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A novel ribosomal RNA in hamster cell mitochondria
538
PRELIMINARY NOTES
BBA 91181 A novel ribosomal R N A
in h a m s t e r cell rnitochondria
Recent studies have revealed the presence of significant amounts of RNA in highly purified mitochondria from several sources 1-4. Differences have also been reported between certain mitochondrial RNA species, and analogous cytoplasmic species, in Neurospora ~ and in yeasO. This communication describes an approach to tile characterization of mitochondrial RNA in a continuously cultured hamster cell line, BHK-2I~,L Using radioactive tracer methods, to which this system lends itself, we have been able to detect differences, in both sedimentation rate and degree of methylation, between a mitochondrial ribosomal RNA (rRNA) species and its cytoplasmic counterpart. Cells were grown in a standard medium (see Fig. i) and harvested and fraetionated into cytoplasmic and mitochondrial fractions essentially as described b y FREEMAN1. Isopycnic centrifugation, tile final mitochondrial purification step, yielded a discrete band at an appropriate density (1.16) (c/. ref. I). When cells had been grown in tile presence of [HCluridine , a sharp peak of labeled RNA coincided exactly with this band. Control experiments, in which labeled cytoplasmic material was added to an unlabeled homogenate, indicated that < 5 % of tile RNA in the mitochondrial band arose from cytoplasmic contamination. When the mitochondrial RNA was purified and fractionated by sucrose density-gradient centrifugation together with unlabeled cytoplasmic RNA, three radioactive peaks were obtained (Fig. I): a "27-S" peak, which invariably sedimented slightly behind the cytoplasmic 28-S RNA peak; a "I7-S" peak, which tended to sediment slightly behind tile cytoplasmic I8-S peak; and a 4-S peak. As shown in Fig. I, tile distribution of mitochondrial RNA among these peaks varied between experiments. In some cases the mitochondrial RNA yielded a relatively "normal" pattern (A), while in others there was a relative excess of the more slowly sedimenting fractions (B). Partial degradation of the larger components, occurring during the prolonged mitochondrial purification procedure, was probably, at least in part, responsible for tile more distorted patterns (c/. ref. 2). Microsomes, when subjected to isopycnic centrifugation, also underwent partial degradation, yielding increased I 7 - I 8 - S and/or 4-S peaks (c/. ref. IO). Tile apparent sedimentation discrepancy between tile cytoplasmic I8-S and the mitochondrial I7-S peaks was variable, and m a y well be an artifact arising from the presence in this region of 27-S breakdown products. However, the discrepancy between cytoplasmic 28-S RNA and mitochondrial 27-S RNA, although never more than one tube in magnitude, appears to be real. I t was found in each of nine runs from six separate experiments, however "normal" the overall mitochondrial RNA pattern. Moreover, partially degraded microsomal RNA yielded no such discrete 27-S peak. The RNA was further characterized b y determining degree of methylation, A b b r e v i a t i o n and terminology: r R N A , ribosomal R N A . The t e r m " c y t o p l a s m i c " is used to refer to the mitochondria-free cell s u p e r n a t a n t fraction. Since only relative s e d i m e n t a t i o n rates have been determined, s-values have been used only as convenient labels.
Biochim. Biophys. dcta, 145 (1967) 538-54 °
PRELIMINARY NOTES
539
B]
i!!i
0,6
O.z
# (3.2
@
16
~@ 26
o
g
16
!oolJ i lg
2'0
o
~:
Froction NO.
