Isolation and characterization of a rapidly labelled RNA from mouse liver mitochondria

394

BIOCHIMICAET BIOPHYSICAACTA

BBA 95967

I S O L A T I O N AND C H A R A C T E R I Z A T I O N OF A R A P I D L Y L A B E L L E D FROM MOUSE L I V E R M I T O C H O N D R I A

RNA

SORIN COMOROSAN, ALEXANDRU GASPAR AND DANA SANDRU Department o/ Biochemistry, University Postgraduate Medical School, Fundeni Clinical Hospital, Bucharest (Rumania)

(Received May 6th, 1968)

SUMMARY

I. The isolation and characterization of a rapidly labelled RNA from mouse liver mitochondria is described. This RNA fraction was shown to have the characteristics of messenger RNA (mRNA). 2. The rapidly labelled RNA fraction exhibits a molecular weight of 1.5 • lO 5 and is continuously synthesized both in vivo and in vitro for 12o rain. 3. The amino acid incorporation into mitochondria in vitro is slightly stimulated b y this RNA fraction. It also prevents the inhibitory effects of chloramphenicol. 4. Hybridization of the rapidly labelled RNA with mitochondrial DNA suggests the mitochondrial origin of this RNA.

INTRODUCTION Mitochondrial protein synthesis, independent of the microsomal system has been reported in m a n y laboratories 1-5. This fact suggests the presence of a particular genetic apparatus e,~, demonstrated b y the isolation of mitochondrial nucleic acids. Mitochondrial DNA was isolated s-i° and its biochemical significance discussed n,12. As for mitochondrial RNA, its role and characteristics are not as clear ~,x~,14. An RNA fraction with ribosomal RNA (rRNA) physico-chemical properties was obtained from yeast mitochondria ~5,1s. Likewise a number of different transfer RNA (tRNA) fractions ~7and aminoacyl-RNA synthetaseslS, 19 have been prepared from these organelles. Concerning the mitochondrial protein, two particular aspects of its synthesis have been demonstrated: (a) whereas amino acid incorporation b y yeast cytoplasmic ribosomal system is not influenced b y chloramphenicol, protein synthesis b y yeast mitochondria is sensitive to this antibiotic2°; (b) mitochondrial protein synthesis is apparently restricted to a limited number of mitochondrial structural proteins ~,~1,2~. In the light of the present evidence, it seems probable that the role of RNA is in principle similar in mitochondrial as well as in microsomal protein synthesis. The experiments of WlNTERSBERGER23 on yeast mitochondria have also suggested the existence of a mitochondrial messenger RNA (mRNA) fraction. Indirect experimental evidence such as the inhibition of the amino acid incorporation into isolated mito.Abbreviations: mRNA, messenger RNA; rRNA, ribosomal RNA, tRNA, transfer RNA; poly U, polyuridylic acid. Biochim. Biophys. Acta, 166 (1968) 394-4o2

395

MITOCHONDRIAL RAPIDLY LABELLED R N A

chondria from tobacco leaves by actinomycin D (ref. 24), may imply its presence. However, a direct attempt to isolate such a fraction has not yet been made. This paper is concerned with the isolation and characterization of a rapidly labelled RNA from mouse-liver mitochondria, possessing the characteristics of a mRNA fraction.

METHODS

Male albino mice, 5 weeks old, weighing about 2o-25 g were used throughout this study.

Preparation o[ mitochondria The animals were killed b y decapitation, bled and the liver was excised, washed and homogenized with a coaxial glass Potter-Elvehjem homogenizer in cold standard buffer (0.25 M sucrose, 2 mM EDTA, 0.02 M Tris-HC1 (pH 7.5))- The homogenate was centrifuged for 5 min at IOOO × g (twice) and the supernatants collected and centrifuged for 20 min at 12 ooo × g. The mitochondrial pellet was suspended in 0.25 M sucrose, 2 mM MgC12, o.o2 M Tris-HC1 (pH 7.5), containing 4 °/~g ribonuclease per ml (EC 2.7.7.16, Mann Research Laboratories) and incubated for IO min at room temperature. The mixture was then cooled in ice and centrifuged for 20 rain at 12 ooo × g. Five washings in 0.34 M sucrose, 2 mM EDTA, 0.02 M Tris-HC1 (pH 7.4) for IO rain at 3200 × g were then performed. The pellet thus obtained was washed in standard buffer, centrifuged at 12 ooo × g and finally resuspended in 5 ml standard buffer. To exclude bacterial contamination, all operations were carried out under sterile conditions. To avoid contamination of the solutions, they were made up with freshly distilled water, stored at --20 ° and thawed just before use. The contamination of isolated mitochondria with microsomes was assayed by determining the RNA/mitochondrial protein ratio, as well as the DPN nucleosidase activity ~5. It must be emphasized that ribonuclease treatment of the isolated mitochondria does not unequivocally exclude microsomal RNA, since membrane-bound ribosomes are not degraded under these conditions and since the RNA indigenous to the smooth microsomal membranes should likewise be expected to be ribonucleaseresistant. Therefore, a RNA/mitochondrial protein ratio should be determined for each mitochondrial preparation. This was performed essentially according to the method of KROON~5. As a criterion for the biochemical activity of the mitochondrial preparation, the cytochrome c oxidase activity was determined 2e.

