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Mitochondrial RNA from rat liver
560
PRELIMINARY NOTES
BBA 91289
Mitochondrial R N A from rot liver Mitochondrial ribosomes sedimenting with an s .... = 73-75 S have been identified in primitive eukaryotes, like Neurospora ~ and yeast 2. The high-molecular weight RNA from these ribosomes cosediments with bacterialrRNA through sucrose gradients and is complementary to mitochondrial DNA 3,4. The search for mitochondrial ribosomes in m a m m a l i a n tissues has led to conflicting results ~-~. In this note we show that rat-liver mitochondria contain 2 major high-molecular weight RNA components, which are complementary to mitochondrial DNA and in the size range expected for the RNA's of the 55-S particle tentatively identified as the rat mitochondrial ribosome s,n. Rat-liver RNA was labeled with I~Hlorotic acid for a 7-day period and the RNA purified from the mitochondrial fraction was analyzed b y polyacrylarnide gel electrophoresis. Relative migration rates in the gel are expressed as apparent molecular weights, using 1.75.IO n for the molecular weight of the RNA from the large
I "285" 1.5
"18S .
.
.
.
~S"
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I
I
300 o'E"
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,
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i
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~ 'L
200~= >I
O
t t
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'~ 0.5
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.//7 20
40 60 FRACTION NUMBER
'._
. 80
'" 100
o~ n~ 120
Fig. I. Hybridization w i t h mitochondrial D N A of mitochondrial R N A fractionated on a polyacrylamide gel. A r a t was given 0.5 mC [3H]orotic acid (Amersham, specific activity 14. 3 C/mmole) b y intraperitoneal injection 7 h after partial h e p a t e c t o m y . The injection was repeated three times at 24-h intervals. The liver was fractionated 7 days after operation and the R N A was extracted as previously described za. C o n t a m i n a t i n g DI~A was r e m o v e d b y t r e a t m e n t with deoxyribonuclease I (W'orthington, ribonuclease-free), followed b y S e p h a d e x G-5o c h r o m a t o g r a p h y . A b o u t 3o/~g mitochondrial R N A (specific activity 23o0 counts/rain per #g) was applied to a p r e r u n 2. 7 % polyacrylamide gel and electrophoresed for 2 h in the cold r o o m (4°). The gel was scanned at 260 nip. and sliced in 12o fractions, the first IOI slices 0. 5 ram, the following 19 slices i.o m m thick. E a c h slice was extracted for 24 h at 25 ° with 1. 5 ml of 0.75 M N a C l + o . o 7 5 M sodium citrate containing o.i °/o sodium dodecyl sulphate. The radioactivity in o.15 ml aliquots of the eluates was used to align the A~n0 nm profile w i t h the gel slices. Recovery of R N A from the gel slices varied between 90 and 95 %. The extracted R N A was hybridized twice, first with 4/~g DNA, t h e n w i t h 2. 5/~g DNA. H y b r i d i z a t i o n was carried out in 0.75 M N a C l + o . o 7 5 M s o d i u m citrate containing o.i °/o sodium dodecyl sulphate, using procedures previously described x3. , A~60 nm; O" - - 0 , total radioactive R N A hybridized to mitochondrial D N A (6.5 #g) in b o t h hybridizations; &- - - &, R N A hybridized to mitochondrial D N A (2"-5 /~g) in the second hybridization only. All values are corrected for b a c k g r o u n d hybridization to the same a m o u n t of Escherichia coli D N A incubated in the same vessel (2o counts/rain including a p p a r a t u s background).
