h a s b e e n c o n f i r m e d s i n c e in s e v e r a l l a b o r a t o r i e s [4-8], a l t h o u g h , due to d i f f e r e n t m e t h o d s of p r e p a r a t i o n s , it is n o t c e r t a i n t h a t t h e s a m e m a t e r i a l w a s s t u d i e d in all cases. H e r e w e r e p o r t t h e r e s u l t s of f u r t h e r s t u d i e s o:n r a t l i v e r dsRNA. It is s h o w n t h a t o n e f r a c t i o n of t h i s R N A is a s s o c i a t e d w i t h m i t o c h o n d r i a a n d hybridizes with purified mitochondrial DNA s t r a n d s , w h i l e a n o t h e r f r a c t i o n is l o c a t e d in t h e nucleus and hybridizes with highly repetitive s e q u e n c e s of c h r o m o s o m a l DNA. MATERIALS AND METHODS. 5-SH o r o t i c a c i d (40 C i / m M ) w a s o b t a i n e d f r o m C.E.A. S a c l a y , F r a n c e . D e o x y r i b o n u c l e a s e (RNase
free) a n d p a n c r e a t i c r i b o n u c l e a s e A ,~'ere p u r chased from Worthington Biochemical Corporation. P r o n a s e a n d p r o p i d i u m d i i o d i d e w e r e f r o m C a l b i o e h e m ; A c t i n o m y e i n D f r o m Merek, S h a r p a n d D o h m e , P a r i s ; S e p h a d e x G200 f r o m P h a r macia, Uppsala, Sweden; Oligo(dT) cellulose from S e a r l e I.aboratoires~ H i g h W y c o m b e , E n g l a n d . Rats were Wistar males or females, weighing b e t w e e n 180 to 230 grs. 1 °) P r e p a r a t i o n o f r a d i o a c t i v e d s R N A . a) L a b e l l i n g : 500 :aCt o r I m C i of 5-3H o r o t i e a c i d w e r e i n o c u l a t e d i n t r a p e r i t o n e a l l y to rats. 3 h o u r s later, a f t e r m i l d a n a e s t h e s i s w i t h e t h e r , rats w e r e s a c r i f i e e d a n d l i v e r s w e r e r e m o v e d . I n e x p e r i m e n t s i n v o l v i n g a e t i n o m y e i n D, a d o s e of 200 i~g/kg w a s a d m i n i s t e r e d 1 h r b e f o r e i n o c u l a t i o n of t h e o r o t i c a c i d . b) F r a c t i o n a t i o n o f r a t l i v e r : all o p e r a t i o n s w e r e c a r r i e d out at 4 ° 4 : - - p u r i f i e d n u c l e i , f r e e of c y t o p l a s m , w e r e p r e p a r e d a c c o r d i n g to t h e m e t h o d of C h a u v e a u [9] a f t e r h o m o g e n e i z a t i o n of l i v e r s in 2.3 M s u c r o s e ; m i t o c h o n d r i a w e r e p u r i f i e d a c c o r d i n g to a t e c h n i q u e a l r e a d y d e s c r i b e d [10] ; -
Abbreviations used : dsRNA : double-stranded RNA. mtDNA, mtRNA : mitochondrial DNA, RNA. nDNA : nuclear DNA. SDS : sodium dodecyl sulfate. SSC : Standard Saline Citrate : 0.15 M NaC1. 0.015 M Sodium Citrate. HnRNA : heterogeneous nuclear RNA. mRNA : messenger RNA. ,~ To whom all correspondence should be addressed.
-
--purified mierosomes were separated from post-mitoehondrial fraction by recentrifugation at 1 2 0 0 0 0 × g for 90 rain of t h e s u p e r n a t a n t w h i c h w a s o b t a i n e d b y c e n t r i f u g a t i o n of l i v e r h o m o g e n a t e in 0.25 M s u c r o s e at 16 000 × g for 10 m i n .
L. Harel, G. Riou and L. Monlagnier.
228
c) Isolation of dsRNA : the method of isolation p r e v i o u s l y described [1, 2] was used in all experiments. F i n a l stage of p u r i f i c a t i o n involved pancreatic RNase t r e a t m e n t a n d filtration on Sephadex G 200 column. The void volume peak was c o n c e n t r a t e d b y e v a p o r a t i o n a n d dialysed against 1000 volumes of 0.1 X SSC.
- - Aliquots i n 1 ml 0.1 X SSC were placed in w a t e r b a t h at t e m p e r a t u r e v a r y i n g from 20 to 100°C for 10 rain, then fast cooled in ice bath. NaC1 5 M was added to make u,p the solution 0.3 M a n d each sample was treated w i t h 10 ~ g / m l of p a n c r e a t i c RNase for 30 rain at 20°C a n d TCA precipitated.
a) Extraction of mtDNA. The m i t o c h o n d r i a were lysed i n S.D.S. to a final c o n c e n t r a t i o n of 1 D. cent in TE buffer (0.01 Tris HC1, 0.001 M EDTA pH 7.5) for 15 m i n at room temperature. The lysate was treated 10 rain at 0°C w i t h 5 M NaC1 to obtain a fin,al c o n c e n t r a t i o n of 1.0 M a n d centrifuged at 16,000 × g i n Sorvall SS 34 rotor. The s u p e r n a t a n t was collected a n d dialysed against TE buffer a n d mIDNA purified in a CsCl-propidium d i i o d i d e (PDI) g r a d i e n t in the following c o n d i t i o n s : mtDNA in 3.3 ml TE b u f f e r ; CsC1 3.53 g r ; PDI (2.5 m g / m l ) 1.0 ml (500 t~g/ml f i n a l ) ; refractive i n d e x n 22°C == 1.3820; e e n t r i f u g a t i o n 24 hours 40 000 r p m at 20°C in a Beckman model L 2 B u ! t r a e e n t r i f u g e (SW 50 rotor). The lower b a n d was collected a n d PDI removed by p a s s i n g t h r o u g h a Dowex 50 resin column. The mtDNA was dialysed against 0.1 x SSC.
The percentage of dsRNA p r e s e n t i n the labelled RNA was estimated a c c o r d i n g to the f o r m u l a :
b) Separation of complementarg strands mtDNA.
The RNase resistance at high i o n i c strength of each prep.aratio,n a n d its m e l t i n g curve i n low i o n i c strength were d e t e r m i n e d i n the following way :
I
1'CAinsolublecountsof 1 t - the sampleheatedat Itt0oEand RNasetreated
x
'100
I'CI ius.hbb' countso[ the samplekept at 20oCand R~asetreated Only p r e p a r a t i o n s w h i c h c o n t a i n e d more than 80 p. cent of dsRNA a n d d i s p l a y e d a sharp m e l t i n g t r a n s i t i o n at a t e m p e r a t u r e above 60°C were used for experiments.
Closed c i r c u l a r mtDNA molecules were n i c k e d by exposure to visible light [20]. Nicked mtDNA was assayed fiuorimetrically in a Zeiss PMQIII fluorescence spectophotometer [21~. The complem e n t a r y strands of mtDNA were p r e p a r e d by cen-
2 °) Preparation of nuclear DNA. Purified n.uclei o b t a i n e d by the t e c h n i q u e of Chauveau [91 were s u s p e n d e d i n buffer saline EDTA (0.15 M NaC1, 0.1 M EDTA pH 8) + 1 p. cent SDS a n d extracted with o,ne volume of saturated phenol. Nucleic acids were p r e c i p i t a t e d from the aqueous phase by ethanol in the p r e s e n c e of 1 p. cent s o d i u m acetate. They were solu.bilized in 1 X SSC and extracted 3 times w i t h chloroform-isoamylie alcohol m i x t u r e a c c o r d i n g to the Marmur method. After p r e c i p i t a t i o n from the aqueous phase, they were treated w i t h RNase and p r o n a s e a c c o r d i n g to Gillespie a n d Spiegelm a n [H]. In e x p e r i m e n t s i n v o l v i n g high Co.t value h y b r i d i z a t i o n , n u c l e i c acids were not treated w i t h RNase, but w i t h 0.3 M KOH for 18 h o n r s at 37°C. After n e u t r a l i z a t i o n , the solm tions was dialysed for 24 h o u r s against 0.1 X SSC solution. 3 °) Preparation and purification of mtDNA. M i t o c h o n d r i a were isolated as p r e v i o u s l y described [12]. Since i n t a c t mtDNA could not be extracted from m i t o c h o n d r i a p r e t r e a t e d w i t h Dnase, this t r e a t m e n t was omitted.
