49
Biochimica et Biophysica Acta, 425 (1976) 49--62 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98522 BASE COMPOSITION STUDIES ON MITOCHONDRIAL 4 S R N A F R O M RAT L I V E R AND MORRIS HEPATOMAS 5 1 2 3 D AND 7777
LI-LI S.Y. CHIA a, H A R O L D P. M O R R I S b K U R T R A N D E R A T H and E R I K A R A N D E R A T H a,,
a
a Department of Pharmacology, Baylor College of Medicine, Texas Medical Center, Houston, Texas 77025 and b Department of Biochemistry, Howard University, School of Medicine, Wash., D.C. 20001 (U.S.A.) (Received August 19th, 1975)
Summary The major and modified base composition of mitochondrial 4 S R N A from rat liver and from Morris hepatomas 5123D and 7777 has been determined for 16 constituents using a chemical tritium-derivative method. The base composition of these mitochondrial 4 S R N A preparations was compared with the base composition of cytoplasmic and bacterial (Escherichia coli B and Bacillus subtilis) 4-S RNAs. The results of these studies are: 1. When compared with cytoplasmic 4 S RNA, the liver and hepatoma mitochondrial 4-S RNAs are, characterized by high (A ~ U)/(G + C) ratios and low overall degrees of base methylation and modification. 2. The mammalian mitochondrial 4-S RNAs are qualitatively even more different from the bacterial 4.S RNAs than from their cytoplasmic counterparts. Thus, several modified constituents found in b o t h cytoplasmic and mitochondrial 4 S R N A are absent from the bacterial 4-S RNAs. 3. Mitochondrial 4 S R N A from both hepatomas was found to be undermethylated and undermodified when compared with normal liver mitochondrial 4 S RNA. This trend is more pronounced for the rapidly growing hepatoma 7777 (i.e., 17% undermethylation) than for the more slowly growing hepat o m a 5123D (i.e., 8% undermethylation). These findings are discussed in relationship to (1) results of other authors on composition of mitochondrial 4 S RNA, (2) special features of structure and
* T o w h o m r e q u e s t s for reprints s h o u l d b e addressed.
A b b r e v i a t i o n s u s e d for n u c l e o s i d a s arc as r e c o m m e n d e d b y t h e I U P A C - I U B C o m m i s s i o n o f B i o c h e m i c a l N o m e n c l a t u r e ( B i o c h i m . B i o p h y s . A c t a ( 1 9 7 1 ) 24"/, 1). X = 3 - ( 3 - a m i n o - 3 - c a r b o x y p r o p y l ) u r i d i n e ; V = u r l d i n e - 5 - o x y a c e t i c acid.
50 biosynthesis of mitochondrial 4 S RNA, (3) the possible evolutionary origin of mitochondria and (4) the possible role played by aberrant mitochondrial 4 S RNA in altered mitochondrial protein synthesis in tumors.
Introduction
There is now ample evidence that mitochondria of various organisms possess their own specific transfer RNAs, which are coded for by mitochondrial DNA and participate in mitochondrial protein synthesis (for reviews, see refs. 1--5). Mitochondrial tRNA of cultured hamster cells [6,7] and HeLa cells [8,9] has been shown to have a relatively high A +.U content and to be considerably less methylated than the respective cytoplasmic tRNA. At present however, no data are available on the base composition of mitochondrial tRNA in intact mammalian organs or in tumors. We have therefore determined the base composition of mitochondrial 4 S RNA from rat liver and from Morris hepatomas 5123D and 7777 using a chemical tritium-derivative method for base analysis of small amounts of unlabeled RNA developed in our laboratory [10--12]. Rat liver cytoplasmic 4 S RNA and 2 bacterial 4-S RNAs have also been analyzed and compared with liver mitochondrial 4 S RNA. In terms of its base composition, rat liver mitochondrial 4 S RNA was found to resemble mitochondrial 4 S RNA from the cultured cells [6--9]. The content of some modified nucleosides that until now had not been determined in mammalian mitochondrial 4 S RNA will be reported. The 2 tumor mitochondrial 4-S RNAs, although being relatively similar to liver mitochondrial 4 S RNA, were found to exhibit certain common alterations of the modified base composition. Materials and Methods Materials. Escherichia coli B tRNA and Bacillus subtilis tRNA (General Biochemicals, Chagrin Falls, Ohio 44022) were purified by polyacrylamide gel electrophoresis (see below). Liver cytoplasmic and mitochondrial 4 S RNA was prepared from Buffalo rats. Morris hepatomas 5123D and 7777 were originally induced chemically and kept by continuous intramuscular transplantation in female Buffalo rats [13]. These hepatomas have growth rates of 5.0 cm/month (7777) and 3.7 cm/month (5123D) and are histologically classified as "poorly differentiated" and "intermediate between well differentiated and poorly differentiated", respectively [14]. Materials used for base analysis of RNA have been described elsewhere [10--12]. Cellulose sheets were mainly Bakerflex 4468, batch 13, and in some experiments Eastman Chromagram 6064, batch 5273 (Figs. 1 and 2) *
* Several r e c e n t b a t c h e s o f b o t h m a t e r i a l s w e r e f o u n d t o give e l o n g a t e d , d i f f u s e , and d i s t o r t e d s p o t s . A c c o r d i n g t o o u r e x p e r i e n c e the s h a r p e s t r e s o l u t i o n o f t h e n u c l e o s i d e derivatives has c o n s i s t e n t l y b e e n o b t a i n e d o n E. M e r c k c e l l u l o s e s h e e t s N o . 5 5 0 2 ( w i t h o u t f l u o r e s c e n t i n d i c a t o r ) supplied b y EM L a b o r a t o r i e s , E l m s f o r d , N.Y. 1 0 5 2 8 . T h e s o l v e n t for t h e first d i m e n s i o n o n t h e s e s h e e t s w a s as d e s c r i b e d in ref. 15.