Biochim. Biophys. Acta, 2.17 (197 o) 5 6 ~ 5 6 2
PRELIMINARY NOTES
561
extramitochondrial ribosomal subunit and the relation between relative migration rate and molecular weight reported b y LOENINGla. The A260 n m and radioactivity profiles of RNA from the mitochondrial fraction (Fig. I) and the microsomal fraction (not shown) differed only in 2 respects: the mitochondrial RNA peak migrating with an apparent molecular weight of 6-lO5-7 -lo 5 ("18 S") was broader than the microsomal peak and the mitochondrial RNA contained an additional peak migrating with an apparent molecular weight of about 3.5" lO5 ("12 S"). A corresponding I2-S peak was also observed in mitochondrial RNA preparations analyzed on sucrose gradients. The gel was sliced and the RNA fractions eluted were hybridized with 4 #g rat-liver mitochondrial DNA for 16 h at 66 °. After removing these DNA filters from the eluates we added fresh filters loaded with 2.5 #g mitochondrial DNA to the eluates and repeated the hybridization procedure. This two-step hybridization assay should allow us to distinguish between quantitatively major and minor RNA components, since the latter will be exhausted by the first hybridization and therefore not contribute to the second. With the unfractionated RNA, 20 °/o of the input radioactive RNA specifically hybridized with mitochondrial DNA. No hybridization with mitochondrial DNA was detectable with the RNA isolated from the microsomes of the same liver, as previously reported a3. Fig. i shows that rat liver contains only 3 components that participate in both hybridizations; these migrate in the gels with apparent molecular weights of approximately 6.6. lO 5, 3.4" lO5 and 27- lO3. The 2 smaller components coincide exactly with peaks in the A2e0 .... profile, the larger component coincides with the leading edge of the "I8-S" peak. We conclude that these 3 components represent the stable mitochondrial RNA species. The additional major RNA components migrating with apparent molecular weights of 1.75. lO6 and 7" lO5 are probably due to contaminating microsomal RNA. We have failed, so far, to find conditions under which this extramitochondrial RNA is removed or enzymically degraded without concomitant degradation of mitochondrial RNA and it seems likely that the conclusions: previously drawn from such experiments are not correct. The bulk of the RNA migrating with an apparent molecular weight of 27" lO3 probably represents mitochondrial t R N A species, identified by NASS AND BUCK14. This peak m a y also contain a mitochondrial equivalent of the 5-S RNA found in all ribosomes. The two major RNA components probably represent the intact structural RNA's from the mitochondrial ribosomes, because the ratio of their molecular weights is about 2 : I and because their combined molecular weights are in the range expected for the 55-S particle, tentatively identified on indirect evidence as the mitochondrial ribosome in rat liver *,e. Further experiments are necessary to exclude the unlikely alternative that the two large RNA components are fragments of larger ribosomal RNA's, produced b y specific enzymic cleavage in situ (c[. ref. 15) or during the isolation of mitochondria. Previous attempts b y ATTARDI AND ATTARDI1° to identify stable mitochondrial RNA components complementary to mitochondrial DNA in m a m m a l i a n cells gave results quite different from ours. After fractionation of H e L a cell mitochondrial RNA on sucrose gradients, most of the hybridizing RNA was in a broad peak sedimenting at approximately 12 S. We attribute this result to a partial degradation of mitochondrial RNA and the poor resolution of mitochondrial RNA components on sucrose gradients. Biochim. Biophys. Acta, 217 (197o) 560-562
562
PRELIMINARY NOTES
SWANSON AND DAWID 1~ h a v e r e c e n t l y i s o l a t e d f r o m X e n o p u s egg m i t o c h o n d r i a a 6o-S r i b o s o m e t h a t is a c t i v e in a s u b m i t o c h o n d r i a l s y s t e m for p r o t e i n synthesis. T h i s r i b o s o m e c o n t a i n s R N A c o m p o n e n t s c o m p l e m e n t a r y to m i t o c h o n d r i a l D N A a n d m i g r a t i n g s l i g h t l y s l o w e r t h a n t h e t w o m a j o r c o m p o n e n t s o b s e r v e d b y us in r a t liver. I t is t h e r e f o r e possible t h a t s u c h m i n i r i b o s o m e s are c h a r a c t e r i s t i c for m i t o c h o n d r i a of h i g h e r a n i m a l s in g e n e r a l . W e t h a n k Dr. A. M. K r o o n for his h e l p a n d a d v i c e , Dr. I. B. DAWlD for s e n d i n g us a p r e p r i n t of ref. 16 a n d Mrs. I. T i e m e r s m a - M e i s n e r for e x p e r t t e c h n i c a l assistance. S u p p o r t e d (in p a r t ) b y t h e N e t h e r l a n d s F o u n d a t i o n for C h e m i c a l R e s e a r c h (S.O.N.) w i t h f i n a n c i a l a i d f r o m t h e N e t h e r l a n d s O r g a n i z a t i o n for t h e A d v a n c e m e n t of P u r e R e s e a r c h (Z.W.O.).