Preparation o[ nuclei Nuclei were prepared essentially according to the procedure of PIHA27.

Incorporation of 3,~p For the incorporation in vivo, 3o #C 3~P/25 g body weight were inoculated intraperitoneally as Na2HszPO~ (specific activity 1.5 rnC/mg.) For the experiments in vitro, Biochim. Biophys..4cta, 166 (1968) 394-4o2

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S. COMOROSAN, A. GASPAR, D. SANDRU

mitochondria were diluted in standard buffer containing I #C s~p/ml in order to obtain a final concentration of 3 mg mitochondrial protein per ml. Incubations were carried out at 37 ° , with gentle stirring.

Incorporation o/[14CJglutamic acid 3ffitochondria were incubated in an incorporation medium (Table I) containing I #C [14C]glutamic acid per ml (initial specific radioactivity, 3 " lOS counts/rain per /zmole) at 37 ° in Warburg flasks, with gentle stirring. The reaction was terminated b y the addition of cold trichloroacetic acid (6 % final concentration). TABLE I E F F E C T OF M I T O C H O N D R I A L R N A F R A C T I O N S A N D O F C H L O R A M P H E N I C O L ON T H E ACID INCORPORATION INTO M O U S E - L I V E R MITOCHONDRIA

[14C~GLUTAMIC

I n c o r p o r a t i o n a c t i v i t y w a s m e a s u r e d b y i n c u b a t i o n at 37 ° for 60 rain, w i t h shaking, in a m e d i u m c o n t a i n i n g 3 ° mM Tris, 50 mM KCI, 5 mM MgCI,. 1 mM E D T A , 3 ° J~.'M p o t a s s i u m p h o s p h a t e , 2 mM ADP, 5 0 / , g l a c t a l b u m i n h y d r o l y s a t e , 0.04 mM [l*C~glutamic acid (specific a c t i v i t y 78.8 m C / m m o l e ) a n d 2 mg m i t o c h o n d r i a l p r o t e i n in a final v o l u m e of I ml. Final p H 7.4-

Addition

Specific activity (counts/min per mg protein)

None + ioo # g RNA F I-fraction + ioo/,g RNA F II-fraction + 5 ° / z g Chloramphenicol + 5 ° / , g Chloramphenicol* + ioo ¢tg R N A F I-fraction + 5 ° / z g chloramphenicol + ioo # g R N A F II-fraction** + 5o/~g c h l o r a m p h e n i c o l

449 580 3oo io o 73 ° 27

* The m i t o c h o n d r i a were ir~cubated for 3 ° mill at 4 ° in a m e d i u m deprived of [14CJglutamic acid. The labelled a m i n o acid and c h l o r a m p h e n i c o l were t h e n added and i n c u b a t i o n performed e x a c t l y as m e n t i o n e d before. ** The m i t o c h o n d r i a were i n c u b a t e d for 3 ° m i n at 4 ° in a m e d i u m deprived of [x4Cjglutamic acid, in the presence of RNA. The labelled amino acid a n d c h l o r a m p h e n i c o l were t h e n added a n d i n c u b a t i o n p e r f o r m e d e x a c t l y as m e n t i o n e d before.

The samples were centrifuged for IO rain at IOOO × g. The pellet was washed with 6 % trichloroacetic acid, the mixture heated for 20 min at 95 ° and recentrifuged. The precipitate was washed wittl ethanol-ether (I : I, v/v) and cold 6 % trichloroacetic acid. The final pellet was dissolved in I ml 0.5 M NaOH and the samples counted in a Selo-liquid scintillation spectrometer, with an 80 % counting efficiency. All determinations were carried out in duplicate with a replication error of less than IO %.

