Volume 4 Number 12 December 1977
Nucleic Acids Research .M
Isolation and partial characterization of poly (A)-containing 7.5S messenger RNA from rat liver mitochondria
O.l.Kisselev, V.S.Gaitskhoki and S.A.Neifakh Laboratory of Biochemical Genetics, Institute of Experimental Medicine, 197022 Leningrad, USSR Received 3 November 1977
ABSTRACT
Poly(A)-containing low molecular weight (7.58) messenger RNA was isolated in a highly purified form from both polyribosomes and post-polysonal supernatant of rat liver sitochondria. Both mRNA's contain rather short pol(A) tracts (40-70 mononucleottdes) according to a profilo of their elution from poly(U)-Sepharose column with a gradient of formamide concentration. Both RNA's when added to a preincubated mitochondrial lysate prograed the synthesis of a hydrophobic polypeptide of a molecular weight about 9000 daltons which was soluble in the neutral chloroform-methanol mixture. INTRODUCTION A great number of data are available concern the primary products of the activity of mitochondrial (mt) genome within an animal cell (1-3). The major RNA classes synthesized within mitochondria were identified and the cistrons coding for rRNA and tRNA were localized along the complementary strands of mtDNA (1). Besides, the partial physical maps were constructed for certain regions of mtDNA from HeLa cells (1,2), Xenopus,
Drosophila and some higher animals (3). Messenger RNA is still one of the-poorly understood components of the mitochondrial system of protein synthesis (2,4,5). The analytical approach is the predominant one in the studies on mt mRNA because the preparative isolation and fractionation of this RNA is a very complicated problem. According to in vivo labelling data poly(A)-containing uRNA of animal mitochondria is represented by 8-18 components of different apparent molecular weights (2,5). Besides, several poly(A)lacking mRNA species were described which did not share common C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
4411
Nucleic Acids Research sequences with poly(A)-containing mRNA (6). Some poly(A)-containg mRNA species were identified as transcripts of mtDNA and the distribution of the corresponding coding sequences between complementary strands of mtDNA was established (2) . The number of polypeptides synthesized by mitochondrial translation machinery is from ten to twenty according to data obtained by different groups (7-9). It seems evident that a complete physical and functional mapping of a mt genome is hardly possible without preparative isolation of individual mt mRNA species concurrent with their functional identification in cell-free translation experiments. In this relation an attempt was undertaken in our laboratory to fractionate the total poly(A)-containing RNA from rat liver mitochondria. In this paper the isolation of a highly enriched low molecular weight mRNA species from both purified mt polysomes and postpolysomal supernatant is described. The product of the cell-free translation of these mRNA's in a homologous system was identified as a low molecular weight bydrophobic polypeptide component of mt membrane.
MATERIAI3 AND METHODS Isolation of mitochondria. 1iitochondria were isolated as described (10) and treated with digitonin to remove cytoplasmic contaminations (11). Isolation and purification of mt Polyribosomes. Mitochondrial pellet after digitonin treatment was washed and resuspended in 50 mM tris HCl, pH 7.6, 50 mM potassium chloride, 5 mM magnesium chloride, 5 mM 2-mercaptoethanol, 200 1g/ml heparin (polysomal buffer). Then 20% Triton X-100 was added up to a final concentration 2%. The lysate obtained after 20 min incubation at 00 was clarified (20000g, 10 min) and then layered on the cushion of 2.0M sucrose (10-12 ml). The polysomes were sedimented through a dense sucrose layer at 35000 rpm in a Spinco rotor No.42.I for 14-16 hours (temperature 40C). The supernatant was carefully decanted and served further as a source of post-polysomal RNA. The sucrose layer containing membrane aggregates was discarded and polysomal 4412
Nucleic Acids Research pellet was resuspended in the polJysomal buffer or in 0.IM acetate buffer, pH 6.0 containing 0.IM sodium chloride and 0.002M EDTA.
Sedimentation of polyribosomes Purified polysom. preparations were layered upon a linear (10-30%, w/w) gradient of sucrose concentration containing polysomal buffer and centrifuged in SW 27.1 bucket rotor of the Spinco L2-65B ultracentrifuge at 25000 rpm, 5 hours, 4°C'. The gradients were fractionated from the bottom and the UV profile was recorded at 260 nm using a flow cell adapted to Hitachi spectrophotometer. Aalytical ultracentrifugation. Polysomes dissociated with 0.68 potassium chloride-0.5 mM puromycin were centrifuged in a MSE analytical ultracentrifuge at 45000rpm and 200C.