Department o/ Medical Enzymolog),, Laboratory o/ Biochemistry, University o / A m s t e r d a m , Amsterdam (The Netherlands)*
C. AAIj P.BoRST
I H. KONTZEL AND I~. •OLL, Nature, 2i 5 (I967) 134o. 2 w . J. STEGEMAN, C. S. COOPER AND C. J. AVERS, Biochem. Biophys. Res. Commun., 39 (197 °) 69. 3 D. D. WOOD AND D. J. L. LUCK, J. Mol. Biol., 41 (1969) 211. 4 E. ~VINTERSBERGER AND G. VIEHHAUSER, Nature, 220 (1968) 699. 5 T. \\7. O'BR1EN AND G. F. KALF, 3/. Biol. Chem., 242 (1967) 2172. 6 M. A. ASHWELL AND T. $. ~'ORK, Biochem. Biophys. Res. Commun., 39 (197 °) 204. 7 A. M. t~ROON AND C. AAIJ, in E. C. SEATER, J. M. TAGER, S. PAPA AND 1~. QUAGLIARIELLO, Biochemical Aspects of the Biogenesis o/ Mitochondria, Adriatica Editrice, Bari, 1968, p. 207. 8 C. VESCO AND S. PENMAN, Proe. Natl. Acad. Sci. U.S., 62 (1969) 218. 9 1. B. DAWlD, in Soe. Expt. Biol. Syrup. 24, The Development and Interrelationships o/ Cell Organelles, in the press. IO B. ATTARDI AND G. ATTARDI, Nature, 224 (1969) lO79. II D. T. DUBIN AND 13. S. MONTENECOURT,J. Mol, Biol.. 48 (197o) 279. 12 U. E. LOEYlNG, J. Mol. Biol., 38 (1968) 355. 13 P. BORST AND C. AAIJ, Biochem. Biophys. Res. Commun., 34 (1969) 358. 14 M. M. K. Nass AND C. A. BUCK, Proe. Natl. Acad. Sci. U.S., 62 (1969) 5o6. 15 B. MARRS AND S. t(-APLAN, .]- Mol. Biol., 49 (197o) 297. 16 R. F. SWANSON AND 1. B. DAWlD, Proc. Natl. Acad. Sci. U.S., 66 (197 o) 117. R e c e i v e d J u l y 7th, 197o " Postal address: Jan Swammerdam Institute, Ie Constantijn Huygensstraat 20, Amsterdam, The Netherlands.
Biochim. Biophys. ,4cta, 2I 7 (197 o) 560-562
PRELIMINARY NOTES
BBA 91289
Mitochondrial R N A from rot liver Mitochondrial ribosomes sedimenting with an s .... = 73-75 S have been identified in primitive eukaryotes, like Neurospora ~ and yeast 2. The high-molecular weight RNA from these ribosomes cosediments with bacterialrRNA through sucrose gradients and is complementary to mitochondrial DNA 3,4. The search for mitochondrial ribosomes in m a m m a l i a n tissues has led to conflicting results ~-~. In this note we show that rat-liver mitochondria contain 2 major high-molecular weight RNA components, which are complementary to mitochondrial DNA and in the size range expected for the RNA's of the 55-S particle tentatively identified as the rat mitochondrial ribosome s,n. Rat-liver RNA was labeled with I~Hlorotic acid for a 7-day period and the RNA purified from the mitochondrial fraction was analyzed b y polyacrylarnide gel electrophoresis. Relative migration rates in the gel are expressed as apparent molecular weights, using 1.75.IO n for the molecular weight of the RNA from the large
I "285" 1.5
"18S .
.
.
.