BIOCHIMIE, 1975, 57, n ° 2.
of
H
0,2
A. ,,
.......
t I ,I t, 15
20
25 Fractions
Separation of heaog and light strands of mt DNA in alkaline caesium chloride gradient. FIG. 1.
-
-
trifugation in an alkaline CsC1 g r a d i e n t i n the following conditions : mtDNA i n 3,6 ml of 0.1 X SSC ; 0.4 ml 0.5 M Na3PO 4 ; 5.35 gr CsC1 ; p H 12.3 ; refractive i n d e x n 22°C = 1.4'035 ; c e n t r i f u g a t i o n for 60 hours at 35,000 r p m at 20°C in the r o t o r 50 of a Beckman ultracentrifuge. 0.5 ml of buffer 0.05 M NaH,,PO4 0.001 M EDTA pH 6.5 was added to each collected fraction (fig. 1). The refractive c o n c e n t r a t i o n s of the heavy and light mtDNA s t r a n d s were d e t e r m i n e d by m e a s u r i n g their opticM d e n s i t y at 260 n m (equivalence 1'0 ~ g / m l = 0.212 O.D. 260 unit) a n d a c o r r e c t i o n factor for h y p e r c h r o m i e i t y at 40 p. cent [13].
Rat liver double-stranded c) Analytical ultracentrifngation.
RNA.
229
centage of dsRNA increases, as p r e v i o u s l y s h o w n for dsRNA from entire cclls [1], especially i n the n u c l e a r a n d m i c r o s o m a l fraction. Only negligeable a m o u n t s of dsRNA were found to be associated w i t h the 100,000 X g s u p e r n a t a n t .
Co.ntrols for p u r i t y of the mtDNA in n e u t r a l CsCI g r a d i e n t and for s t r a n d s e p a r a t i o n in alkaline CsCI gradient (0.05 M NaaPO 4 0.05 M NaOH pH 12.5) were p e r f o r m e d in a B e c k m a n model E u l t r a c e n t r i f u g e (25°C - - 44770 r p m for 22 hours).
2 °) Characterization of m i t o c h o n d r i a l and nuclear dsRNA.
RESULTS.
a) H y b r i d i z a t i o n w i t h m i t o c h o n d r i a l and nuclear rat liver DNA.
1 °) Distribution of radioactive dsRNA in rat liver fractions. In p r e l i m i n a r y experiments, a rat liver homogenate made in 0.25 M sucrose was separated i n three fractions by differential centrifugation. 80 p. cent of the total r a d i o a c t i v i t y associated w i t h dsRNA was f o u n d in the pellet obtained b y c e n t r i f u g a t i o n at 16,000 × g for 10 rain. This pellet c o n t a i n e d nuclei, m i t o c h o n d r i a , a n d some u n b r o k e n cells. The r e m a i n i n g 20 p. cent of
E v i d e n c e that dsRNA associated w i t h the nfitoc h a n d r i a l fraction arises i n d e e d from t r a n s c r i p tion of mtDNA was given by its h y b r i d i z a t i o n with purified mtDNA separated into light (L) a n d heavy (It) strands. Owing to the small a m o u n t s of either pure mtDNA and dsRNA used, p r o p e r s a t u r a t i o n curves could not be obtained. However, as s h o w n in table II, it is clear that melted mt-dsRNA h y b r i -
TABLE I.
Subcellalar localization of dsRNA. Naclei Experiment n °
cpm
o 1-7
t
lI
p. cent total radi( activity in RNA
18551
0.20
Mitochondria
cpm
Microsomes
p. cent
cpm
p. cent
0.12
800
0.02t
512 2300
0.07 0.012
ill
1460 I 0 047
860
vt(*)
8987 I 0.51
624 I
o.2o
Supernatant 100,000 ~" cpm
201
p. cent
O.OOl
n~gligeable
0.09
(*) Rats treated with 0,2'80 mg/kg of Actinomycin D 1 hour before orotic acid injection. dsRNA was f o u n d associated w i t h microsomes obtained by r e c e n t r i f u g i n g at 120 000 g for 1 h r the s u p e r n a t a n t of the 16 000 × g centrifugation. I n subsequent experiments, the rat liver fractions were purified as described in Materials a n d Methods. Table I sho~-s the results of 4 experiments. It clearly appears that the m a j o r i t y of radioactive dsRNA is p r e s e n t i n both n u c l e i a n d mitochondria. This is true for the absolute n u m b e r of counts a n d also for the percentage of the total labelling of RNA in each fraction. W h e n rats are p r e t r e a t e d w i t h a c t i n o m y c i n D at a c o n c e n t r a t i o n k n o w n to i n h i b i t p r e f e r e n t i a l l y ribosomal RNA synthesis [14] the relative per-
BIOCHIMIE, 1975, 57, n* 2.
dizes to small a m o u n t s of mtDNA, the h y b r i d i zation percentage b e i n g h i g h e r for the light s t r a n d t h a n for the heavy strand. The percentage h y b r i dized w i t h s i m i l a r a m o u n t s of rat liver nDNA was in the range of unspecific h y b r i d i z a t i o n , as c o m p a r e d to E. colt DNA. Even w i t h 40 ,~g of rat liver nDNA, the h y b r i d i z a t i o n percentage was lower t h a n that o b t a i n e d w i t h 1,7 ag of mtDNA. I n a control experiment, dsRNA extracted from an i m p u r e n u c l e a r p r e p a r a t i o n (600 × g pellet of the same liver homogenate used for m i t o c h o n drial dsRNA p r e p a r a t i o n ) was h y b r i d i z e d to mt and nDNA (table III). C o m p a r i s o n of tables II and III shows that this dsRNA h y b r i d i z e s to a higher extent than mt-dsRNA with the same a m o u n t s of nDNA (40 ~g). Conversely, its p e r c e n 17
L. HareI, G. R i o u and L. Montagnier.
230
TArtLY. II.
Homology of mitochondrial dsRNA to mitochondrial DNA.
Experiment n*
IsRNA mt-
Mitochondrial DNA '
L Strand
E. coli
Nuclear DNA
control DNA
H Strand
added I - - [cpm) 9g added P" cent dsRNA p. cent dsRNA . . ~ Ip. cent dsRNA . . . . . Ip. cent dsRNA hybridized (') i~g added hybridized (*) - - l ~ gaaaea [ hybridized ('j ~g auueu hybridized (*)
I
700
II
860 860
0.6 ~ 0.8 1.7
860
11.0 (77) 9.3 (80) 11.2 (96) 14.1/121)
0.6
11.8 (83)
~
5.9 (51) 8.9 177)
,}.8 1.7
9.8
(84)
2.8 (20) 40 -~----
'
40
0.2 ( 2 ) 8.1 (70)
0.3 (2l 1.0 (9)
(*) In bracket, epm. tage of h y b r i d i z a t i o n w i t h m t D N A is l o w e r t h a n that observed with mt-dsRNA. The small percentage h y b r i d i z e d w i t h m t D N A m a y be e x p l a i n e d
It m a y be c o n c l u d e d t h a t one p a r t of d s R N A h y b r i d i z e s w i t h r e p e t i t i v e s e q u e n c e s of D N A a n d t h a t t h e o t h e r p a r t d o e s not.
TABLE III.