Isolation of RNA. Polysomal or post-polysomal RNA was isolated according to Perry et al. (12). Fractionation of RNA. Ethanol-precipitated RNA was washed with 3.0M lithium chloride and with 75% ethanol to remove the salts and then dissolved in bidistilled water. After short-time (1-2 hours) dialysis against 10 mM tris-HCl, pH 7.0. the RNA solution (no more then 750 ,gml) was adjusted to 1.5m with sodium chloride and allowed to stand overnight at 00. The precipitate formed was collected at 10000g, dissolved in bidistilled water and reprecipitated with ethanol. The supernatant containing low molecular weight RNA was dialysed shortly and then used for pdlr(U)-Sepharose fractionation. In some cases low molecular weight RNA fractions from several identical experiments were precipitated with ethanol and then pooled for further fractionation.
Poly(U )-Sepharose chromatograpby Poly(U)-Sbpharose chromatography was performed essentially as described earlier (11). RNA was dissolved in 20 mM tris-HCl 4413
Nucleic Acids Research buffer, pH 7.5 to adjust a final RNA concentration to 250-500 pg/ml. Then sodium chloride, EDTA and sodium dodecyl sulphate were added up to final concentrations 0.75M, 0.0021 and 0.2% respectively. All the solutions were treated with diethyl pyrocarbonate before use (13). Poly(U)-Sepharose columns (0.5x3.0 cm) were used for the fractionation. The retained RNA fraction was eluted with 0.2% sodium dodecyl sulphate. In some experiments the elution was made with a linear gradient of formamide concentration (0-80%, v/v) in 20 mM tris-HlC, pH 7.5 - 2 mM EDTA as described by Jelinek et al. (14) in order to estimate the length heterogeneity of poly(A) tracts. Poly(A)-containing RNA from cytoplasmic polysomes isolated as described earlier (15) or synthetic poly(A) were used as standards in these experiments. Eluate was concentrated with polyethylene glycol and RNA was reprecipitated with ethanol. For the cell-free translation experiments RNA was precipitated with ethanol directly from eluate using E.coli tRNA as a carrier. Sucrose gradient centrifugation of RNA RNA preparations were layered upon a linear (5-20%, w/w) sucrose concentration gradient prepared with 0.IM sodium acetate buffer, pH 6.0, 0.IM sodium chloride and 0.002U EDTA. The centrifugation was performed in a SW27.I bucket rotor at 25000 rpm and 40C. The duration of centrifugation is specified in the legends to figures.
Electrophoresis of RNA Electrophoresis of RNA was performed according to Peacock and Dingman (16) using 2.7% or 5% polyacrylamide gels. The gels were either scanned at 260 na or stained with methylene blue. Cell-free translation in a mitochondrial lysate RNA preparations were translated in a mitochondrial lysate from rat liver preincubated for 30 min to deplete the endogenous mlNA activity. The preparation of lysate and conditions for incubation were described earlier (11). The incubation was stopped with the addition of 80% methanol. Hydrophobic material of the precipitate was extracted with neutral chloroform-methanol (2:1, v/v). 4414
Nucleic Acids Research Electrophoresis of the product of cell-free translation
The electrophoresis of polypeptides synthesized in a cell-free system was performed in 7.5% polyacrylamide gels containing sodium dodecyl sulphate using tris-glycine buffer (11).
]RESULTS The sedimentation profile of mt polyribosomes is given in the figure IA. It is seen that mt polysomes sediment in a sucrose gradient rather slowly. Their sedimentation coefficients are in the range of 80-1908 corresponding to 2-9 monoribosomes per one mRNA molecule (17). On dissociation mt polysomes release 28S and 40S subparticles which were demonstrated with the aid of analytical ultracentrifugation (fig. IB). It is evident from this figure that there is no contaminating subunits of cytoplasmic ribosomes in the dissociated mt polysomal material. RNA extracted from mt polysomes is represented by two major components (11S and 16S) which were separated with sucrose gradient centrifugation (fig. 2). The 288 and 18S rRNA's of cytoplasmic origin were absent completely. Thus a conclusion could be drawn that polysomal preparations which served further as a starting material for mRNA isolation are in a highly purified state and practically free from cytoplasmic contaminations. Besides, it should be noted that neither mt lysate nor post-polysomal fraction contained ribonucleoprotein material with the properties of monosomes or ribosomal subunits. After 1.5M sodium chloride treatment about 20% of the total mt polysomal RNA remained in a supernatant. The sedimentation analysis showed that this fraction contained a bulk of 4S tRNA and a material sedimenting in a range of 5-8S (fig. 3A). A similar sedimentation behavior was also a characteristic of the RNA isolated from post-polysomal supernatant which contained a homogeneous 7.5S peak (fig. 3B). The electrophoresis in 2.7% polyacrylamide gel showed that both polysomal and post-polysomal low molecular weight RNA's contain a homogeneous 7.5S component differing significantly from rRNA in the electrophoretic mobility (Fig. 4).