51
Preparation o f liver cytoplasmic 4 S RNA. Liver cytoplasmic 4 S RNA was prepared from a . p H 5 precipitate as detailed previously [16]. Purified RNA was obtained by polyacrylamide gel electrophoresis (see below). Preparation o f liver and tumor mitochondrial 4 S RNA. Mitochondria were prepared from 10--20 g of tissue essentially as described by Malkin [17]. Contaminating cytoplasmic ribosomes were removed by digitonin treatment at a concentration of 1 mg digitonin/100 mg mitochondrial protein [17,18]. Recovery of mitochondrial protein was 19.5 mg/g for liver, 9 mg/g for Morris hepatoma 5123D, and 4.1 mg/g for Morris hepatoma 7777. Nucleic acids were prepared from mitochondria by extraction with 80% phenol containing 0.1% 8-hydroxyquinoline and subsequent precipitation from the aqueous phase by the addition of an equal volume of isopropanol. 4 S RNA was isolated by polyacrylamide gel electrophoresis (see below). Purification of 4 S R N A by polyacrylamide gel electrophoresis. For purification, the crude nucleic acid preparations were subjected to polyacrylamide gel electrophoresis [16]. The 4 S RNA band was excised from the gel and extracted by homogenization and phenol treatment as described previously [ 16]. The recovery of mitochondrial 4 S RNA per mg of mitochondrial protein was about 0.8 pg for liver, 1.5 pg for Morris hepatoma 5123D, and 1.0 gg for Morris hepatoma 7777. These values have been corrected for about 20% loss occurring during the isolation of the RNA from the gel. Base analysis o f R N A by tritium labeling. The base constituency of 4 S RNA was determined by the tritium derivative method previously described in detail [10--12]. The method entails enzymatic digestion of RNA to nucleosides, treatment with NaIO4 and KB3H4, two-dimensional thin-layer chromatography on cellulose and film detection by fluorography [19] of 3H-labeled nucleoside trialcohols, followed by liquid scintillation counting of individual derivatives. Several replicate analyses were carried out for each labeled digest. Base compositions were calculated from the count rates of the radioactive derivatives [10--12]. Standard deviations were in the same range as reported earlier for the base composition of 4 S RNA from various sources [12,20], except as indicated in footnote g of Table II. Results
Isolation o f 4 S R N A Mitochondria were isolated from liver and Morris hepatomas 5123D and 7777 by a procedure entailing differential centrifugation and digitonin treatment [17,18], which had been shown to yield mitochondria free of cytoplasmic ribosomes but with an intact outer membrane as judged by the respiratory control ratio and adenylate kinase activity [17]. The digitonin procedure was chosen because the removal of contaminating cytoplasmic ribosomes should also result in the elimination from the mitochondria of extramitochondrial 4 S RNA bound to endoplasmic reticulum. As judged by polyacrylamide gel electrophoresis, a small amount of 4 S RNA corresponding to 10--15% of 4 S RNA isolated from the purified mitochondria was present in the supernatant solution after the digitonin treatment in addition to various RNAs of higher molecular
52 weight. All 4 S RNA preparations to be compared were freed from contaminating RNAs by polyacrylamide gel electrophoresis and subsequent isolation from the gel [16,20]. In no case was background staining with methylene blue detectable on the gels, indicating that the RNA preparations were free of R N A degradation products originating from nuclease action. Thus, there were no degradation products of high molecular weight RNA present in the 4 S R N A bands. Morris hepatomas 5123D and 7777 appear to be particularly suited for comparative investigations of t R N A because intact RNA can be easily obtained from these tumors as well as from liver whereas, even under the strictest precautions, we were unable to obtain undegraded R N A from certain other Morris hepatomas [20]. Our recovery value of a b o u t 20 mg mitochondrial protein/g liver is in agreement with a value reported for liver mit0chondria [21]. Recovery of mito-
Fig. 1. F l u o r o g m m [19] of 3H-labeled digest of rat liver m l t o c h o n d r i a l 4 S RNA c hroma t ogra phe d on cellulose [ 1 0 , 1 1 ] . N', a nucleoside trlalcohol, t6A, N-(9-(~D-Ribofuranosyl)-purln-6-yl-carbamoyl)threonine (not analyzed quantitatively); Y! and Y2, u n k n o w n labeled produc t s originating from the RNA; ~-D, decomposition product of ~ ; gly, glycerol; B, labeled c o m p o u n d s not derived from t he RNA.
53 chondrial protein was found to be lower for the tumor tissues than for liver and also to correlate negatively with the degree of malignancy of the tumors, being about 45% for Morris hepatoma 5123D and about 20% for Morris hepatoma 7777 when compared with liver. Recovery of mitochondrial 4 S RNA per weight of mitochondrial protein was found to be higher for both tumors than for liver (see Materials and Methods). The recovery of mitochondrial 4 S RNA of about 12 ttg/g liver corresponds to about 2.5% of the total 4 S RNA.