Hybridization of dsRNA from nuclear pellet. MitoehondriaI DNA Experiment no
I
II
~uelea IsRNA added (epm)
1000
E. coli
Nuclear DNA
control DNA
H Strand
L Strand
- - , , [p. cent dsR____NA ~g added p'cent dsRNA ~g added P' e,en.t,.ds~l~,.A ~g added p. eentdsRNA hybridized I') rtg aaaea hybridized (*) hybridized (') - - l l y n Z l g l Z e ~ [ ) _- - -_0.6 ~ - - -
2.7 (27) (*) 4.4 (70)
0.6
-%sU-,
3.0 (30)
40
14.3 (143)
40
3.0 (30)
(50,
(*) cpm. b y t h e c o n t a m i n a t i o n of t h e n u c l e a r p e l l e t b y l a r g e m i t o c h o n d r i a (cf. M a t e r i a l s a n d M e t h o d s ) . b) Homology of dsRNA with repetitive and non repetitive sequences of nuclear DNA. U n d e r c o n d i t i o n s in w h i c h o n l y h y b r i d i z a t i o n w i t h r e p e t i t i v e s e q u e n c e s of D N A c a n t a k e p l a c e , a b o u t 20 p. c e n t of m e l t e d n u c l e a r d s R N A f o r m R N a s e r e s i s t a n t c o m p l e x e s -with n u c l e a r DNA. T h e p o s s i b i l i t y w a s c o n s i d e r e d t h a t R N A self hybridization, which has already been reported to o c c u r Eli, m i g h t be a l i m i t i n g f a c t o r of t h e D N A - R N A h y b r i d i z a t i o n rate. T h e r e f o r e t w o e x p e r i m e n t s w e r e p e r f o r m e d , in w h i c h t h e n o n h y b r i d i z e d p a r t of n u c l e a r d s R N A (the M i l l i p o r e filtrate) w a s h e a t e d a g a i n at 100°C for 5 rain a n d r e i n e u b a t e d w i t h t h e s a m e a m o u n t of d e n a t u r e d r a t l i v e r D N A at 65°C. R e s u l t s in table IV s h o w that the second hybridization level was much l o w e r t h a n t h e first one.
BIOCHIM1E, 1975, 57, n ° 2.
For the latter fraction, the question was asked w h e t e r it c o u l d b e h y b r i d i z e d to n o n r e p e t i t i v e f r a c t i o n of D N A o,r w h e t h e r it w a s not h o m o l o g o u s at all to DNA. Three hybridization experiments were perf o r m e d in c o n d i t i o n s of h i g h Co.t v a l u e (Co.t = 7.5 × 103) a c c o r d i n g to. t h e m e t h o d of Melli a n d B i s h o p [1'5]. As sh(ywn in t a b l e V, t h e formatio.n of R N a s e r e s i s t a n t c o m p l e x e s r e a c h e d up to 88 p. c e n t of t h e i n p u t m e l t e d dsRNA, b u t n e a r l y s i m i l a r l e v e l s w e r e o b t a i n e d in u s i n g n o n - s p e c i f i c D N A (E. coli) i n s t e a d of rat l i v e r dsRNA. T h e p o s s i b i l i t y w a s c o n s i d e r e d that, in s u c h c o n d i tions, a f r a c t i o n of R N a s e r e s i s t a n t c o m p l e x e s a r o s e f r o m s e l f - h y b r i d i z a l i o n of R N A s t r a n d s [1]. E q u i l i b r i u m d e n s i t y a n a l y s i s in c a e s i u m s u l f a t e s h o w e d i n d e e d t h a t the t o t a l i t y of the R N a s e res i s t a n t m a t e r i a l b a n d e d at t h e d e n s i t y of dsRNA, w h e n E. coli D N A w a s p r e s e n t in t h e i n c u b a t i o n medium. When rat liver dsRNA was present,
R a t liver d o u b l e - s t r a n d e d R N A . a p p r o x i m a t e l y 60 p. c e n t of R N a s e r e s i s t a n t m a terial formed bands in the RNA region and the D N A - R N A h y b r i d r e g i o n , w h e r e a s 40 p. c e n t rem a i n e d i n t h e r e g i o n of R N A d u p l e x e s .
i m p o s s i b l e to o b t a i n a d e f i n i t e a n s w e r to t h e q u e s t i o n as t o w h e t h e r d s D N A is h o m o l o g o u s t o m o d e r a t e l y r e p e t i t i v e a n d u n i q u e s e q u e n c e s of n u c l e a r RNA, o r not.
TABLE I'V.
]OO
Incubation ds melted RNA added {cpm)
lI
P. cent hybridized
2d
1140 420
280 22
24 5 5
1st 9d
5000 1120
700 50
14 5
1st
I
RNA hybridized {cpm)
p .....
!
Homology of dsRNA with repetitive sequences of DNA. Exp.
231
I//
//
.
T h u s , i n o u r c o n d i t i o n s of h i g h Co.t v a l u e , t h e r e w a s a c o m p e t i t i o n b e t w e e n f o r m a t i o n of h y b r i d s a n d R N A - B N A self h y b r i d i z a t i o n . I t w a s t h e r e f o r e
20
60
80
lO0 12D Temperature rc)
Fit;. 2. - - Melting curve of nuclear ( © - - © ) and mitochondrial (e .... el. dsRNA in 0.1 × SSC (as described in
TABLE V.
Materials a n d Methods).
Search [or hybridization of dsRNA with non repetitive sequences of DNA. (See conditions of h y b r i d i z a t i o n in t h e text). cpm acid precipitable Exp.
I
II Ill
DNA
R a t liver
E. colt Rat liver R a t liver
E. colt
--RNase
+ RNase
1100 450
996 33O
88 75
670
43(') 42(')
670 1600
E. colt
P. cent RNase resistant
1550 1550
~ 1180 1100
76 71
(*) Material f u r t h e r analyzed in Caesium sulfate density gradient.
c) Melting curves of nuclear and mitochondrial dsRNAs. As s h o w n i n f i g u r e 2, n o l a r g e d i f f e r e n c e c o u l d b e o b s e r v e d i n t h e m e l t i n g c u r v e s of d s R N A s of nuclear and mitochondrial origin. This suggests t h a t t h e b a s e c o m p o s i t i o n s of b o t h d s R N A s d o n o t g r e a t l y differ. d) Polg A content : t h e p r e s e n c e of l o n g p o l y A s e q u e n c e w h i c h m i g h t b e a t t a c h e d to d s R N A , as i n m o s t of m e s s e n g e r RNAs, w a s i n v e s t i g a t e d b y passing the purified dsRNAs in oligo(dT) cellulose column.
TABLE VI.
Chromatography of rat liver nuclear and mitochondrial dsRNAs on oligo(dT)cellulose column. Origin
Fraction
Not heated
Heated (')
n-dsRNA
Filtrate 0.1M Tris pH 7.4, 0.5M KCl Eluate 0.1M Tris 0.1M KCI Eluate 0.1M Tris
71 p. ccnt 19 p. cent 9 p. cent
74 p. cent 13 p. cent 12 p. cent
mt-dsRNA
Filtrate 0.1M Tris p H 7.4, 0.5M KC1 Eluate 0.1M Tris 0.1M KC1 Eluate 0.1M Tris
84 p. cent 10 p. eent 6 p. cent
83 p. cent 7 p. cent 9 p. cent
(*) Heated 100°C 10 min in 0.1 × SSC, fast-cooled in ice b a t h , e t h a n o l precipit a t e d a n d redissolved in 0.I M TPis, 0.5 M KC1.
BIOCH1MIE, 1975, 57, n o 2.