Extraction of mitochondrial RNA The mitochondria were diluted i :5 (v/v) with o.14 M NaCh An equal volume of phenol saturated with water was added and the mixture shaken for i h at 65 °. After 30 rain at 15oo × g, the aqueous phase was deproteinized with 0.5 vol. of chloroform-isoamyl alcohol (24 : I, v/v) and recentrifuged. The RNA from the supernatant was precipitated overnight with 2 vols. of cold ethanol. Biochim. Biophys. Mcta, 166 (1968) 394-4o2

MITOCHONDRIAL RAPIDLY LABELLED R N A

397

Chromatographic ]ractionation RNA was chromatographed on a 1. 5 cm × 18 cm DEAE-Sephadex A 50coarse column, equilibrated with 5 mM MgC12-o.o2 M Tris-HC1 (pH 7.4). A sample of i mg RNA in 2 ml buffer was applied to the column, which was then washed with IOO ml buffer and subsequently developed with increasing molar concentrations of NaC1 in o . I - I M buffer, as illustrated in Figs. I and 3. Flow rate: 0. 7 ml/min. Samples of 5 ml were collected.

D N A / R N A hybridization Nuclear DNA was extracted according to the method of KAY2s and mitochondrial DNA according to that of ]~ARMUR29. In order to obtain pure samples devoid of RNA, repeated washings with isopropanol were made, according to the procedure of SAITO AND IV[uRAa°.

Hybridization was performed according to the technique of GILLESPIE AND SPIEGELMAN31 in 2 × SSC (o.15 M NaCl-o.oI5 M sodium citrate (pH 7.0)) containing 4.5 #g ~2p labelled RNA per ml. In order to obtain an RNA with high specific radioactivity for hybridization experiments, a series of animals were inoculated intraperitoneally with lOO-2OO #C n,p and killed b y decapitation after 6 h. The RNA was prepared from the mitochondrial pellet as described in METHODS.Samples of IOO ooo15o ooo counts/min per #g thus obtained were utilized. DNA was denatured with NaOH at pH 13 for IO min, neutralized with HC1 and then retained on Millipore filters H.A. For hybridization with nuclear DNA and mitochondrial DNA samples of IiO #g/ml and 70 #g/ml, respectively were utilized. Results are expressed as °/o of hybridized RNA.

Molecular weight The molecular weight of the RNA fractions was determined by light-scattering measurements, using a Sofica light-scattering photometer, with unpolarized light of 2 ---- 5460 •. The variation of refractive index with concentration (the dx/dc ratio) was 1.71, as estimated b y interferometric methods. Data were treated in terms of Zimm's procedurO 2 by plotting the concentration] light-scattered intensity versus sin 2 0/2 (where 0 is the scattered angle) and from the resulting diagrams I/M was computed.

Chemical determination Protein was estimated according to the procedure of O¥OMA AND EAGLE 33, DNA by the diphenylamine reaction and RNA by the orcinol method ~.

RESULTS

Amount o] mitochondrial RNA In our experiments the amount of RNA in mouse liver mitochondria was 15 #g/mg mitochondrial protein. ROODYN, REIS AND WORK~ found about 8-14 #g RNA/ Biochim. Biophys. Acta, 166 (1968) 394-402

398

S. COMOROSAN, A. GASPAR, D. SANDRU

mg mitochondrial protein in rat liver and WINTERSBERGER AND TUPP¥ 15 about 12-48 pg RNA/mg mitochondrial protein in yeast. Two kinds of errors are generally acknowledged when RNA is extracted from mitochondria: contamination with cytoplasmic rRNA fractions or bacterial contamination. In our experiments we avoided the contamination with cytoplasmic RNA b y pre-treatment of mitochondria with ribonuclease and b y centrifugation in 0.34 M sucrose, which, according to O'BRIEN AND KALF35, reduced this contamination to a minimum. Likewise we avoided bacterial contamination by working under sterile conditions.

Fractionation o/ mitochondrial R N A Two RNA fractions were separated b y fractionation on DEAE-Sephadex (Fig. I). We designate the RNA obtained in the chromatographic profile of Fig. I from Samples 5-1o, the F I-peak, and the RNA obtained from Samples 14-2o, the F II-peak.

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WINTERSBERGER AND TUPeY15 obtained a similar result with Saccharomyces cerevisiae mitochondria. In our experiment, the bulk of RNA is found in the F II-peak. By fractionation of the ~2P-labelled RNA in vivo or in vitro, 86 % of the radioactivity is found in the F I-peak, so that the specific activity of the F I I - p e a k appears to be practically insignificant.