Comparison between base composition o f liver mitochondrial and cytoplasmic 4 S RNA Figs. 1 and 2 show fiuorograms [19] of 2-dimensional thin-layer chromatograms of 3H-labeled nucleoside trialcohols [10--12] obtained from enzymatic digests of mitochondrial and cytoplasmic 4 S RNA of rat liver, respectively. All compounds derived from cytoplasmic 4 S RNA are also present in the labeled digest of mitochondrial 4 S RNA. However, intensities of individual spots differ
Fig. 2. F l u o r o g r a m o f 3 H o l a b e l e d d i g e s t o f rat liver c y t o p l a s m i c 4 S R N A c h r o m a t o g r a p h e d o n cellulose. F o r c o m p o u n d s , c o n s u l t l e g e n d t o Fig. 1.
54 considerably between the two R N A preparations. In particular, intensities of nucleoside trialcohols of guanine and all modified constituents are weak for mitochondrial 4 S RNA when compared with cytoplasmic 4 S RNA. However, mitochondrial 4 S RNA was found to give rise to two additional 3H-labeled derivatives (Y 1 and Y 2, see Fig. 1) not present in the labeled digest of cytoplasmic 4 S RNA. By following the time course of the enzymatic digestion of mitochondrial 4 S RNA we have established that these 2 additional labeled c o m p o u n d s originate from the RNA rather than from impurities (data not shown). These c o m p o u n d s may represent trialcohols derived from u n k n o w n modified nucleosides with highly hydrophilic properties. Alternatively, but less likely, they may be derived from short mitochondria-specific oligonucleotides resistant to enzymatic degradation by a mixture of RNAase A, snake venom phosphodiesterase, and alkaline phosphatase [10--12]. In Table I {columns 1--3), total base compositions of 4 S RNA from rat liver mitochondria and cytoplasm have been compared. Sixteen constituents were analyzed as trialcohol derivatives by the tritium derivative method [10--12] including most base-modified and all base-methylated nucleosides known to occur in mammalian tRNA. As can be seen from Table I, the 2 R N A populations differ in all 16 constituents assayed. The differences are statistically highly significant. The mitochondrial 4 S R N A is characterized by a particularly high A and U content and a relatively low degree of modification and methylation when compared with cytoplasmic 4 S RNA, the amounts of A and U being a b o u t 50% higher and the degrees of modification and methylation more than 50% lower for the mitochondrial 4 S RNA. G and C are a b o u t 25% and 12% lower, respectively, in the mitochondrial 4 S RNA. The high proportion of A and U in the mitochondrial RNA is also reflected by the ratios (A + U)/(G + C) and ( A t o t a 1 + Vtotal)/(Gtota 1 + C t o t a l ) , which respectively are a b o u t 1.9 and 1.7 times higher for the mitochondrial 4 S RNA. The proportions of all modified nucleosides are diminished in the mitochondrial R N A when compared with the cytoplasmic RNA, b u t to a varying degree, ranging from a b o u t 20% for m~A to a b o u t 70% for m SU, with the other values falling in between. The amounts of seven modified constituents (mSU, hU, m3C, mSC, I, mTG, and X) were found to be reduced by > 5 0 % when compared with the values for cytoplasmic 4 S RNA {Table I). Different 4 S RNA preparations from the same source were found to give essentially the same results. This was shown previously for various cytoplasmic 4 S RNA preparations [20]. The base composition of different mitochondrial 4 S RNA preparations also varies only slightly, deviations from the values of Table I, column 1, being in a range of a b o u t + 5%. Comparison between base composition o f bacterial 4 S R N A and liver mitochondrial and cytoplasmic 4 S R N A In Table I the base composition of E. coli and B. subtilis 4 S R N A is compared with that of liver mitochondrial and cytoplasmic 4 S RNA. The 2 bacterial 4-S RNAs are relatively more similar to each other than to liver cytoplasmic and mitochondrial 4 S RNA. If the base compositions of all 4 R N A populations in Table I are compared it is apparent that the greatest differences exist between liver mitochondrial 4 S R N A and the bacterial RNAs. Both bacterial
55 TABLE I T O T A L BASE C O M P O S I T I O N OF 4 S RNA. C O M P A R I S O N OF R A T L I V E R M I T O C H O N D R I A L 4 S RNA WITH CYTOPLASMIC AND BACTERIAL 4 S RNA 4 S R N A w a s p r e p a r e d as d e s c r i b e d in t h e t e x t . Base a n a l y s i s w a s c a r r i e d o u t b y c h e m i c a l t r i t i u m derivat i z a t i o n o f n u c l e o s i d e s in e n z y m a t i c digests [ 1 0 - - 1 2 ] . Base c o m p o s i t i o n d a t a w e r e c a l c u l a t e d f r o m c o u n t r a t e s o f t r i t i u m - l a b e l e d n u c l e o s i d e t r i a l c o h o l s o b t a i n e d f r o m several r e p l i c a t e c h r o m a t o g r a p h i c a n a l y s e s of e a c h labeled digest. Nucleoside
(1) R a t liver mitochondria
(2) R a t liver cytoplasm
X(1) (%)
X(2) (%)
21.05 26.90 22.63 20.58 1.03
13.98 17.28 25.72 27.62 . 1.32
0.001
mSU hU
0.19 1.20
0.61 2.99
0.001 0.001
m]G m2G mlG m3C c
0.38 0.98 0.54 0.14
0.62 1.38 0.77 0.35
0.001 0.001 0.001 0.001
mSC
1.01
2.27
I mTG d
2.80 0.14 0.28
3.77 0.31 0.65
U A C G m6A mlA b
.
X
.
0.17
V m2A
. .
Total
100.00
(ZNMelT, N T) X 1 0 0 e (~=NMo/ZN T) X 1 0 0 f (A + U ) I ( G + C) (A T + U T ) / ( G T + C T ) g
1.15
. .