L. Harel, G. Riou and L. Montagnier.
232
Control e x p e r i m e n t s (not sho~m) i n d i c a t e d that m o r e thant 70 p. cent of v a c c i n i a virus early mRNA w a s r e t a i n e d by the c o l u m n and eluted in 0.1 M Tris buffer. U n d e r the same conditions, the m a j o r i t y of either n u c l e a r or m i t o e h o n d r i a l dsRNA was not r e t a i n e d by the column, less t h a n 10 p. cent b ei n g eluted in 0.1 M Tris (table VI). S e p a r a t i o n by heat of dsRNA strands did not i n c r e a s e the binding, suggesting that t h e r e w e r e no poly A stretches h i d d e n by the s e c o n d a r y structure. DISCUSSION. An u n e x p e c t e d result of this w o r k ~ a s that a non-negligeable f r a c t i o n of rat l iv e r dsRNA is located in m i t o c h o n d r i a and is h o m o l o g o u s to p u r i f i e d IntDNA. P r e l i m i n a r y results suggest that a s i m i l a r situation also exists in c u l t u r e d cells and that synthesis of mt-dsRNA is s p e c i f i c a l l y i n h i b i t e d by e t h i d i u m b r o m i d e , as is the synthesis of the w h o l e mtRNA. T h e most likely e x p l a n a t i o n is that this dsRNA f r a c t i o n arises f r o m s y m e t r i c t r a n s c r i p t i o n of m i t o c h o n d r i a l DNA. In this respect, it is i n t e r e s t i n g to r e c a l l the w o r k of At t ard i [16], s h o w i n g that both strands of m i t o c h o n d r i a l DNA are t r a n s c r i b e d , the h e a v y strand b e i n g p r e f e r e n t i a l l y degraded. It is t h e r e f o r e c o n c e i v a b l e that mt-dsRNA is d e r i v e d f r o m the association of single c o m p l e m e n t a r y 13NA strands m a i n t a i n e d in close c o n t a c t by p r o t e i n s s h o rt l y after t h e i r synthesis. T h e ~ h o l e d u p l e x s t r u ct u r e w o u l d then be f o r m e d only after d e p r o t e i n i z a t i o n by p h e n o l e x tr a c t io n . Alternatively, as assumed e a r l i e r [:2], dsRNA loops m ay o ri g i n a te f r o m the t r a n s c r i p t i o n of a d j a c e n t c o m p l e m e n t a r y sequences on the same DNA strand. E v i d e n c e that such p a l i n d r o m i c se q u en ces exist in c h r o m o s o m a l DNA has r e c e n t l y been p u t f o r w a r d by W i l s o n and T h o m a s [17] but no data are available c o n c e r n i n g the o c c u r e n c e of s i m i l ar sequences in mtDNA. The results of Monckton and N o o r a E8] are at first sight in conflict w i t h ours, since these a u t h o r s f o u n d a ds!RNA e x c l u s i v e l y in the n u c l e a r f r a c t i o n of rat l i v e r and no dsRNA in the cytop l a s m i c fraction. H o w e v e r the latter was the supernatan.t r e m a i n i n g after c e n t r i f u g a t i o n at 20 000 × g. U n d e r these c o n d i t i o n s most of the m i t o c h o n d r i a should be in the pellet with nuclei. Moreover the time of labelling was s h o r t e r (60 m i n u t es i n s t e a d of 180 m i n u t e s in o u r expert-
BIOCHIMIE, 1975, 57, n ° 2.
ments) so that a dsRNA s y n t h e t i z e d in the nucleus m ay have not passed yet into the cytoplasm. Th e dsRNA that w e f o u n d associated w i t h the m i c r o s o m a l f r a c t i o n m ay i n d e e d h a v e a n u c l e a r origin. P r e l i m i n a r y e x p e r i m e n t s i n d i c a t e that it h y b r i d i z e s w i t h n u c l e a r DNA to the same extent than n u c l e a r dsRNA and it m a y t h e r e f o r e d e r i v e from the latter. THE
NATURE OF NUCLEAR
dsRNA.
Th e same r e a s o n i n g used above for the origin of m i t o c h o n d r i a l dsRNA m a y be a p p l i e d to nucl ear dsRN£. It could be f o r m e d by s y m c i r i c t r a n s c r i p t i o n of DNA strands and subsequent associ at i o n of the c o m p l e m e n t a r y RNA strands, or o r i g i n at e f r o m the t r a n s c r i p t i o n of p a l i n d r o m i c DNA. The r eg i o n s of p a l i n d r o m i c DNA isolated by Wilson and T h o m a s E171 are long enough to be t r a n s c r i b e d in a dsRNA h a v i n g the size that we Have p r e v i o u s l y r e p o r t e d (8-9 S in s e d i m e n t a t i o n ) . In contrast, it seems that our dsRNA m a t e r i a l differs f r o m that isolated in nuclei by two different groups [18, 19] ; the latter m a t e r i a l is shorter, since it w as not isolated by ex cl u si o n in Sephadex G 200, and r e p r e s e n t s i n t r a m o l e c u l a r h e l i c a l loops of HnRNA. Our o w n e x p e r i m e n t s i n d i c a t e that HnRNA isolated as an aggregate does not y i el d dsRNA e x c l u d e d in G 200 c o l u m n (L. Mont ag n i er and H. Collandre, in p r e p a r a t i o n ) . A t h i r d p o ssi b i l i t y w h i c h cannot be by the p r e s e n t w o r k , is that n u c l e a r and m i c dsRNAs r ef l ect the self-replication n u c l e a r FIN,As f o l l o w i n g t h e i r synthesis
excluded cytoplasof some on DNA.
Acknowledgments. We thank the able technical assistance of H. Collanclre, S. Chamaret, M. Gabillot and G. Frezoul. RfiSUM~. On a dtudi~ la l ocalisation intracellulaire du R NA bicat~naire du foie de rat. La majoritd de ce RNA est distribute clans les fractions nucldaircs et mitochondriales et la fraction restante est associ6e aux microsomes. Le RNA bicat~naire mitochondrial hybridise sp~cifiquemcnt avec les brins purifi~s de DNA: mitochondrial, et scmble donc provenir cle la transcription de ce dernier. Une fraction du RNA bicat~naire nucl~aire hybridise avec des s~quences rSpdtitives du DNA nucl~aire. Par suite de l'antohybridation d.e RNA, il n'a pas ~t~ possi,ble de d~terminer si une autre fraction est ~galement homologue de s~quences non rdpdtitives du DNA. Les deux types d~e RNA bicat~naire, mitochondrial et nucl4aire, ne sont pas retenus par des colonnes d'oligo (dT)~ceHulose et semble.nt donc ~tre ddpourvus de sSquence poly A longues.
Rat
liver double-stranded
Le mdcanisme de f o r m a t i o n du RNA bicatdnaire est discutd. REFERENCES. 1. Montagnier, L. (196.8) C. R. Acad. Sci., 267, 14171420. 2. Harel, L. & M~ontagnier, L. (1971) Nature New Biol., 229, 106-108. 3. De Maeyer, E., De Maeyer, J. & Montagnier L. (1971) Nature N e w Biol., 229, 109-110. 4. Kronenberg, L. H. & Humphrey, T. (1972) Biochemistry, 11, 202,0-2026. 5. Stern, R. a Fr~e4man, R. M. (1970) Nature, 226, 612616. 6. Patnaik, R. ,¢ Taylor, M. W. (1973) Biochemistry, 12, 1990-1994. 7. Bases, R. & Kaplan, B. H. (1973) Biochim. Biophys. Acta, 312, 574~-580. 8. Monckton, R. P. a Naora, H. (1974) Biochim. Biophys. Acta, 335, 139-154. 9. Chauveau, J., Mould, Y. & Rouiller, C. (1957) Bull. Soc. Chim. Biol., 39, 1521-1533.
BIOCHIMIE, 1975,
57, n °
2.
RNA.
233
10. Harel, L., Jacob, A. & Mould, Y. (1957) Bull. Soc. chim. Biol., 39, 819-832. 11. Gillespie, D. ~ Spiegelman, S. A. (1965) J. Mol. Biol., 12, 829-842. 12. Riou, G. a Dela.in, E. (1971) Biochimie, 53, 831-836. 13. Nass, M. M. K. (1969) J. Mol. Biol., 42, 529=545. 14. Harel, L. a Harel, J., Boer, A., Imbenotte, J. & Carpeni, N. (1964) Biochim. Biophgs. Acta, 87, 212218. 15. Melli, M., Whitsfield, C., Rao, R. V., Ricchardson, M. Bishop, J. O. (1971) Nature N e w Biol., 231, 8-12. 16. Attardi, G. (1972) J. Mol. Biol., 70, 363-373. 17. Wilson, D. A. ,¢ Thomas, C. A. Jr. (1974) J. Mol. Biol., 84, 115-144. 18. Jelinek, W. ~ Darnell, J. E. (1972) P r o c . Nat. Acad. Sci. U.S.A., 69, 2537-2541. 19. Ryskow, A. P., Saunders, G. F., F a r a s h y a n , V. R. & Goergiev, G. P. (1973) Biochim. Biophys. Acta, 312, 152-164. 20. Clayton, D. A., Davis, R. W. & Vinograd, J. (1970) J. Mol. Biol., 47, 137-153. 21. Le Pecq, J. B. & Paoletti, C. (1966) Analyt. Biochem., 17, 100-107.