Fig. I. Velocity s e d i m e n t a t i o n p a t t e r n s of mitochondrial R N A f r o m h a m s t e r cells. P a t t e r n s f r o m two s e p a r a t e e x p e r i m e n t s ( " A " and " B " ) are shown. B H K - 2 I cells% ~ were g r o w n in s p i n n e r flasks ~ in EAGLE'S 8 m e d i u m modified for s u s p e n s i o n culture and s u p p l e m e n t e d w i t h io % calf s e r u m , non-essential a m i n o acids, twice the usual c o n c e n t r a t i o n of vitamins, s t r e p t o m y c i n and penicillin, adenosine and guanosine (IO-* M), and N a H C O 3 (2.8 g/l), and equilibrated w i t h 5 % CO s in air. Cells were labeled b y diluting into m e d i u m containing [t4C]uridine, 0.033 mM (0.08/zC/ ml for culture A, 0.07/zC/ml for B), and densities were k e p t b e t w e e n 2. Io 5 and 12. lO 6 cells per ml b y f u r t h e r dilution. Cultures (approx. 4" l ° s cells in 400 ml) were h a r v e s t e d after 46-48 h (equivalent to a 9-fold increase in cell n u m b e r ) , and the cells were processed as described in the text. I m m e d i a t e l y after the isopycnic centrifugation step (39 ooo rev./min, 7 ° nlin, Spinco SW 39 rotor), R N A was purified f r o m the mitochondrial band, t o g e t h e r w i t h unlabeled cytoplasmic RNA, as previously described 9 except t h a t the e x t r a c t i n g m i x t u r e contained 0.2 M s o d i u m acetate, p H 5.1. The R N A w a s fractionated by sucrose d e n s i t y - g r a d i e n t s e d i m e n t a t i o n (38 ooo rev./min, 5 h, Spinco S \ ¥ 39 rotor), and fractions were assayed as previously described 9. O - C ) , a b s o r b a n c e at 260 mff; Q - O , counts/rain in RNA.
using cells doubly labeled with [Me-14C]methionine and a~Pt (c/. ref. 9). The values for cytoplasmic RNA (Table I) were similar to those reported earlier for HeLa cells n. Mitochondrial 27-S RNA resembled, but was slightly more highly methylated than, cytoplasmic 28-S RNA. We conclude that hamster mitochondrial ~,7-S RNA is ribosomal but is not identical with its cytoplasmic counterpart. The marked undermethylation of mitochondrial I7-S RNA is our most puzzling finding; degradation products of 27-S RNA could not per se have lowered the TABLE I DEGREES OF METHYLATION OF MITOCHONDRIAL AND CYTOPLASMIC R N A FROM HAMSTER CELLS Cells were g r o w n as for Fig. I, except t h a t the m e d i u m contained 3~P1 (o. 5 F C / I . 2 / , m o l e per ml) and [~!e-14C]methionine, 0.25/~C/6.8/~g per ml. Cells were t h e n fractionated into cytoplasmic a n d mitochondrial fractions, and R N A w a s purified and fractionated as for Fig. i. The degree of m e t h y l a t i o n of each R N A fraction was calculated f r o m 14C/32P ratios, as previously described 9. The mitochondrial R N A in this e x p e r i m e n t (as detected b y 3~p) h a d relatively high I7-S and 4-S peaks (see Fig. IB); the cytoplasmic 32p p a t t e r n followed the 26o mff a b s o r b a n c e profile precisely.
Methyl groups per zoo nucleotides
Cytoplasmic R N A Mitochondrial R N A
28(27) S
18(17) S
4 S
1.49 1.7o
2.05 0.77
6.3 ° 2.53
Biochim. Biophys. Acta, 145 (1967) 538-54 °
540
PRELIMINARY NOTES
apparent methylation of a conventional mammalian rRNA species to this extent. On the other hand, the apparent relative undermethylation of the mitochondrial 4-S peak can be attribated to the presence of degradation products of the larger mitochondrial RNA species. It should be noted that, while mitochondria127-S rRNA is more "mammalian" than "bacterial" in both sedimentation rate 12 and degree of methylation, mitochondrial I7-S RNA, if reasonably homogeneous and, in fact, ribosomal, would be classified as "bacterial" in its degree of methylation 9. The present findings are interesting in the light of other recent observations: (i) mitochondrial ribosomes are smaller than cytoplasmic ribosomes~, 13, and (2) mitochondrial protein synthesis is sensitive to certain antibacterial agents 14-18. Our results suggest that these properties may, in part, reside in the rRNA. Further studies are planned, to characterize mitochondrial rRNA more definitively, and to decide whether its special properties reflect synthesis on a special (presumably mitochondrial) DNA template, or, rather, reflect modifications imposed on "maturing" polynucleotide chains withirt mitochondria. This work was performed during the tenure of a U. S. Public Health Service Career Development Award to D.T.D., while visiting the laboratory of Professor T. S. WORK. The hospitality of Professor T. S. WORK and his colleagues is gratefully acknowledged.
Division o/ Biochemistry, National Institute/or Medicat Research, Mill Hill, London (Great Britain).