(4) (1)/(2)
(5)
(6)
E. coli B
B. subtilis
X(1)/X(2)
2 ( 5 ) (%)
X(8) (%)
1.51 1.57 0.88 0.74 0.78
14.77 18.87 29.48 28.79 0.12 --
18.52 18.39 28.92 29.47 0.19 0.37
0.31 0.40
1.25 2.32
1.26 1.89
0.58 0.71 0.70 0.40
--0.19 --
--0.24 --
0.001
0.44
--
--
0.001 0.001 0.001
0.74 0.45 0.43
2.47 0.11 0.81
1.84 0.10 0.69
0.001
0.46
0.29
0.10
0.001 0.001 0.001 0.001 .
0.37 . .
4.53% 8.84% 1.11
(3) P(1),(2) a
. .
0.22 0.30
100.01
99.99
99.98
7.97% 15.41% 0.59
0.001 0.001 0.001
0.57 0.57 1.88
2.67% 8.08% 0.58
2.75% 6.68% 0.60
0.68
0.001
1.69
0.69
0.69
a P r o b a b i l i t y values e s t i m a t e d o n t h e basis of S t u d e n t ' s t test: FAT1 N 2 ( N I _ + N 2 - - 2
t ~ t_
! I 1/2 X
N , + N~
~I --~2
,/N,d
+ N~s~
= m e a n of 4 a n a l y s e s ( N = 4)
b
c d e f g
s = s t a n d a r d d e v i a t i o n = ~/}=(x - - ~ ) 2 / ( N - 1) P values f o r t h e c o m p a r i s o n of t h e b a c t e r i a l 4-S R N A s w i t h e a c h o t h e r a n d w i t h t h e liver s a m p l e s are n o t listed. G e n e r a l l y , a d i f f e r e n c e w a s f o u n d to b e statistically significant (P ~ 0 . 0 5 ) if t h e r a t i o o f t h e 2 c o n s t i t u e n t s c o m p a r e d d i f f e r e d b y m o r e t h a n +-10% f r o m 1 f o r t h e m o d i f i e d n u c l e o s i d e s or b y m o r e t h a n -+5% for t h e m a j o r n u c l e o s i d e s . T h e v a l u e o f m 1A w a s o b t a i n e d f r o m t h e s u m o f t h e c o u n t r a t e s of t h e 3 H - l a b e l e d t r i a l c o b o l s o f m I A a n d m 6 A [ 1 1 ] . I n t h e case of B. subtilis 4 S R N A , t h e c a l c u l a t i o n of t h e v a l u e s for m 1A a n d m 6 A is b a s e d o n a 20% c o n v e r s i o n of m 1A t o m 6 A [ 1 1 ] . C o r r e c t e d f o r 85% r e c o v e r y [ 1 1 ] . C o r r e c t e d for 65% r e c o v e r y [ 1 1 ] . Z Base m e t h y l a t e d nucleosides/~: T o t a l n u c l e o s i d e s ~ d e g r e e o f base m e t h y l a t i o n . ~: M o d i f i e d nucleo6ides/]~ T o t a l n u c l e o s i d e s ~ d e g r e e o f m o d i f i c a t i o n . ( A t o t a l + U t o t a l ) / ( G t o t a l + C t o t a l ) ; A t o t a1 = A + ~ : A m o d i f i e d etc.
56 RNAs do not contain m2G, m~G, m3C, and mSC. In addition, E. coli B 4 S RNA lacks m ~A. These components are present in liver mitochondrial and cytoplasmic 4 S RNA. Both bacterial RNAs contain m 6A, which is not present in mitochondrial and cytoplasmic 4 S RNA. Two constituents, i.e., uridine5-oxyacetic acid and m 2 A, are unique to E. coli 4 S RNA. Whereas the proportions of the major constituents in the bacterial 4-S RNAs resemble those of the liver cytoplasmic 4 S RNA, they are very different from those of mitochondrial 4 S RNA. The high content of A and U and the high ratio of ( A t o t a 1 + V t o t a l ) / ( G t o t a I + Ctotal) are unique to mitochondrial 4 S RNA. The mSU content, which is highest in the bacterial RNAs and intermediate in liver cytoplasmic RNA, is very low in the liver mitochondrial 4 S R N A [22]. m 7G is also low in the mitochondrial RNA. On the other hand, m I G is low in the bacterial RNAs, intermediate in the mitochondrial RNA, and high in the cytoplasmic RNA. It is interesting to note that in the bacterial RNA m2G and m~G are absent and m~G is low b u t mTG is rather high. Of the 4 RNA populations compared in Table I, cytoplasmic 4 S RNA has the highest degree of modification and base methylation. The only apparent c o m m o n characteristic of liver mitochondrial 4 S RNA and the bacterial RNAs is a similar overall degree of modification, which is a b o u t 2-times lower than that of liver cytoplasmic 4 S RNA. The degree of base methylation of the mitochondrial 4 S RNA is almost twice as high as that of the bacterial 4-S RNAs but only a b o u t half that of the cytoplasmic 4 S RNA. Comparison between mitochondrial 4 S R N A o f liver and hepatomas 5123D and 7777 The total base composition of mitochondrial 4 S R N A from both hepatomas was found to resemble that o f liver mitochondrial 4 S R N A (data not shown). Regarding the major base compositions, the 4 S R N A from hepatoma 5123D mitochondria contained somewhat more A and U b u t less G and C than the corresponding liver R N A preparation whereas the opposite was found to be true for hepatoma 7777 mitochondrial 4 S RNA. In each case, deviations for the individual major nucleosides were
Biochimica et Biophysica Acta, 425 (1976) 49--62 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98522 BASE COMPOSITION STUDIES ON MITOCHONDRIAL 4 S R N A F R O M RAT L I V E R AND MORRIS HEPATOMAS 5 1 2 3 D AND 7777
LI-LI S.Y. CHIA a, H A R O L D P. M O R R I S b K U R T R A N D E R A T H and E R I K A R A N D E R A T H a,,
a
a Department of Pharmacology, Baylor College of Medicine, Texas Medical Center, Houston, Texas 77025 and b Department of Biochemistry, Howard University, School of Medicine, Wash., D.C. 20001 (U.S.A.) (Received August 19th, 1975)