4415
Nucleic Acids Research
I.
Fig. 1. Sedimentation of mitochondrial polyribosomes in sucrose density gradient and analytical ultracentrifugation of the dissociated polysomes (insert)
0,5
) Fig. 2. Sedimentation of mitochondrial polysomal RNA ( and cytoplasmic RNA ( - - -) in a sucrose concentration gradient. Centrifugation time - 18 hours. 4416
Nucleic Acids Research
F r a c t I ns
Fig. 3. Gradient centrifugation of a low molecular weight RNA isIoated from mitochondrial polysomes (A) and from post-polysomal supernatant (B). Centrifugation time - 21 .5 hours. 21 s
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Electrophoresis of mitochondrial RNA preparations in 27W 6lyacrylamide gel. A. Total po lysomal RNA ( - -- ) and ). B. Total post-polysoits 1.51M NaCl-soluble fraction ( mal RNA.
Fig. 4
4417
Nucleic Acids Research In an attempt to fractionate further a low molecular weight mt RNA preparations the poly(U)-Sepharose chromatography was used. A certa±n poly(A)-containing fraction was found which was retained on the columns and eluted with 15-30% formamide (Fig. 5). In this respect mt poly(A)-containing RNA differed from cytoplasmic one which was resolved into three fractions eluted with higher formamide concentrations (30-40%) (Fig. 6). The synthetic poly(A) (average chain length about 300 nucleotides) was eluted from the poly(U)-Sepharose columns only with very high formamide concentrations (60-70%). The length of a poly(A)- tract in mt mRNA was calculated to be as short as 40-70 AMP residues, while a cytoplasmic mRNA is represented by three subfractions having poly(A)-tracts of different average length (50, 100 and 150-200 mononucleotides). As is shown in Fig. 7, poly(U)-chromatography resulted in a complete removal of tRNA from the retained fractions which sedimented in a sucrose gradient as 7.5S components. Poat-polysomal 7.5S RNA was more homogeneous than 7.5S RNS from mt polysomes. Gradient fractions containing 7.5S RNA were pooled and RNA was precipitated with ethanol using E.coli tRNA as a carrier and then added to a preincubated mt lysate. It was shown that polysomal 7.5S poly(A)-containing mRNA produced 4-6-fold stimulation of 14C-amino acid incorporation into acidE260
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ig. 5. Poly(U)-Sepharose chromatography of 1.5M NaCl-soluble RNA from mitochondrial polysomes (A) and from post-polysomal
supernatant (B).
4418
Nucleic Acids Research -insoluble material ( fig. 8A). Post-polysomal poly(A)-containing 7.5S mRNA produced 3-4-fold stimulation (fi,g. 8B). In both cases about 70-80% of the product of translation could be extracted from the incubation medium with a neutral chloroform-me-
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somalRNA (
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Fig. 7. Sucrose gradient sedimentation of poly(A)-containing 7A5SRNA from mitochondrial polysomes (A) and from post-polysomal supernatant (B). Centrifugation time - 34 hours. 4419
Nucleic Acids Research
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Fig;. 8. The template activity of 7.5S mPRNA from polysomes (A) and from post-polysomal supernatant (B) in the preincubated mitochondrial lysate. I - total acid-insoluble radioactivity; 2 - radioactivity of chloroform-methanol extracted polypeptides; 3 - the same without RNA &dded.
thanol mixture. The moderate stimulating effect of the RNA's added could be due to a high activity of exogeneous nucleases in the mt lysate (18). The gel electrophoresis under denaturing conditions revealed that chloroform-metbanol-extractable translation product was similar to a hydrophobic polypeptide extracted from liver mitochondria with chloroform-methanol according to Sierra and Tzagoloff (19). The molecular weigth of this polypeptide estimated in several independent experiments was in a range of 8000-9000 daltons (fig. 9).
The control experiments were performed in which the preincubated mt lysate was incubated further with 14C-amino acids and the newly formed polypeptides extractable with chloroformmethanol were analysed electrophoretically. It was shown that only a minor fraction of endogeneous product co-migrated with the marker hydrophobic polypeptide.