DONALD T. DUBIN* RONALD E. BROWN
i K. B. FREEMAN, Biochem. J., 94 (1965) 494. 2 E. WlNTERSBER6ER, in J. M. TAGER, S. PAPA, E. QUAGLIARIELLOAND E. C. SLATER, Regulation of Metabolic Processes in Mitochondria (BBA L i b r a r y vol. 7), Elsevier, A m s t e r d a m , 1966, p. 439. 3 T. W. O'BRIEN AND a . F. KALF, J. Biol. Chem., 242 (1967) 2172, 218o. 4 P- J. ROGERS, g. N. PRESTON, E. B. TITCHENER AND A. W. LINNANE, Biochem. Biophys. Res. Commun., 27 (1967) 405 . 5 W. E. BARNETT AND D. H. BROWN, Proc. Natl, Acad. Sci. U.S., 57 (1967) 452. 6 M. STOKER ANn I. MAcPHERSON, Nature, 203 (1964) 1355. 7 P- B. CAPSTICK, I{. C. TELLING, W. O. CHAPMAN AND D. L. STEWART, Nature, 195 (1962) 1163. 8 H. EAGLE, Science, 13o (1959) 432. 9 D. T. DUBIN AND A. G. GI3NALP, Biochim. Biophys. Acta, 134 (1967) lO6. io S. J. MARTIN, Biochem. J., i o i (1966) 721. i i G. M. BROWN AND G. ATTARDI, Biochem. Biophys. Res. Commun., 20 (1965) 298. 12 E. STUTZ AND H. 1N~OLL, Proc. Natl. Acad. Sci. U.S., 57 (1967) 774. 13 J. ANDR£ AND V. MARINOZZI, J. Microscop., 4 (1965) 615. 14 R. RENDI, Exptl. Cell Res., 18 (1959) 187. 15 O. D. CLARK-WALKER ANn A. W. LINNANE, Biochem. Biophys. Res. Cornmun., 25 (1966) 8. 16 D. HALDAR, K. B. FREEMAN AND T. S. WORK, Biochem. J., lO2 (1967) 684.
Received July 6th, 1967 P r e s e n t address: D e p a r t m e n t of Microbiology, R u t g e r s Medical School, New Brunswick, N.J., U.S.A.
Biochim. Biophys. Acta, 145 (1967) 538-54 °
PRELIMINARY NOTES
BBA 91181 A novel ribosomal R N A
in h a m s t e r cell rnitochondria
Recent studies have revealed the presence of significant amounts of RNA in highly purified mitochondria from several sources 1-4. Differences have also been reported between certain mitochondrial RNA species, and analogous cytoplasmic species, in Neurospora ~ and in yeasO. This communication describes an approach to tile characterization of mitochondrial RNA in a continuously cultured hamster cell line, BHK-2I~,L Using radioactive tracer methods, to which this system lends itself, we have been able to detect differences, in both sedimentation rate and degree of methylation, between a mitochondrial ribosomal RNA (rRNA) species and its cytoplasmic counterpart. Cells were grown in a standard medium (see Fig. i) and harvested and fraetionated into cytoplasmic and mitochondrial fractions essentially as described b y FREEMAN1. Isopycnic centrifugation, tile final mitochondrial purification step, yielded a discrete band at an appropriate density (1.16) (c/. ref. I). When cells had been grown in tile presence of [HCluridine , a sharp peak of labeled RNA coincided exactly with this band. Control experiments, in which labeled cytoplasmic material was added to an unlabeled homogenate, indicated that < 5 % of tile RNA in the mitochondrial band arose from cytoplasmic contamination. When the mitochondrial RNA was purified and fractionated by sucrose density-gradient centrifugation together with unlabeled cytoplasmic RNA, three radioactive peaks were obtained (Fig. I): a "27-S" peak, which invariably sedimented slightly behind the cytoplasmic 28-S RNA peak; a "I7-S" peak, which tended to sediment slightly behind tile cytoplasmic I8-S peak; and a 4-S peak. As shown in Fig. I, tile distribution of mitochondrial RNA among these peaks varied between experiments. In some cases the mitochondrial RNA yielded a relatively "normal" pattern (A), while in others there was a relative excess of the more slowly sedimenting fractions (B). Partial degradation of the larger components, occurring during the prolonged mitochondrial purification procedure, was probably, at least in part, responsible for tile more distorted patterns (c/. ref. 2). Microsomes, when subjected to isopycnic centrifugation, also underwent partial degradation, yielding increased I 7 - I 8 - S and/or 4-S peaks (c/. ref. IO). Tile apparent sedimentation discrepancy between tile cytoplasmic I8-S and the mitochondrial I7-S peaks was variable, and m a y well be an artifact arising from the presence in this region of 27-S breakdown products. However, the discrepancy between cytoplasmic 28-S RNA and mitochondrial 27-S RNA, although never more than one tube in magnitude, appears to be real. I t was found in each of nine runs from six separate experiments, however "normal" the overall mitochondrial RNA pattern. Moreover, partially degraded microsomal RNA yielded no such discrete 27-S peak. The RNA was further characterized b y determining degree of methylation, A b b r e v i a t i o n and terminology: r R N A , ribosomal R N A . The t e r m " c y t o p l a s m i c " is used to refer to the mitochondria-free cell s u p e r n a t a n t fraction. Since only relative s e d i m e n t a t i o n rates have been determined, s-values have been used only as convenient labels.