Summary The major and modified base composition of mitochondrial 4 S R N A from rat liver and from Morris hepatomas 5123D and 7777 has been determined for 16 constituents using a chemical tritium-derivative method. The base composition of these mitochondrial 4 S R N A preparations was compared with the base composition of cytoplasmic and bacterial (Escherichia coli B and Bacillus subtilis) 4-S RNAs. The results of these studies are: 1. When compared with cytoplasmic 4 S RNA, the liver and hepatoma mitochondrial 4-S RNAs are, characterized by high (A ~ U)/(G + C) ratios and low overall degrees of base methylation and modification. 2. The mammalian mitochondrial 4-S RNAs are qualitatively even more different from the bacterial 4.S RNAs than from their cytoplasmic counterparts. Thus, several modified constituents found in b o t h cytoplasmic and mitochondrial 4 S R N A are absent from the bacterial 4-S RNAs. 3. Mitochondrial 4 S R N A from both hepatomas was found to be undermethylated and undermodified when compared with normal liver mitochondrial 4 S RNA. This trend is more pronounced for the rapidly growing hepatoma 7777 (i.e., 17% undermethylation) than for the more slowly growing hepat o m a 5123D (i.e., 8% undermethylation). These findings are discussed in relationship to (1) results of other authors on composition of mitochondrial 4 S RNA, (2) special features of structure and
* T o w h o m r e q u e s t s for reprints s h o u l d b e addressed.
A b b r e v i a t i o n s u s e d for n u c l e o s i d a s arc as r e c o m m e n d e d b y t h e I U P A C - I U B C o m m i s s i o n o f B i o c h e m i c a l N o m e n c l a t u r e ( B i o c h i m . B i o p h y s . A c t a ( 1 9 7 1 ) 24"/, 1). X = 3 - ( 3 - a m i n o - 3 - c a r b o x y p r o p y l ) u r i d i n e ; V = u r l d i n e - 5 - o x y a c e t i c acid.
50 biosynthesis of mitochondrial 4 S RNA, (3) the possible evolutionary origin of mitochondria and (4) the possible role played by aberrant mitochondrial 4 S RNA in altered mitochondrial protein synthesis in tumors.
Introduction
There is now ample evidence that mitochondria of various organisms possess their own specific transfer RNAs, which are coded for by mitochondrial DNA and participate in mitochondrial protein synthesis (for reviews, see refs. 1--5). Mitochondrial tRNA of cultured hamster cells [6,7] and HeLa cells [8,9] has been shown to have a relatively high A +.U content and to be considerably less methylated than the respective cytoplasmic tRNA. At present however, no data are available on the base composition of mitochondrial tRNA in intact mammalian organs or in tumors. We have therefore determined the base composition of mitochondrial 4 S RNA from rat liver and from Morris hepatomas 5123D and 7777 using a chemical tritium-derivative method for base analysis of small amounts of unlabeled RNA developed in our laboratory [10--12]. Rat liver cytoplasmic 4 S RNA and 2 bacterial 4-S RNAs have also been analyzed and compared with liver mitochondrial 4 S RNA. In terms of its base composition, rat liver mitochondrial 4 S RNA was found to resemble mitochondrial 4 S RNA from the cultured cells [6--9]. The content of some modified nucleosides that until now had not been determined in mammalian mitochondrial 4 S RNA will be reported. The 2 tumor mitochondrial 4-S RNAs, although being relatively similar to liver mitochondrial 4 S RNA, were found to exhibit certain common alterations of the modified base composition. Materials and Methods Materials. Escherichia coli B tRNA and Bacillus subtilis tRNA (General Biochemicals, Chagrin Falls, Ohio 44022) were purified by polyacrylamide gel electrophoresis (see below). Liver cytoplasmic and mitochondrial 4 S RNA was prepared from Buffalo rats. Morris hepatomas 5123D and 7777 were originally induced chemically and kept by continuous intramuscular transplantation in female Buffalo rats [13]. These hepatomas have growth rates of 5.0 cm/month (7777) and 3.7 cm/month (5123D) and are histologically classified as "poorly differentiated" and "intermediate between well differentiated and poorly differentiated", respectively [14]. Materials used for base analysis of RNA have been described elsewhere [10--12]. Cellulose sheets were mainly Bakerflex 4468, batch 13, and in some experiments Eastman Chromagram 6064, batch 5273 (Figs. 1 and 2) *
* Several r e c e n t b a t c h e s o f b o t h m a t e r i a l s w e r e f o u n d t o give e l o n g a t e d , d i f f u s e , and d i s t o r t e d s p o t s . A c c o r d i n g t o o u r e x p e r i e n c e the s h a r p e s t r e s o l u t i o n o f t h e n u c l e o s i d e derivatives has c o n s i s t e n t l y b e e n o b t a i n e d o n E. M e r c k c e l l u l o s e s h e e t s N o . 5 5 0 2 ( w i t h o u t f l u o r e s c e n t i n d i c a t o r ) supplied b y EM L a b o r a t o r i e s , E l m s f o r d , N.Y. 1 0 5 2 8 . T h e s o l v e n t for t h e first d i m e n s i o n o n t h e s e s h e e t s w a s as d e s c r i b e d in ref. 15.