Nuclear and mitochondrial origin of rat liver double-stranded RNA. L o u i s e HAREL, (*) Guy Rxou ( * ' ) (*) l n s t i t u t d e R e c h e r c h e s s u r (*') Institut Gustave (*'*) D~partement de ViroIogie,
and L u c MONTAGNIER ( . . . . /). le C a p e e r - - B.P. 8 V i I l e j u i f . Roussg -- Villejuif. I n s t i t u t P a s t e u r - - P a r i s (XV~).
(4-12-1974).
S u m m a r y . - - We have stud.ted the intraeellular location of double-stranded RNA from rat liver. The m a j o r i t y of this dsRNA is associated with nuclear and mitochondrial fractions and the remaining portion is associated w i t h microsomes. Mitochondrial dsRNA hybridizes specifically with purified strands of mitochondrial DNA and seems therefore to arise from transcription of the latter. A fraction of nuclear dsRNA hybridizes with repetitive sequences of nuclear DNA. Because of self-hybridization of dsRNA strands, it was not possible to determine whether another fraction of dsRNA is homologous to nonrepetitivc sequences of DNA. Both mitochond'rial and nuclear types of dsRNA are not retained on oligo(dT) cellulose columns and therefore seem to lack long po]y A sequences. The mechanism of formation of dsRNA is discussed.
INTRODUCTION. W e h a v e p r e v i o u s l y r e p o r t e d t h e i s o l a t i o n of s m a l l a m o u n t s of d o u b l e - s t r a n d e d R N A in cells o r tissues n o t i n f e c t e d b y v i r u s e s [1]. T h e d s R N A e x t r a c t e d f r o m a d u l t r a t l i v e r w a s f o u n d to h y b r i dize to a l a r g e e x t e n t w i t h c e l l u l a r D N A of t h e h o m o l o g o u s tissue [2], a n d to be an i n d u c e r of i n t e r f e r o n [3]. T h e g e n e r a l o c c u r e n c e of d s R N A s e q u e n c e s h a s b e e n c o n f i r m e d s i n c e in s e v e r a l l a b o r a t o r i e s [4-8], a l t h o u g h , due to d i f f e r e n t m e t h o d s of p r e p a r a t i o n s , it is n o t c e r t a i n t h a t t h e s a m e m a t e r i a l w a s s t u d i e d in all cases. H e r e w e r e p o r t t h e r e s u l t s of f u r t h e r s t u d i e s o:n r a t l i v e r dsRNA. It is s h o w n t h a t o n e f r a c t i o n of t h i s R N A is a s s o c i a t e d w i t h m i t o c h o n d r i a a n d hybridizes with purified mitochondrial DNA s t r a n d s , w h i l e a n o t h e r f r a c t i o n is l o c a t e d in t h e nucleus and hybridizes with highly repetitive s e q u e n c e s of c h r o m o s o m a l DNA. MATERIALS AND METHODS. 5-SH o r o t i c a c i d (40 C i / m M ) w a s o b t a i n e d f r o m C.E.A. S a c l a y , F r a n c e . D e o x y r i b o n u c l e a s e (RNase
free) a n d p a n c r e a t i c r i b o n u c l e a s e A ,~'ere p u r chased from Worthington Biochemical Corporation. P r o n a s e a n d p r o p i d i u m d i i o d i d e w e r e f r o m C a l b i o e h e m ; A c t i n o m y e i n D f r o m Merek, S h a r p a n d D o h m e , P a r i s ; S e p h a d e x G200 f r o m P h a r macia, Uppsala, Sweden; Oligo(dT) cellulose from S e a r l e I.aboratoires~ H i g h W y c o m b e , E n g l a n d . Rats were Wistar males or females, weighing b e t w e e n 180 to 230 grs. 1 °) P r e p a r a t i o n o f r a d i o a c t i v e d s R N A . a) L a b e l l i n g : 500 :aCt o r I m C i of 5-3H o r o t i e a c i d w e r e i n o c u l a t e d i n t r a p e r i t o n e a l l y to rats. 3 h o u r s later, a f t e r m i l d a n a e s t h e s i s w i t h e t h e r , rats w e r e s a c r i f i e e d a n d l i v e r s w e r e r e m o v e d . I n e x p e r i m e n t s i n v o l v i n g a e t i n o m y e i n D, a d o s e of 200 i~g/kg w a s a d m i n i s t e r e d 1 h r b e f o r e i n o c u l a t i o n of t h e o r o t i c a c i d . b) F r a c t i o n a t i o n o f r a t l i v e r : all o p e r a t i o n s w e r e c a r r i e d out at 4 ° 4 : - - p u r i f i e d n u c l e i , f r e e of c y t o p l a s m , w e r e p r e p a r e d a c c o r d i n g to t h e m e t h o d of C h a u v e a u [9] a f t e r h o m o g e n e i z a t i o n of l i v e r s in 2.3 M s u c r o s e ; m i t o c h o n d r i a w e r e p u r i f i e d a c c o r d i n g to a t e c h n i q u e a l r e a d y d e s c r i b e d [10] ; -
Abbreviations used : dsRNA : double-stranded RNA. mtDNA, mtRNA : mitochondrial DNA, RNA. nDNA : nuclear DNA. SDS : sodium dodecyl sulfate. SSC : Standard Saline Citrate : 0.15 M NaC1. 0.015 M Sodium Citrate. HnRNA : heterogeneous nuclear RNA. mRNA : messenger RNA. ,~ To whom all correspondence should be addressed.
-
--purified mierosomes were separated from post-mitoehondrial fraction by recentrifugation at 1 2 0 0 0 0 × g for 90 rain of t h e s u p e r n a t a n t w h i c h w a s o b t a i n e d b y c e n t r i f u g a t i o n of l i v e r h o m o g e n a t e in 0.25 M s u c r o s e at 16 000 × g for 10 m i n .
L. Harel, G. Riou and L. Monlagnier.
228
c) Isolation of dsRNA : the method of isolation p r e v i o u s l y described [1, 2] was used in all experiments. F i n a l stage of p u r i f i c a t i o n involved pancreatic RNase t r e a t m e n t a n d filtration on Sephadex G 200 column. The void volume peak was c o n c e n t r a t e d b y e v a p o r a t i o n a n d dialysed against 1000 volumes of 0.1 X SSC.
- - Aliquots i n 1 ml 0.1 X SSC were placed in w a t e r b a t h at t e m p e r a t u r e v a r y i n g from 20 to 100°C for 10 rain, then fast cooled in ice bath. NaC1 5 M was added to make u,p the solution 0.3 M a n d each sample was treated w i t h 10 ~ g / m l of p a n c r e a t i c RNase for 30 rain at 20°C a n d TCA precipitated.
a) Extraction of mtDNA. The m i t o c h o n d r i a were lysed i n S.D.S. to a final c o n c e n t r a t i o n of 1 D. cent in TE buffer (0.01 Tris HC1, 0.001 M EDTA pH 7.5) for 15 m i n at room temperature. The lysate was treated 10 rain at 0°C w i t h 5 M NaC1 to obtain a fin,al c o n c e n t r a t i o n of 1.0 M a n d centrifuged at 16,000 × g i n Sorvall SS 34 rotor. The s u p e r n a t a n t was collected a n d dialysed against TE buffer a n d mIDNA purified in a CsCl-propidium d i i o d i d e (PDI) g r a d i e n t in the following c o n d i t i o n s : mtDNA in 3.3 ml TE b u f f e r ; CsC1 3.53 g r ; PDI (2.5 m g / m l ) 1.0 ml (500 t~g/ml f i n a l ) ; refractive i n d e x n 22°C == 1.3820; e e n t r i f u g a t i o n 24 hours 40 000 r p m at 20°C in a Beckman model L 2 B u ! t r a e e n t r i f u g e (SW 50 rotor). The lower b a n d was collected a n d PDI removed by p a s s i n g t h r o u g h a Dowex 50 resin column. The mtDNA was dialysed against 0.1 x SSC.