Kinetics o/8~p incorporation into mitochondrial R N A The incorporation of 3~p into the mitochondria] RNA was followed for 12o rain. Our results show a linear increase of 32p uptake both in vitro and in vivo, with a slight difference after 90 rain (Fig. 2). During the first 15 rain of 3~p incorporation, 85-9 o % of the radioactivity is rapidly retained b y the F 1-peak and maintained during the Biochim. Biophys. Acta, 166 (1968) 394-402

MITOCHONDRIAL RAPIDLY LABELLED R N A

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Fig. 2. I n c o r p o r a t i o n of 3ap into m i t o c h o n d r i a l R N A in vivo ( A - - A ) a n d in vitro ( A - & ) . I n c o r p o r a t i o n in vivo: 3 °/*C s2P/2 5 g b o d y w e i g h t 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 as Na~Hs2PO~ (specific a c t i v i t y , 1. 5 m C / m g ) . R N A f r o m liver e x t r a c t e d a n d r a d i o a c t i v i t y d e t e r m i n e d as d e s c r i b e d in METHODS. I n c o r p o r a t i o n in vitro: I/*C 3~P/ml s t a n d a r d b u f f e r i n c u b a t e d w i t h 3 m g m i t o c h o n d r i a l p r o t e i n p e r m l s t a n d a r d buffer. I n c u b a t i o n s were c a r r i e d o u t a t 37 ° w i t h g e n t l e stirring. Fig. 3. D i s t r i b u t i o n of r a d i o a c t i v i t y in t h e c h r o m a t o g r a p h i c profile of m i t o c h o n d r i a l R N A , labelled in vivo w i t h 32p (3 0 #C/25 g a n i m a l b o d y w e i g h t ) for 6 h. - , A z60 rap; ..... , c o u n t s / m i n ; - - -, m o l a r c o n c e n t r a t i o n of NaC1.

whole period of the experiment in vitro. In vivo, after 6 h the radioactivity increases in the F II-peak, presenting a homogeneous distribution in the chromatographic profile (Fig. 3). We designate the rapidly 32P-labelled RNA obtained in the chromatographic profile of Fig. I from Samples 6 and 7 the F I-fraction and the RNA obtained in the chromatographic profile of Fig. I from Samples 16-17 the F I[-fraction.

Molecular weight The molecular weight of the rapidly labelled RNA (F 1-fraction) was 1.5 • lO5, as determined by the light-scattering method. By converting this value to sedimentation units 38, this would represent a 9.7-S RNA species. Taking into account the codon length, our rapidly labelled RNA would be able to code for nearly 15o amino acids corresponding to a polypeptide chain of 17 ooo average mol. wt. Incorporation o/ [14C]glutamic acid As seen in Table I, our rapidly labelled RNA (F 1-fraction) slightly stimulates the uptake of [14Clglutamic acid into the mitochondrial protein in vitro. This effect is specific, since the F II-fraction proves to be inactive, presenting a slightly inhibitory action. As expected, [14C]glutamic acid incorporation is strongly inhibited by chloramphenicol. This effect may be prevented, if the mitochondria are pre-incubated for 30 min with the F I-fraction. This aspect seems to be significant since the inactive fraction F II is incapable of preventing inhibition by chloramphenicol. The effects described in Table I are highly reproducible. In a series of experiments, the F 1-fraction consistently presented a clear-cut effect, whereas the F II-fraction proved to be inactive. Biochim. Biophys. Acta, 166 (1968) 394-402

400

S. COMOROSAN, A. GASPAR, D. SANDRU

TABLE II HYBRIDIZATION OF MITOCHONDRIAL R N A WITH NUCLEAR AND MITOCHONDRIAL D N A The nuclear D N A ( i i o / , g / m l ) and m i t o c h o n d r i a l D N A (7o/2g/ml) were retained on Millipore filters, H.A., a n d t h e n hybridized w i t h 4.2/zg s2P-labelled R N A p e r ml (specific activity, 15o ooo) in 2 × SSC (o.15 M N a C l - o . o i 5 M s o d i u m citrate (pH 7.o)), for 16 h at 65 °, F o r e x p e r i m e n t a l details see METHODS.