51
Preparation o f liver cytoplasmic 4 S RNA. Liver cytoplasmic 4 S RNA was prepared from a . p H 5 precipitate as detailed previously [16]. Purified RNA was obtained by polyacrylamide gel electrophoresis (see below). Preparation o f liver and tumor mitochondrial 4 S RNA. Mitochondria were prepared from 10--20 g of tissue essentially as described by Malkin [17]. Contaminating cytoplasmic ribosomes were removed by digitonin treatment at a concentration of 1 mg digitonin/100 mg mitochondrial protein [17,18]. Recovery of mitochondrial protein was 19.5 mg/g for liver, 9 mg/g for Morris hepatoma 5123D, and 4.1 mg/g for Morris hepatoma 7777. Nucleic acids were prepared from mitochondria by extraction with 80% phenol containing 0.1% 8-hydroxyquinoline and subsequent precipitation from the aqueous phase by the addition of an equal volume of isopropanol. 4 S RNA was isolated by polyacrylamide gel electrophoresis (see below). Purification of 4 S R N A by polyacrylamide gel electrophoresis. For purification, the crude nucleic acid preparations were subjected to polyacrylamide gel electrophoresis [16]. The 4 S RNA band was excised from the gel and extracted by homogenization and phenol treatment as described previously [ 16]. The recovery of mitochondrial 4 S RNA per mg of mitochondrial protein was about 0.8 pg for liver, 1.5 pg for Morris hepatoma 5123D, and 1.0 gg for Morris hepatoma 7777. These values have been corrected for about 20% loss occurring during the isolation of the RNA from the gel. Base analysis o f R N A by tritium labeling. The base constituency of 4 S RNA was determined by the tritium derivative method previously described in detail [10--12]. The method entails enzymatic digestion of RNA to nucleosides, treatment with NaIO4 and KB3H4, two-dimensional thin-layer chromatography on cellulose and film detection by fluorography [19] of 3H-labeled nucleoside trialcohols, followed by liquid scintillation counting of individual derivatives. Several replicate analyses were carried out for each labeled digest. Base compositions were calculated from the count rates of the radioactive derivatives [10--12]. Standard deviations were in the same range as reported earlier for the base composition of 4 S RNA from various sources [12,20], except as indicated in footnote g of Table II. Results
Isolation o f 4 S R N A Mitochondria were isolated from liver and Morris hepatomas 5123D and 7777 by a procedure entailing differential centrifugation and digitonin treatment [17,18], which had been shown to yield mitochondria free of cytoplasmic ribosomes but with an intact outer membrane as judged by the respiratory control ratio and adenylate kinase activity [17]. The digitonin procedure was chosen because the removal of contaminating cytoplasmic ribosomes should also result in the elimination from the mitochondria of extramitochondrial 4 S RNA bound to endoplasmic reticulum. As judged by polyacrylamide gel electrophoresis, a small amount of 4 S RNA corresponding to 10--15% of 4 S RNA isolated from the purified mitochondria was present in the supernatant solution after the digitonin treatment in addition to various RNAs of higher molecular
52 weight. All 4 S RNA preparations to be compared were freed from contaminating RNAs by polyacrylamide gel electrophoresis and subsequent isolation from the gel [16,20]. In no case was background staining with methylene blue detectable on the gels, indicating that the RNA preparations were free of R N A degradation products originating from nuclease action. Thus, there were no degradation products of high molecular weight RNA present in the 4 S R N A bands. Morris hepatomas 5123D and 7777 appear to be particularly suited for comparative investigations of t R N A because intact RNA can be easily obtained from these tumors as well as from liver whereas, even under the strictest precautions, we were unable to obtain undegraded R N A from certain other Morris hepatomas [20]. Our recovery value of a b o u t 20 mg mitochondrial protein/g liver is in agreement with a value reported for liver mit0chondria [21]. Recovery of mito-
Fig. 1. F l u o r o g m m [19] of 3H-labeled digest of rat liver m l t o c h o n d r i a l 4 S RNA c hroma t ogra phe d on cellulose [ 1 0 , 1 1 ] . N', a nucleoside trlalcohol, t6A, N-(9-(~D-Ribofuranosyl)-purln-6-yl-carbamoyl)threonine (not analyzed quantitatively); Y! and Y2, u n k n o w n labeled produc t s originating from the RNA; ~-D, decomposition product of ~ ; gly, glycerol; B, labeled c o m p o u n d s not derived from t he RNA.
53 chondrial protein was found to be lower for the tumor tissues than for liver and also to correlate negatively with the degree of malignancy of the tumors, being about 45% for Morris hepatoma 5123D and about 20% for Morris hepatoma 7777 when compared with liver. Recovery of mitochondrial 4 S RNA per weight of mitochondrial protein was found to be higher for both tumors than for liver (see Materials and Methods). The recovery of mitochondrial 4 S RNA of about 12 ttg/g liver corresponds to about 2.5% of the total 4 S RNA.