The percentage of dsRNA p r e s e n t i n the labelled RNA was estimated a c c o r d i n g to the f o r m u l a :
b) Separation of complementarg strands mtDNA.
The RNase resistance at high i o n i c strength of each prep.aratio,n a n d its m e l t i n g curve i n low i o n i c strength were d e t e r m i n e d i n the following way :
I
1'CAinsolublecountsof 1 t - the sampleheatedat Itt0oEand RNasetreated
x
'100
I'CI ius.hbb' countso[ the samplekept at 20oCand R~asetreated Only p r e p a r a t i o n s w h i c h c o n t a i n e d more than 80 p. cent of dsRNA a n d d i s p l a y e d a sharp m e l t i n g t r a n s i t i o n at a t e m p e r a t u r e above 60°C were used for experiments.
Closed c i r c u l a r mtDNA molecules were n i c k e d by exposure to visible light [20]. Nicked mtDNA was assayed fiuorimetrically in a Zeiss PMQIII fluorescence spectophotometer [21~. The complem e n t a r y strands of mtDNA were p r e p a r e d by cen-
2 °) Preparation of nuclear DNA. Purified n.uclei o b t a i n e d by the t e c h n i q u e of Chauveau [91 were s u s p e n d e d i n buffer saline EDTA (0.15 M NaC1, 0.1 M EDTA pH 8) + 1 p. cent SDS a n d extracted with o,ne volume of saturated phenol. Nucleic acids were p r e c i p i t a t e d from the aqueous phase by ethanol in the p r e s e n c e of 1 p. cent s o d i u m acetate. They were solu.bilized in 1 X SSC and extracted 3 times w i t h chloroform-isoamylie alcohol m i x t u r e a c c o r d i n g to the Marmur method. After p r e c i p i t a t i o n from the aqueous phase, they were treated w i t h RNase and p r o n a s e a c c o r d i n g to Gillespie a n d Spiegelm a n [H]. In e x p e r i m e n t s i n v o l v i n g high Co.t value h y b r i d i z a t i o n , n u c l e i c acids were not treated w i t h RNase, but w i t h 0.3 M KOH for 18 h o n r s at 37°C. After n e u t r a l i z a t i o n , the solm tions was dialysed for 24 h o u r s against 0.1 X SSC solution. 3 °) Preparation and purification of mtDNA. M i t o c h o n d r i a were isolated as p r e v i o u s l y described [12]. Since i n t a c t mtDNA could not be extracted from m i t o c h o n d r i a p r e t r e a t e d w i t h Dnase, this t r e a t m e n t was omitted.
BIOCHIMIE, 1975, 57, n ° 2.
of
H
0,2
A. ,,
.......
t I ,I t, 15
20
25 Fractions
Separation of heaog and light strands of mt DNA in alkaline caesium chloride gradient. FIG. 1.
-
-
trifugation in an alkaline CsC1 g r a d i e n t i n the following conditions : mtDNA i n 3,6 ml of 0.1 X SSC ; 0.4 ml 0.5 M Na3PO 4 ; 5.35 gr CsC1 ; p H 12.3 ; refractive i n d e x n 22°C = 1.4'035 ; c e n t r i f u g a t i o n for 60 hours at 35,000 r p m at 20°C in the r o t o r 50 of a Beckman ultracentrifuge. 0.5 ml of buffer 0.05 M NaH,,PO4 0.001 M EDTA pH 6.5 was added to each collected fraction (fig. 1). The refractive c o n c e n t r a t i o n s of the heavy and light mtDNA s t r a n d s were d e t e r m i n e d by m e a s u r i n g their opticM d e n s i t y at 260 n m (equivalence 1'0 ~ g / m l = 0.212 O.D. 260 unit) a n d a c o r r e c t i o n factor for h y p e r c h r o m i e i t y at 40 p. cent [13].
Rat liver double-stranded c) Analytical ultracentrifngation.
RNA.
229
centage of dsRNA increases, as p r e v i o u s l y s h o w n for dsRNA from entire cclls [1], especially i n the n u c l e a r a n d m i c r o s o m a l fraction. Only negligeable a m o u n t s of dsRNA were found to be associated w i t h the 100,000 X g s u p e r n a t a n t .
Co.ntrols for p u r i t y of the mtDNA in n e u t r a l CsCI g r a d i e n t and for s t r a n d s e p a r a t i o n in alkaline CsCI gradient (0.05 M NaaPO 4 0.05 M NaOH pH 12.5) were p e r f o r m e d in a B e c k m a n model E u l t r a c e n t r i f u g e (25°C - - 44770 r p m for 22 hours).
2 °) Characterization of m i t o c h o n d r i a l and nuclear dsRNA.
RESULTS.
a) H y b r i d i z a t i o n w i t h m i t o c h o n d r i a l and nuclear rat liver DNA.
1 °) Distribution of radioactive dsRNA in rat liver fractions. In p r e l i m i n a r y experiments, a rat liver homogenate made in 0.25 M sucrose was separated i n three fractions by differential centrifugation. 80 p. cent of the total r a d i o a c t i v i t y associated w i t h dsRNA was f o u n d in the pellet obtained b y c e n t r i f u g a t i o n at 16,000 × g for 10 rain. This pellet c o n t a i n e d nuclei, m i t o c h o n d r i a , a n d some u n b r o k e n cells. The r e m a i n i n g 20 p. cent of
E v i d e n c e that dsRNA associated w i t h the nfitoc h a n d r i a l fraction arises i n d e e d from t r a n s c r i p tion of mtDNA was given by its h y b r i d i z a t i o n with purified mtDNA separated into light (L) a n d heavy (It) strands. Owing to the small a m o u n t s of either pure mtDNA and dsRNA used, p r o p e r s a t u r a t i o n curves could not be obtained. However, as s h o w n in table II, it is clear that melted mt-dsRNA h y b r i -
TABLE I.
Subcellalar localization of dsRNA. Naclei Experiment n °
cpm
o 1-7
t
lI
p. cent total radi( activity in RNA
18551
0.20
Mitochondria
cpm
Microsomes
p. cent
cpm
p. cent
0.12
800
0.02t
512 2300
0.07 0.012
ill
1460 I 0 047
860
vt(*)
8987 I 0.51
624 I
o.2o
Supernatant 100,000 ~" cpm
201
p. cent
O.OOl
n~gligeable
0.09
(*) Rats treated with 0,2'80 mg/kg of Actinomycin D 1 hour before orotic acid injection. dsRNA was f o u n d associated w i t h microsomes obtained by r e c e n t r i f u g i n g at 120 000 g for 1 h r the s u p e r n a t a n t of the 16 000 × g centrifugation. I n subsequent experiments, the rat liver fractions were purified as described in Materials a n d Methods. Table I sho~-s the results of 4 experiments. It clearly appears that the m a j o r i t y of radioactive dsRNA is p r e s e n t i n both n u c l e i a n d mitochondria. This is true for the absolute n u m b e r of counts a n d also for the percentage of the total labelling of RNA in each fraction. W h e n rats are p r e t r e a t e d w i t h a c t i n o m y c i n D at a c o n c e n t r a t i o n k n o w n to i n h i b i t p r e f e r e n t i a l l y ribosomal RNA synthesis [14] the relative per-
BIOCHIMIE, 1975, 57, n* 2.
dizes to small a m o u n t s of mtDNA, the h y b r i d i zation percentage b e i n g h i g h e r for the light s t r a n d t h a n for the heavy strand. The percentage h y b r i dized w i t h s i m i l a r a m o u n t s of rat liver nDNA was in the range of unspecific h y b r i d i z a t i o n , as c o m p a r e d to E. colt DNA. Even w i t h 40 ,~g of rat liver nDNA, the h y b r i d i z a t i o n percentage was lower t h a n that o b t a i n e d w i t h 1,7 ag of mtDNA. I n a control experiment, dsRNA extracted from an i m p u r e n u c l e a r p r e p a r a t i o n (600 × g pellet of the same liver homogenate used for m i t o c h o n drial dsRNA p r e p a r a t i o n ) was h y b r i d i z e d to mt and nDNA (table III). C o m p a r i s o n of tables II and III shows that this dsRNA h y b r i d i z e s to a higher extent than mt-dsRNA with the same a m o u n t s of nDNA (40 ~g). Conversely, its p e r c e n 17
L. HareI, G. R i o u and L. Montagnier.
230
TArtLY. II.