~S"
I
I
I
300 o'E"
,o) ~,o ,n
"i
¢,7,t
r
1.0 ,
E
J i
,
~
I
i
L
N
~,
~ 'L
200~= >I
O
t t
e,i
I00;
'~ 0.5
,-,o
_/ o
0
•
.//7 20
40 60 FRACTION NUMBER
'._
. 80
'" 100
o~ n~ 120
Fig. I. Hybridization w i t h mitochondrial D N A of mitochondrial R N A fractionated on a polyacrylamide gel. A r a t was given 0.5 mC [3H]orotic acid (Amersham, specific activity 14. 3 C/mmole) b y intraperitoneal injection 7 h after partial h e p a t e c t o m y . The injection was repeated three times at 24-h intervals. The liver was fractionated 7 days after operation and the R N A was extracted as previously described za. C o n t a m i n a t i n g DI~A was r e m o v e d b y t r e a t m e n t with deoxyribonuclease I (W'orthington, ribonuclease-free), followed b y S e p h a d e x G-5o c h r o m a t o g r a p h y . A b o u t 3o/~g mitochondrial R N A (specific activity 23o0 counts/rain per #g) was applied to a p r e r u n 2. 7 % polyacrylamide gel and electrophoresed for 2 h in the cold r o o m (4°). The gel was scanned at 260 nip. and sliced in 12o fractions, the first IOI slices 0. 5 ram, the following 19 slices i.o m m thick. E a c h slice was extracted for 24 h at 25 ° with 1. 5 ml of 0.75 M N a C l + o . o 7 5 M sodium citrate containing o.i °/o sodium dodecyl sulphate. The radioactivity in o.15 ml aliquots of the eluates was used to align the A~n0 nm profile w i t h the gel slices. Recovery of R N A from the gel slices varied between 90 and 95 %. The extracted R N A was hybridized twice, first with 4/~g DNA, t h e n w i t h 2. 5/~g DNA. H y b r i d i z a t i o n was carried out in 0.75 M N a C l + o . o 7 5 M s o d i u m citrate containing o.i °/o sodium dodecyl sulphate, using procedures previously described x3. , A~60 nm; O" - - 0 , total radioactive R N A hybridized to mitochondrial D N A (6.5 #g) in b o t h hybridizations; &- - - &, R N A hybridized to mitochondrial D N A (2"-5 /~g) in the second hybridization only. All values are corrected for b a c k g r o u n d hybridization to the same a m o u n t of Escherichia coli D N A incubated in the same vessel (2o counts/rain including a p p a r a t u s background).
Biochim. Biophys. Acta, 2.17 (197 o) 5 6 ~ 5 6 2
PRELIMINARY NOTES
561
extramitochondrial ribosomal subunit and the relation between relative migration rate and molecular weight reported b y LOENINGla. The A260 n m and radioactivity profiles of RNA from the mitochondrial fraction (Fig. I) and the microsomal fraction (not shown) differed only in 2 respects: the mitochondrial RNA peak migrating with an apparent molecular weight of 6-lO5-7 -lo 5 ("18 S") was broader than the microsomal peak and the mitochondrial RNA contained an additional peak migrating with an apparent molecular weight of about 3.5" lO5 ("12 S"). A corresponding I2-S peak was also observed in mitochondrial RNA preparations analyzed on sucrose gradients. The gel was sliced and the RNA fractions eluted were hybridized with 4 #g rat-liver mitochondrial DNA for 16 h at 66 °. After removing these DNA filters from the eluates we added fresh filters loaded with 2.5 #g mitochondrial DNA to the eluates and repeated the hybridization procedure. This two-step hybridization assay should allow us to distinguish between quantitatively major and minor RNA components, since the latter will be exhausted by the first hybridization and therefore not contribute to the second. With the unfractionated RNA, 20 °/o of the input radioactive RNA specifically hybridized with mitochondrial DNA. No hybridization with mitochondrial DNA was detectable with the RNA isolated from the microsomes of the same liver, as previously reported a3. Fig. i shows that rat liver contains only 3 components that participate in both hybridizations; these migrate in the gels with apparent molecular weights of approximately 6.6. lO 5, 3.4" lO5 and 27- lO3. The 2 smaller components coincide exactly with peaks in the A2e0 .... profile, the larger component coincides with the leading edge of the "I8-S" peak. We conclude that these 3 components represent the stable mitochondrial RNA species. The additional major RNA components migrating with apparent molecular weights of 1.75. lO6 and 7" lO5 are probably due to contaminating microsomal RNA. We have failed, so far, to find conditions under which this extramitochondrial RNA is removed or enzymically degraded without