Comparison between base composition o f liver mitochondrial and cytoplasmic 4 S RNA Figs. 1 and 2 show fiuorograms [19] of 2-dimensional thin-layer chromatograms of 3H-labeled nucleoside trialcohols [10--12] obtained from enzymatic digests of mitochondrial and cytoplasmic 4 S RNA of rat liver, respectively. All compounds derived from cytoplasmic 4 S RNA are also present in the labeled digest of mitochondrial 4 S RNA. However, intensities of individual spots differ
Fig. 2. F l u o r o g r a m o f 3 H o l a b e l e d d i g e s t o f rat liver c y t o p l a s m i c 4 S R N A c h r o m a t o g r a p h e d o n cellulose. F o r c o m p o u n d s , c o n s u l t l e g e n d t o Fig. 1.
54 considerably between the two R N A preparations. In particular, intensities of nucleoside trialcohols of guanine and all modified constituents are weak for mitochondrial 4 S RNA when compared with cytoplasmic 4 S RNA. However, mitochondrial 4 S RNA was found to give rise to two additional 3H-labeled derivatives (Y 1 and Y 2, see Fig. 1) not present in the labeled digest of cytoplasmic 4 S RNA. By following the time course of the enzymatic digestion of mitochondrial 4 S RNA we have established that these 2 additional labeled c o m p o u n d s originate from the RNA rather than from impurities (data not shown). These c o m p o u n d s may represent trialcohols derived from u n k n o w n modified nucleosides with highly hydrophilic properties. Alternatively, but less likely, they may be derived from short mitochondria-specific oligonucleotides resistant to enzymatic degradation by a mixture of RNAase A, snake venom phosphodiesterase, and alkaline phosphatase [10--12]. In Table I {columns 1--3), total base compositions of 4 S RNA from rat liver mitochondria and cytoplasm have been compared. Sixteen constituents were analyzed as trialcohol derivatives by the tritium derivative method [10--12] including most base-modified and all base-methylated nucleosides known to occur in mammalian tRNA. As can be seen from Table I, the 2 R N A populations differ in all 16 constituents assayed. The differences are statistically highly significant. The mitochondrial 4 S R N A is characterized by a particularly high A and U content and a relatively low degree of modification and methylation when compared with cytoplasmic 4 S RNA, the amounts of A and U being a b o u t 50% higher and the degrees of modification and methylation more than 50% lower for the mitochondrial 4 S RNA. G and C are a b o u t 25% and 12% lower, respectively, in the mitochondrial 4 S RNA. The high proportion of A and U in the mitochondrial RNA is also reflected by the ratios (A + U)/(G + C) and ( A t o t a 1 + Vtotal)/(Gtota 1 + C t o t a l ) , which respectively are a b o u t 1.9 and 1.7 times higher for the mitochondrial 4 S RNA. The proportions of all modified nucleosides are diminished in the mitochondrial R N A when compared with the cytoplasmic RNA, b u t to a varying degree, ranging from a b o u t 20% for m~A to a b o u t 70% for m SU, with the other values falling in between. The amounts of seven modified constituents (mSU, hU, m3C, mSC, I, mTG, and X) were found to be reduced by > 5 0 % when compared with the values for cytoplasmic 4 S RNA {Table I). Different 4 S RNA preparations from the same source were found to give essentially the same results. This was shown previously for various cytoplasmic 4 S RNA preparations [20]. The base composition of different mitochondrial 4 S RNA preparations also varies only slightly, deviations from the values of Table I, column 1, being in a range of a b o u t + 5%. Comparison between base composition o f bacterial 4 S R N A and liver mitochondrial and cytoplasmic 4 S R N A In Table I the base composition of E. coli and B. subtilis 4 S R N A is compared with that of liver mitochondrial and cytoplasmic 4 S RNA. The 2 bacterial 4-S RNAs are relatively more similar to each other than to liver cytoplasmic and mitochondrial 4 S RNA. If the base compositions of all 4 R N A populations in Table I are compared it is apparent that the greatest differences exist between liver mitochondrial 4 S R N A and the bacterial RNAs. Both bacterial
55 TABLE I T O T A L BASE C O M P O S I T I O N OF 4 S RNA. C O M P A R I S O N OF R A T L I V E R M I T O C H O N D R I A L 4 S RNA WITH CYTOPLASMIC AND BACTERIAL 4 S RNA 4 S R N A w a s p r e p a r e d as d e s c r i b e d in t h e t e x t . Base a n a l y s i s w a s c a r r i e d o u t b y c h e m i c a l t r i t i u m derivat i z a t i o n o f n u c l e o s i d e s in e n z y m a t i c digests [ 1 0 - - 1 2 ] . Base c o m p o s i t i o n d a t a w e r e c a l c u l a t e d f r o m c o u n t r a t e s o f t r i t i u m - l a b e l e d n u c l e o s i d e t r i a l c o h o l s o b t a i n e d f r o m several r e p l i c a t e c h r o m a t o g r a p h i c a n a l y s e s of e a c h labeled digest. Nucleoside
(1) R a t liver mitochondria
(2) R a t liver cytoplasm
X(1) (%)
X(2) (%)
21.05 26.90 22.63 20.58 1.03
13.98 17.28 25.72 27.62 . 1.32
0.001
mSU hU
0.19 1.20
0.61 2.99
0.001 0.001
m]G m2G mlG m3C c
0.38 0.98 0.54 0.14
0.62 1.38 0.77 0.35
0.001 0.001 0.001 0.001
mSC
1.01
2.27
I mTG d
2.80 0.14 0.28
3.77 0.31 0.65
U A C G m6A mlA b
.
X
.
0.17
V m2A
. .
Total
100.00
(ZNMelT, N T) X 1 0 0 e (~=NMo/ZN T) X 1 0 0 f (A + U ) I ( G + C) (A T + U T ) / ( G T + C T ) g
1.15
. .