Homology of mitochondrial dsRNA to mitochondrial DNA.
Experiment n*
IsRNA mt-
Mitochondrial DNA '
L Strand
E. coli
Nuclear DNA
control DNA
H Strand
added I - - [cpm) 9g added P" cent dsRNA p. cent dsRNA . . ~ Ip. cent dsRNA . . . . . Ip. cent dsRNA hybridized (') i~g added hybridized (*) - - l ~ gaaaea [ hybridized ('j ~g auueu hybridized (*)
I
700
II
860 860
0.6 ~ 0.8 1.7
860
11.0 (77) 9.3 (80) 11.2 (96) 14.1/121)
0.6
11.8 (83)
~
5.9 (51) 8.9 177)
,}.8 1.7
9.8
(84)
2.8 (20) 40 -~----
'
40
0.2 ( 2 ) 8.1 (70)
0.3 (2l 1.0 (9)
(*) In bracket, epm. tage of h y b r i d i z a t i o n w i t h m t D N A is l o w e r t h a n that observed with mt-dsRNA. The small percentage h y b r i d i z e d w i t h m t D N A m a y be e x p l a i n e d
It m a y be c o n c l u d e d t h a t one p a r t of d s R N A h y b r i d i z e s w i t h r e p e t i t i v e s e q u e n c e s of D N A a n d t h a t t h e o t h e r p a r t d o e s not.
TABLE III.
Hybridization of dsRNA from nuclear pellet. MitoehondriaI DNA Experiment no
I
II
~uelea IsRNA added (epm)
1000
E. coli
Nuclear DNA
control DNA
H Strand
L Strand
- - , , [p. cent dsR____NA ~g added p'cent dsRNA ~g added P' e,en.t,.ds~l~,.A ~g added p. eentdsRNA hybridized I') rtg aaaea hybridized (*) hybridized (') - - l l y n Z l g l Z e ~ [ ) _- - -_0.6 ~ - - -
2.7 (27) (*) 4.4 (70)
0.6
-%sU-,
3.0 (30)
40
14.3 (143)
40
3.0 (30)
(50,
(*) cpm. b y t h e c o n t a m i n a t i o n of t h e n u c l e a r p e l l e t b y l a r g e m i t o c h o n d r i a (cf. M a t e r i a l s a n d M e t h o d s ) . b) Homology of dsRNA with repetitive and non repetitive sequences of nuclear DNA. U n d e r c o n d i t i o n s in w h i c h o n l y h y b r i d i z a t i o n w i t h r e p e t i t i v e s e q u e n c e s of D N A c a n t a k e p l a c e , a b o u t 20 p. c e n t of m e l t e d n u c l e a r d s R N A f o r m R N a s e r e s i s t a n t c o m p l e x e s -with n u c l e a r DNA. T h e p o s s i b i l i t y w a s c o n s i d e r e d t h a t R N A self hybridization, which has already been reported to o c c u r Eli, m i g h t be a l i m i t i n g f a c t o r of t h e D N A - R N A h y b r i d i z a t i o n rate. T h e r e f o r e t w o e x p e r i m e n t s w e r e p e r f o r m e d , in w h i c h t h e n o n h y b r i d i z e d p a r t of n u c l e a r d s R N A (the M i l l i p o r e filtrate) w a s h e a t e d a g a i n at 100°C for 5 rain a n d r e i n e u b a t e d w i t h t h e s a m e a m o u n t of d e n a t u r e d r a t l i v e r D N A at 65°C. R e s u l t s in table IV s h o w that the second hybridization level was much l o w e r t h a n t h e first one.
BIOCHIM1E, 1975, 57, n ° 2.
For the latter fraction, the question was asked w h e t e r it c o u l d b e h y b r i d i z e d to n o n r e p e t i t i v e f r a c t i o n of D N A o,r w h e t h e r it w a s not h o m o l o g o u s at all to DNA. Three hybridization experiments were perf o r m e d in c o n d i t i o n s of h i g h Co.t v a l u e (Co.t = 7.5 × 103) a c c o r d i n g to. t h e m e t h o d of Melli a n d B i s h o p [1'5]. As sh(ywn in t a b l e V, t h e formatio.n of R N a s e r e s i s t a n t c o m p l e x e s r e a c h e d up to 88 p. c e n t of t h e i n p u t m e l t e d dsRNA, b u t n e a r l y s i m i l a r l e v e l s w e r e o b t a i n e d in u s i n g n o n - s p e c i f i c D N A (E. coli) i n s t e a d of rat l i v e r dsRNA. T h e p o s s i b i l i t y w a s c o n s i d e r e d that, in s u c h c o n d i tions, a f r a c t i o n of R N a s e r e s i s t a n t c o m p l e x e s a r o s e f r o m s e l f - h y b r i d i z a l i o n of R N A s t r a n d s [1]. E q u i l i b r i u m d e n s i t y a n a l y s i s in c a e s i u m s u l f a t e s h o w e d i n d e e d t h a t the t o t a l i t y of the R N a s e res i s t a n t m a t e r i a l b a n d e d at t h e d e n s i t y of dsRNA, w h e n E. coli D N A w a s p r e s e n t in t h e i n c u b a t i o n medium. When rat liver dsRNA was present,
R a t liver d o u b l e - s t r a n d e d R N A . a p p r o x i m a t e l y 60 p. c e n t of R N a s e r e s i s t a n t m a terial formed bands in the RNA region and the D N A - R N A h y b r i d r e g i o n , w h e r e a s 40 p. c e n t rem a i n e d i n t h e r e g i o n of R N A d u p l e x e s .
i m p o s s i b l e to o b t a i n a d e f i n i t e a n s w e r to t h e q u e s t i o n as t o w h e t h e r d s D N A is h o m o l o g o u s t o m o d e r a t e l y r e p e t i t i v e a n d u n i q u e s e q u e n c e s of n u c l e a r RNA, o r not.
TABLE I'V.
]OO
Incubation ds melted RNA added {cpm)
lI
P. cent hybridized
2d
1140 420
280 22
24 5 5
1st 9d
5000 1120
700 50
14 5
1st
I
RNA hybridized {cpm)
p .....
!
Homology of dsRNA with repetitive sequences of DNA. Exp.
231
I//
//
.
T h u s , i n o u r c o n d i t i o n s of h i g h Co.t v a l u e , t h e r e w a s a c o m p e t i t i o n b e t w e e n f o r m a t i o n of h y b r i d s a n d R N A - B N A self h y b r i d i z a t i o n . I t w a s t h e r e f o r e
20
60
80
lO0 12D Temperature rc)
Fit;. 2. - - Melting curve of nuclear ( © - - © ) and mitochondrial (e .... el. dsRNA in 0.1 × SSC (as described in
TABLE V.
Materials a n d Methods).
Search [or hybridization of dsRNA with non repetitive sequences of DNA. (See conditions of h y b r i d i z a t i o n in t h e text). cpm acid precipitable Exp.
I
II Ill
DNA
R a t liver
E. colt Rat liver R a t liver
E. colt
--RNase
+ RNase
1100 450
996 33O
88 75
670
43(') 42(')
670 1600
E. colt
P. cent RNase resistant
1550 1550
~ 1180 1100
76 71
(*) Material f u r t h e r analyzed in Caesium sulfate density gradient.
c) Melting curves of nuclear and mitochondrial dsRNAs. As s h o w n i n f i g u r e 2, n o l a r g e d i f f e r e n c e c o u l d b e o b s e r v e d i n t h e m e l t i n g c u r v e s of d s R N A s of nuclear and mitochondrial origin. This suggests t h a t t h e b a s e c o m p o s i t i o n s of b o t h d s R N A s d o n o t g r e a t l y differ. d) Polg A content : t h e p r e s e n c e of l o n g p o l y A s e q u e n c e w h i c h m i g h t b e a t t a c h e d to d s R N A , as i n m o s t of m e s s e n g e r RNAs, w a s i n v e s t i g a t e d b y passing the purified dsRNAs in oligo(dT) cellulose column.