Biochim. Biophys. dcta, 145 (1967) 538-54 °
PRELIMINARY NOTES
539
B]
i!!i
0,6
O.z
# (3.2
@
16
~@ 26
o
g
16
!oolJ i lg
2'0
o
~:
Froction NO.
Fig. I. Velocity s e d i m e n t a t i o n p a t t e r n s of mitochondrial R N A f r o m h a m s t e r cells. P a t t e r n s f r o m two s e p a r a t e e x p e r i m e n t s ( " A " and " B " ) are shown. B H K - 2 I cells% ~ were g r o w n in s p i n n e r flasks ~ in EAGLE'S 8 m e d i u m modified for s u s p e n s i o n culture and s u p p l e m e n t e d w i t h io % calf s e r u m , non-essential a m i n o acids, twice the usual c o n c e n t r a t i o n of vitamins, s t r e p t o m y c i n and penicillin, adenosine and guanosine (IO-* M), and N a H C O 3 (2.8 g/l), and equilibrated w i t h 5 % CO s in air. Cells were labeled b y diluting into m e d i u m containing [t4C]uridine, 0.033 mM (0.08/zC/ ml for culture A, 0.07/zC/ml for B), and densities were k e p t b e t w e e n 2. Io 5 and 12. lO 6 cells per ml b y f u r t h e r dilution. Cultures (approx. 4" l ° s cells in 400 ml) were h a r v e s t e d after 46-48 h (equivalent to a 9-fold increase in cell n u m b e r ) , and the cells were processed as described in the text. I m m e d i a t e l y after the isopycnic centrifugation step (39 ooo rev./min, 7 ° nlin, Spinco SW 39 rotor), R N A was purified f r o m the mitochondrial band, t o g e t h e r w i t h unlabeled cytoplasmic RNA, as previously described 9 except t h a t the e x t r a c t i n g m i x t u r e contained 0.2 M s o d i u m acetate, p H 5.1. The R N A w a s fractionated by sucrose d e n s i t y - g r a d i e n t s e d i m e n t a t i o n (38 ooo rev./min, 5 h, Spinco S \ ¥ 39 rotor), and fractions were assayed as previously described 9. O - C ) , a b s o r b a n c e at 260 mff; Q - O , counts/rain in RNA.
using cells doubly labeled with [Me-14C]methionine and a~Pt (c/. ref. 9). The values for cytoplasmic RNA (Table I) were similar to those reported earlier for HeLa cells n. Mitochondrial 27-S RNA resembled, but was slightly more highly methylated than, cytoplasmic 28-S RNA. We conclude that hamster mitochondrial ~,7-S RNA is ribosomal but is not identical with its cytoplasmic counterpart. The marked undermethylation of mitochondrial I7-S RNA is our most puzzling finding; degradation products of 27-S RNA could not per se have lowered the TABLE I DEGREES OF METHYLATION OF MITOCHONDRIAL AND CYTOPLASMIC R N A FROM HAMSTER CELLS Cells were g r o w n as for Fig. I, except t h a t the m e d i u m contained 3~P1 (o. 5 F C / I . 2 / , m o l e per ml) and [~!e-14C]methionine, 0.25/~C/6.8/~g per ml. Cells were t h e n fractionated into cytoplasmic a n d mitochondrial fractions, and R N A w a s purified and fractionated as for Fig. i. The degree of m e t h y l a t i o n of each R N A fraction was calculated f r o m 14C/32P ratios, as previously described 9. The mitochondrial R N A in this e x p e r i m e n t (as detected b y 3~p) h a d relatively high I7-S and 4-S peaks (see Fig. IB); the cytoplasmic 32p p a t t e r n followed the 26o mff a b s o r b a n c e profile precisely.