concomitant degradation of mitochondrial RNA and it seems likely that the conclusions: previously drawn from such experiments are not correct. The bulk of the RNA migrating with an apparent molecular weight of 27" lO3 probably represents mitochondrial t R N A species, identified by NASS AND BUCK14. This peak m a y also contain a mitochondrial equivalent of the 5-S RNA found in all ribosomes. The two major RNA components probably represent the intact structural RNA's from the mitochondrial ribosomes, because the ratio of their molecular weights is about 2 : I and because their combined molecular weights are in the range expected for the 55-S particle, tentatively identified on indirect evidence as the mitochondrial ribosome in rat liver *,e. Further experiments are necessary to exclude the unlikely alternative that the two large RNA components are fragments of larger ribosomal RNA's, produced b y specific enzymic cleavage in situ (c[. ref. 15) or during the isolation of mitochondria. Previous attempts b y ATTARDI AND ATTARDI1° to identify stable mitochondrial RNA components complementary to mitochondrial DNA in m a m m a l i a n cells gave results quite different from ours. After fractionation of H e L a cell mitochondrial RNA on sucrose gradients, most of the hybridizing RNA was in a broad peak sedimenting at approximately 12 S. We attribute this result to a partial degradation of mitochondrial RNA and the poor resolution of mitochondrial RNA components on sucrose gradients. Biochim. Biophys. Acta, 217 (197o) 560-562
562
PRELIMINARY NOTES
SWANSON AND DAWID 1~ h a v e r e c e n t l y i s o l a t e d f r o m X e n o p u s egg m i t o c h o n d r i a a 6o-S r i b o s o m e t h a t is a c t i v e in a s u b m i t o c h o n d r i a l s y s t e m for p r o t e i n synthesis. T h i s r i b o s o m e c o n t a i n s R N A c o m p o n e n t s c o m p l e m e n t a r y to m i t o c h o n d r i a l D N A a n d m i g r a t i n g s l i g h t l y s l o w e r t h a n t h e t w o m a j o r c o m p o n e n t s o b s e r v e d b y us in r a t liver. I t is t h e r e f o r e possible t h a t s u c h m i n i r i b o s o m e s are c h a r a c t e r i s t i c for m i t o c h o n d r i a of h i g h e r a n i m a l s in g e n e r a l . W e t h a n k Dr. A. M. K r o o n for his h e l p a n d a d v i c e , Dr. I. B. DAWlD for s e n d i n g us a p r e p r i n t of ref. 16 a n d Mrs. I. T i e m e r s m a - M e i s n e r for e x p e r t t e c h n i c a l assistance. S u p p o r t e d (in p a r t ) b y t h e N e t h e r l a n d s F o u n d a t i o n for C h e m i c a l R e s e a r c h (S.O.N.) w i t h f i n a n c i a l a i d f r o m t h e N e t h e r l a n d s O r g a n i z a t i o n for t h e A d v a n c e m e n t of P u r e R e s e a r c h (Z.W.O.).
Department o/ Medical Enzymolog),, Laboratory o/ Biochemistry, University o / A m s t e r d a m , Amsterdam (The Netherlands)*
C. AAIj P.BoRST
I H. KONTZEL AND I~. •OLL, Nature, 2i 5 (I967) 134o. 2 w . J. STEGEMAN, C. S. COOPER AND C. J. AVERS, Biochem. Biophys. Res. Commun., 39 (197 °) 69. 3 D. D. WOOD AND D. J. L. LUCK, J. Mol. Biol., 41 (1969) 211. 4 E. ~VINTERSBERGER AND G. VIEHHAUSER, Nature, 220 (1968) 699. 5 T. \\7. O'BR1EN AND G. F. KALF, 3/. Biol. Chem., 242 (1967) 2172. 6 M. A. ASHWELL AND T. $. ~'ORK, Biochem. Biophys. Res. Commun., 39 (197 °) 204. 7 A. M. t~ROON AND C. AAIJ, in E. C. SEATER, J. M. TAGER, S. PAPA AND 1~. QUAGLIARIELLO, Biochemical Aspects of the Biogenesis o/ Mitochondria, Adriatica Editrice, Bari, 1968, p. 207. 8 C. VESCO AND S. PENMAN, Proe. Natl. Acad. Sci. U.S., 62 (1969) 218. 9 1. B. DAWlD, in Soe. Expt. Biol. Syrup. 24, The Development and Interrelationships o/ Cell Organelles, in the press. IO B. ATTARDI AND G. ATTARDI, Nature, 224 (1969) lO79. II D. T. DUBIN AND 13. S. MONTENECOURT,J. Mol, Biol.. 48 (197o) 279. 12 U. E. LOEYlNG, J. Mol. Biol., 38 (1968) 355. 13 P. BORST AND C. AAIJ, Biochem. Biophys. Res. Commun., 34 (1969) 358. 14 M. M. K. Nass AND C. A. BUCK, Proe. Natl. Acad. Sci. U.S., 62 (1969) 5o6. 15 B. MARRS AND S. t(-APLAN, .]- Mol. Biol., 49 (197o) 297. 16 R. F. SWANSON AND 1. B. DAWlD, Proc. Natl. Acad. Sci. U.S., 66 (197 o) 117. R e c e i v e d J u l y 7th, 197o " Postal address: Jan Swammerdam Institute, Ie Constantijn Huygensstraat 20, Amsterdam, The Netherlands.
Biochim. Biophys. ,4cta, 2I 7 (197 o) 560-562