(4) (1)/(2)
(5)
(6)
E. coli B
B. subtilis
X(1)/X(2)
2 ( 5 ) (%)
X(8) (%)
1.51 1.57 0.88 0.74 0.78
14.77 18.87 29.48 28.79 0.12 --
18.52 18.39 28.92 29.47 0.19 0.37
0.31 0.40
1.25 2.32
1.26 1.89
0.58 0.71 0.70 0.40
--0.19 --
--0.24 --
0.001
0.44
--
--
0.001 0.001 0.001
0.74 0.45 0.43
2.47 0.11 0.81
1.84 0.10 0.69
0.001
0.46
0.29
0.10
0.001 0.001 0.001 0.001 .
0.37 . .
4.53% 8.84% 1.11
(3) P(1),(2) a
. .
0.22 0.30
100.01
99.99
99.98
7.97% 15.41% 0.59
0.001 0.001 0.001
0.57 0.57 1.88
2.67% 8.08% 0.58
2.75% 6.68% 0.60
0.68
0.001
1.69
0.69
0.69
a P r o b a b i l i t y values e s t i m a t e d o n t h e basis of S t u d e n t ' s t test: FAT1 N 2 ( N I _ + N 2 - - 2
t ~ t_
! I 1/2 X
N , + N~
~I --~2
,/N,d
+ N~s~
= m e a n of 4 a n a l y s e s ( N = 4)
b
c d e f g
s = s t a n d a r d d e v i a t i o n = ~/}=(x - - ~ ) 2 / ( N - 1) P values f o r t h e c o m p a r i s o n of t h e b a c t e r i a l 4-S R N A s w i t h e a c h o t h e r a n d w i t h t h e liver s a m p l e s are n o t listed. G e n e r a l l y , a d i f f e r e n c e w a s f o u n d to b e statistically significant (P ~ 0 . 0 5 ) if t h e r a t i o o f t h e 2 c o n s t i t u e n t s c o m p a r e d d i f f e r e d b y m o r e t h a n +-10% f r o m 1 f o r t h e m o d i f i e d n u c l e o s i d e s or b y m o r e t h a n -+5% for t h e m a j o r n u c l e o s i d e s . T h e v a l u e o f m 1A w a s o b t a i n e d f r o m t h e s u m o f t h e c o u n t r a t e s of t h e 3 H - l a b e l e d t r i a l c o b o l s o f m I A a n d m 6 A [ 1 1 ] . I n t h e case of B. subtilis 4 S R N A , t h e c a l c u l a t i o n of t h e v a l u e s for m 1A a n d m 6 A is b a s e d o n a 20% c o n v e r s i o n of m 1A t o m 6 A [ 1 1 ] . C o r r e c t e d f o r 85% r e c o v e r y [ 1 1 ] . C o r r e c t e d for 65% r e c o v e r y [ 1 1 ] . Z Base m e t h y l a t e d nucleosides/~: T o t a l n u c l e o s i d e s ~ d e g r e e o f base m e t h y l a t i o n . ~: M o d i f i e d nucleo6ides/]~ T o t a l n u c l e o s i d e s ~ d e g r e e o f m o d i f i c a t i o n . ( A t o t a l + U t o t a l ) / ( G t o t a l + C t o t a l ) ; A t o t a1 = A + ~ : A m o d i f i e d etc.
56 RNAs do not contain m2G, m~G, m3C, and mSC. In addition, E. coli B 4 S RNA lacks m ~A. These components are present in liver mitochondrial and cytoplasmic 4 S RNA. Both bacterial RNAs contain m 6A, which is not present in mitochondrial and cytoplasmic 4 S RNA. Two constituents, i.e., uridine5-oxyacetic acid and m 2 A, are unique to E. coli 4 S RNA. Whereas the proportions of the major constituents in the bacterial 4-S RNAs resemble those of the liver cytoplasmic 4 S RNA, they are very different from those of mitochondrial 4 S RNA. The high content of A and U and the high ratio of ( A t o t a 1 + V t o t a l ) / ( G t o t a I + Ctotal) are unique to mitochondrial 4 S RNA. The mSU content, which is highest in the bacterial RNAs and intermediate in liver cytoplasmic RNA, is very low in the liver mitochondrial 4 S R N A [22]. m 7G is also low in the mitochondrial RNA. On the other hand, m I G is low in the bacterial RNAs, intermediate in the mitochondrial RNA, and high in the cytoplasmic RNA. It is interesting to note that in the bacterial RNA m2G and m~G are absent and m~G is low b u t mTG is rather high. Of the 4 RNA populations compared in Table I, cytoplasmic 4 S RNA has the highest degree of modification and base methylation. The only apparent c o m m o n characteristic of liver mitochondrial 4 S RNA and the bacterial RNAs is a similar overall degree of modification, which is a b o u t 2-times lower than that of liver cytoplasmic 4 S RNA. The degree of base methylation of the mitochondrial 4 S RNA is almost twice as high as that of the bacterial 4-S RNAs but only a b o u t half that of the cytoplasmic 4 S RNA. Comparison between mitochondrial 4 S R N A o f liver and hepatomas 5123D and 7777 The total base composition of mitochondrial 4 S R N A from both hepatomas was found to resemble that o f liver mitochondrial 4 S R N A (data not shown). Regarding the major base compositions, the 4 S R N A from hepatoma 5123D mitochondria contained somewhat more A and U b u t less G and C than the corresponding liver R N A preparation whereas the opposite was found to be true for hepatoma 7777 mitochondrial 4 S RNA. In each case, deviations for the individual major nucleosides were