DNA

R N A /DNA × loo

Mitochondrial D N A Nuc]eax D N A

12 o. 7

D N A - R N A complexes As may be seen in Table II, it is evident that the rapidly labelled mitochondrial RNA hybridizes with mitochondrial DNA (12 %) and only at a very low rate (0. 7 %) with nuclear DNA. In this respect, HUMM AND HUMM8~ obtained lower values, but they used unfractionated RNA rather than a specific fraction as in our experiment. Along the same line of evidence, a series of reports 3s-t° have described hybridization experiments between mitochondrial RNA and mitochondrial DNA.

DISCUSSION

The results presented in this paper indicate the existence of a mitochondrialRNA fraction with mRNA characteristics. Contamination with cytoplasmic or bacterial RNA was avoided by ribonuclease treatment and sterile conditions, respectively. The mitochondrial mRNA fraction is rapidly labelled with 3,p, both in vitro and in vivo. Its synthesis seems continuous, a fact supported by the linear incorporation of L14C]leucine in the experiment in vitro for 3 h (ref. 41) and also confirmed by WINTERSBERGER AND TUPPYls using [3H]uracil incorporation into yeast mitochondria. In this respect it may be emphasized that the kinetics of 3zp incorporation into mitochondrial RNA support the point of view that a rapidly synthesized RNA is also made by the isolated mitochondria, i.e., under conditions which preclude mitochondrial multiplication. Indeed, from the study of our chromatographic profiles, it is clear that, during the first 15 min of ~ P incorporation, 85-9 ° % of the radioactivity is already retained by F 1-fraction and maintained during the whole period of the experiment in vitro. Thus, compensation by increased synthesis of other RNA components in the data of Fig. 2 seems unlikely. Our hypothesis suggests that only the F I-fraction represents the "rapidly labelled" RNA, and that the F II-fraction contains a ribosomal-like RNA similar to the fraction described by WINTERSBERGERAND TUPPY1~. Its origin may be nucleolar, and it may be transported to the cytoplasm and taken in by mitochondria. However, the hybridization experiments 39 demonstrated that at least a large portion of the rRN-A of mitochondria is complementary to mitochondrial DNA. Thus, the extramitochondrial origin of this fraction is still open to discussion. Biochim. Biophys. Acta, 166 (1968) 394-402

MIYOCHONDRIAL RAPIDLY LABELLED

RNA

4Ol

The molecular weight of the F I-fraction lies in the range of rat-liver mRN'A: 30 000-800 ooo (see ref. 42) and Escherichia coti mRNA: 8-30 S (see ref. 36). The effect of this fraction on the amino acid incorporation into the mitochondrial protein strongly suggests mRNA characteristics. It is also known that chloramphenicol inhibits amino acid uptake in a cell-free system by preventing the binding of mRNA to ribosomes. When rabbit reticulocyte ribosomes are pre-incubated with polyuridylic acid (poly U), this effect is removed4s. In this respect, our results seem significant: by pre-incubation of mitochondria with a sufficient amount of rapidly labelled RNA, the inhibitory effect of chloramphenicol could be prevented. The inactive fraction F II is incapable of preventing this effect. Yet, in some respects a number of uncertainties remain. Since addition of the rapidly labelled RNA stimulates amino acid incorporation only slightly, the amount of mRNA bound to the mitochondrial ribosomes is presumably large enough to mediate maximal protein synthesis. However, such an addition abolishes the chloramphenicol effect, whereas, otherwise, incorporation is fully chloramphenicolsensitive. In this context the problem of the penetration of small molecules, i.e., tRNA or mRNA, through the mitochondrial membranes is still at issue. BARNETT AND BROWN44 presented evidence that translational macromolecular tRNA's and aminoacyl-RNA synthetases, found in Neurospora mitochondria, are different from those isolated from the cytoplasm. Then again, TAKAHASHIAND HIRAI ~a reported direct evidence for the presence in mitochondria of TMV-infected tobacco leaves of both DNA and assocated polymerase. The possibility of hybrid formation between the rapidly labelled RNA and the mitochondrial DNA suggests the mitochondrial origin of the F I-fraction. The very low percentage of hybridization with nuclear DNA (0.7 %) also supports this point of view. In the light of the above discussion it seems reasonable to conclude that our F I-fraction is a mitochondrial RNA with the characteristics of an mRNA.

REFERENCES I 2 3 4 5 6 7 8 9 io Ii 12 13 14 15 16 17 ]8

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38 39 4° 41 42 43 44

s. COMOROSAN, ,~. GASPAR, D. SANDRU

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Biochim. Biophys. Acta, 166 (1968) 394-4o2