Methyl groups per zoo nucleotides
Cytoplasmic R N A Mitochondrial R N A
28(27) S
18(17) S
4 S
1.49 1.7o
2.05 0.77
6.3 ° 2.53
Biochim. Biophys. Acta, 145 (1967) 538-54 °
540
PRELIMINARY NOTES
apparent methylation of a conventional mammalian rRNA species to this extent. On the other hand, the apparent relative undermethylation of the mitochondrial 4-S peak can be attribated to the presence of degradation products of the larger mitochondrial RNA species. It should be noted that, while mitochondria127-S rRNA is more "mammalian" than "bacterial" in both sedimentation rate 12 and degree of methylation, mitochondrial I7-S RNA, if reasonably homogeneous and, in fact, ribosomal, would be classified as "bacterial" in its degree of methylation 9. The present findings are interesting in the light of other recent observations: (i) mitochondrial ribosomes are smaller than cytoplasmic ribosomes~, 13, and (2) mitochondrial protein synthesis is sensitive to certain antibacterial agents 14-18. Our results suggest that these properties may, in part, reside in the rRNA. Further studies are planned, to characterize mitochondrial rRNA more definitively, and to decide whether its special properties reflect synthesis on a special (presumably mitochondrial) DNA template, or, rather, reflect modifications imposed on "maturing" polynucleotide chains withirt mitochondria. This work was performed during the tenure of a U. S. Public Health Service Career Development Award to D.T.D., while visiting the laboratory of Professor T. S. WORK. The hospitality of Professor T. S. WORK and his colleagues is gratefully acknowledged.
Division o/ Biochemistry, National Institute/or Medicat Research, Mill Hill, London (Great Britain).
DONALD T. DUBIN* RONALD E. BROWN
i K. B. FREEMAN, Biochem. J., 94 (1965) 494. 2 E. WlNTERSBER6ER, in J. M. TAGER, S. PAPA, E. QUAGLIARIELLOAND E. C. SLATER, Regulation of Metabolic Processes in Mitochondria (BBA L i b r a r y vol. 7), Elsevier, A m s t e r d a m , 1966, p. 439. 3 T. W. O'BRIEN AND a . F. KALF, J. Biol. Chem., 242 (1967) 2172, 218o. 4 P- J. ROGERS, g. N. PRESTON, E. B. TITCHENER AND A. W. LINNANE, Biochem. Biophys. Res. Commun., 27 (1967) 405 . 5 W. E. BARNETT AND D. H. BROWN, Proc. Natl, Acad. Sci. U.S., 57 (1967) 452. 6 M. STOKER ANn I. MAcPHERSON, Nature, 203 (1964) 1355. 7 P- B. CAPSTICK, I{. C. TELLING, W. O. CHAPMAN AND D. L. STEWART, Nature, 195 (1962) 1163. 8 H. EAGLE, Science, 13o (1959) 432. 9 D. T. DUBIN AND A. G. GI3NALP, Biochim. Biophys. Acta, 134 (1967) lO6. io S. J. MARTIN, Biochem. J., i o i (1966) 721. i i G. M. BROWN AND G. ATTARDI, Biochem. Biophys. Res. Commun., 20 (1965) 298. 12 E. STUTZ AND H. 1N~OLL, Proc. Natl. Acad. Sci. U.S., 57 (1967) 774. 13 J. ANDR£ AND V. MARINOZZI, J. Microscop., 4 (1965) 615. 14 R. RENDI, Exptl. Cell Res., 18 (1959) 187. 15 O. D. CLARK-WALKER ANn A. W. LINNANE, Biochem. Biophys. Res. Cornmun., 25 (1966) 8. 16 D. HALDAR, K. B. FREEMAN AND T. S. WORK, Biochem. J., lO2 (1967) 684.
Received July 6th, 1967 P r e s e n t address: D e p a r t m e n t of Microbiology, R u t g e r s Medical School, New Brunswick, N.J., U.S.A.
Biochim. Biophys. Acta, 145 (1967) 538-54 °