TABLE VI.
Chromatography of rat liver nuclear and mitochondrial dsRNAs on oligo(dT)cellulose column. Origin
Fraction
Not heated
Heated (')
n-dsRNA
Filtrate 0.1M Tris pH 7.4, 0.5M KCl Eluate 0.1M Tris 0.1M KCI Eluate 0.1M Tris
71 p. ccnt 19 p. cent 9 p. cent
74 p. cent 13 p. cent 12 p. cent
mt-dsRNA
Filtrate 0.1M Tris p H 7.4, 0.5M KC1 Eluate 0.1M Tris 0.1M KC1 Eluate 0.1M Tris
84 p. cent 10 p. eent 6 p. cent
83 p. cent 7 p. cent 9 p. cent
(*) Heated 100°C 10 min in 0.1 × SSC, fast-cooled in ice b a t h , e t h a n o l precipit a t e d a n d redissolved in 0.I M TPis, 0.5 M KC1.
BIOCH1MIE, 1975, 57, n o 2.
L. Harel, G. Riou and L. Montagnier.
232
Control e x p e r i m e n t s (not sho~m) i n d i c a t e d that m o r e thant 70 p. cent of v a c c i n i a virus early mRNA w a s r e t a i n e d by the c o l u m n and eluted in 0.1 M Tris buffer. U n d e r the same conditions, the m a j o r i t y of either n u c l e a r or m i t o e h o n d r i a l dsRNA was not r e t a i n e d by the column, less t h a n 10 p. cent b ei n g eluted in 0.1 M Tris (table VI). S e p a r a t i o n by heat of dsRNA strands did not i n c r e a s e the binding, suggesting that t h e r e w e r e no poly A stretches h i d d e n by the s e c o n d a r y structure. DISCUSSION. An u n e x p e c t e d result of this w o r k ~ a s that a non-negligeable f r a c t i o n of rat l iv e r dsRNA is located in m i t o c h o n d r i a and is h o m o l o g o u s to p u r i f i e d IntDNA. P r e l i m i n a r y results suggest that a s i m i l a r situation also exists in c u l t u r e d cells and that synthesis of mt-dsRNA is s p e c i f i c a l l y i n h i b i t e d by e t h i d i u m b r o m i d e , as is the synthesis of the w h o l e mtRNA. T h e most likely e x p l a n a t i o n is that this dsRNA f r a c t i o n arises f r o m s y m e t r i c t r a n s c r i p t i o n of m i t o c h o n d r i a l DNA. In this respect, it is i n t e r e s t i n g to r e c a l l the w o r k of At t ard i [16], s h o w i n g that both strands of m i t o c h o n d r i a l DNA are t r a n s c r i b e d , the h e a v y strand b e i n g p r e f e r e n t i a l l y degraded. It is t h e r e f o r e c o n c e i v a b l e that mt-dsRNA is d e r i v e d f r o m the association of single c o m p l e m e n t a r y 13NA strands m a i n t a i n e d in close c o n t a c t by p r o t e i n s s h o rt l y after t h e i r synthesis. T h e ~ h o l e d u p l e x s t r u ct u r e w o u l d then be f o r m e d only after d e p r o t e i n i z a t i o n by p h e n o l e x tr a c t io n . Alternatively, as assumed e a r l i e r [:2], dsRNA loops m ay o ri g i n a te f r o m the t r a n s c r i p t i o n of a d j a c e n t c o m p l e m e n t a r y sequences on the same DNA strand. E v i d e n c e that such p a l i n d r o m i c se q u en ces exist in c h r o m o s o m a l DNA has r e c e n t l y been p u t f o r w a r d by W i l s o n and T h o m a s [17] but no data are available c o n c e r n i n g the o c c u r e n c e of s i m i l ar sequences in mtDNA. The results of Monckton and N o o r a E8] are at first sight in conflict w i t h ours, since these a u t h o r s f o u n d a ds!RNA e x c l u s i v e l y in the n u c l e a r f r a c t i o n of rat l i v e r and no dsRNA in the cytop l a s m i c fraction. H o w e v e r the latter was the supernatan.t r e m a i n i n g after c e n t r i f u g a t i o n at 20 000 × g. U n d e r these c o n d i t i o n s most of the m i t o c h o n d r i a should be in the pellet with nuclei. Moreover the time of labelling was s h o r t e r (60 m i n u t es i n s t e a d of 180 m i n u t e s in o u r expert-
BIOCHIMIE, 1975, 57, n ° 2.
ments) so that a dsRNA s y n t h e t i z e d in the nucleus m ay have not passed yet into the cytoplasm. Th e dsRNA that w e f o u n d associated w i t h the m i c r o s o m a l f r a c t i o n m ay i n d e e d h a v e a n u c l e a r origin. P r e l i m i n a r y e x p e r i m e n t s i n d i c a t e that it h y b r i d i z e s w i t h n u c l e a r DNA to the same extent than n u c l e a r dsRNA and it m a y t h e r e f o r e d e r i v e from the latter. THE
NATURE OF NUCLEAR
dsRNA.
Th e same r e a s o n i n g used above for the origin of m i t o c h o n d r i a l dsRNA m a y be a p p l i e d to nucl ear dsRN£. It could be f o r m e d by s y m c i r i c t r a n s c r i p t i o n of DNA strands and subsequent associ at i o n of the c o m p l e m e n t a r y RNA strands, or o r i g i n at e f r o m the t r a n s c r i p t i o n of p a l i n d r o m i c DNA. The r eg i o n s of p a l i n d r o m i c DNA isolated by Wilson and T h o m a s E171 are long enough to be t r a n s c r i b e d in a dsRNA h a v i n g the size that we Have p r e v i o u s l y r e p o r t e d (8-9 S in s e d i m e n t a t i o n ) . In contrast, it seems that our dsRNA m a t e r i a l differs f r o m that isolated in nuclei by two different groups [18, 19] ; the latter m a t e r i a l is shorter, since it w as not isolated by ex cl u si o n in Sephadex G 200, and r e p r e s e n t s i n t r a m o l e c u l a r h e l i c a l loops of HnRNA. Our o w n e x p e r i m e n t s i n d i c a t e that HnRNA isolated as an aggregate does not y i el d dsRNA e x c l u d e d in G 200 c o l u m n (L. Mont ag n i er and H. Collandre, in p r e p a r a t i o n ) . A t h i r d p o ssi b i l i t y w h i c h cannot be by the p r e s e n t w o r k , is that n u c l e a r and m i c dsRNAs r ef l ect the self-replication n u c l e a r FIN,As f o l l o w i n g t h e i r synthesis
excluded cytoplasof some on DNA.
Acknowledgments. We thank the able technical assistance of H. Collanclre, S. Chamaret, M. Gabillot and G. Frezoul. RfiSUM~. On a dtudi~ la l ocalisation intracellulaire du R NA bicat~naire du foie de rat. La majoritd de ce RNA est distribute clans les fractions nucldaircs et mitochondriales et la fraction restante est associ6e aux microsomes. Le RNA bicat~naire mitochondrial hybridise sp~cifiquemcnt avec les brins purifi~s de DNA: mitochondrial, et scmble donc provenir cle la transcription de ce dernier. Une fraction du RNA bicat~naire nucl~aire hybridise avec des s~quences rSpdtitives du DNA nucl~aire. Par suite de l'antohybridation d.e RNA, il n'a pas ~t~ possi,ble de d~terminer si une autre fraction est ~galement homologue de s~quences non rdpdtitives du DNA. Les deux types d~e RNA bicat~naire, mitochondrial et nucl4aire, ne sont pas retenus par des colonnes d'oligo (dT)~ceHulose et semble.nt donc ~tre ddpourvus de sSquence poly A longues.
Rat
liver double-stranded
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