4420
Nucleic Acids Research
1~~~~~~~~~~~~~C
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20
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20
30
40
SI ce s
Fig 9. Gel electrophoresis of a marker hydrophobic polypep), of endogeneous translation tide from mitochondria (A, the products of translation of 7.5S product (A,. ).of mRNA from potYsomes (B) and from post-polysomal supernatant (C). DISCUSSION We did not find aay data on the preparative fractionation of mt mRNA and isolation of its individual species in the literature available. Principally, two approaches could be used in the studies of such a kind: i) the immunochemical fractionation of at polysomes with the subsequent isolation of the mRNA concerned (17) and ii) the direct fractionation of the total mRNA into individ,ual species and their identification in the cell-free tranalation experiments. It should be noted that immunoprecipitation of at polysomes is significantly hampered by the presence of contaminatin hydrophobic proteins causing a non-specific aggregation of polysomes. Hence, the second approach was chosen in our experiments. 7.5S poly(A)-containing mRNA was isolated from both mt lolysomes and post-polysomal fraction. Polysoma 7.5S mRNA contained additional minor RNA components while post-polysomal 7.5S mRNA was-highly homogeneous according to sedimentation data. Tlhe preparations of 7.5S mRNA from both sources programmed the synthesis of a hydrophobic polypeptide with a molecular 4421
Nucleic Acids Research weight about 9000 daltons when added to a preincubated mitochondrial lysate. A similar mRNA fraction was found by Attardi et al. (2) in the preparations of in vivo labelled total mt poly(A)-containing RNA from HeLa cells. A peculiar polypeptide fraction of the apparent molecular weight about 8000-10000 daltons was identified as a product of both in vivo and in vitro mitochondrial translation (20-23). At least a part of the polypeptide components of this fraction could be extracted with neutral chloroform-methanol (22). The functions of this polypeptide in mitochondria remain to be understood. It is unknown also whether this polypeptide is a part of any oligomeric enzymatic complex of the inner mt membrane. With respect' to both molecular size and marked hydrophobicity it is similar to the subunit IX of the ATPase complex which was described in detail in the studies on the composition of ATPase and subcellular localization of the synthesis of its subunits in an yeast cell (19). In a number of works it was shown that polypeptide fraction synthesized within mitochondria and extractable with neutral chloroform-methanol is composed of at least two components (23). Hence, the identification of the product coded for by 7.5S mRNA from mitochondria could be hardly unequivocal. The product of the cell-free translation of 7.5S mRNA could be well different from the corresponding in vivo synthesized polypeptide. Firstly, the newly formed polypeptide could contain some additional amino acid sequences, in particular "anchor" NH2-terminal extensions which are known to be extremely hydrophobic (24). Such sequences could alter markedly the solubility of this polypeptide in both aqueous and organic solvents. Secondly, the product of in vitro translation of 7.5S mRNA could be well a fragment of a native polypeptide formed as a result of either proteolytic attack (25) or a premature termination which seems to be less probable in a homologous cell-free system. Of significant interest is the comparison of the molecular size of the poly(A)-containing 7.5S mRNA described here with that of the encoded polypeptide. From the molecular weight of the translation product it could be calculated that it is 4422
Nucleic Acids Research composed of 80-90 amino acid residues. The expected length of the coding part of the corresponding mRNA is 240-370 nucleotides. The apparent molecular weight of 7.58 mRNA calculated from its sedimentation and electrophoretic behaviour is about 1.1-1.2x105 daltons corresponding to a chain length about 360 nucleotides. This value could well accomodate a coding sequence of 240-270 nucleotides leaving 100 nucleotides in excess for poly(A) and other untranslated sequences. It has been shown in our earlier work (10) that the dissociation of mt polyribosomes with potassium chloride - puromycin resulted in the release of mRIA-containing ribonucleoprotein (mRNP); IOS mRNP being a major component. The size of this predominant mRNP corresponds well to the size of 7.5S mRNA. It seems possible that 7.5S mRNA coding for a hydrophobic polypeptide is a major mRNA species both in mt polysomes and in the non-polysomal RNP containing untranslated mRNA. Now we are studying a homology relationships between polysomal and post-polysomal poly(A)-containing 7.5S mRNA's using complementary DNA probe synthesized with reverse transcriptase. Some properties of post-polysomal mRNP from mitochondria are under study as well. The work was supported by grant No. 03/181/38 from the World Health Organization.
REFERENCES 1. Wu, M., Davidson, N., Attardi, G. and Aloni, J. (1972) J. Mol. Biol. 71, 81-93. 2. Attardi, G., Albring, M., AmalriO, F., Ge$and, R., Griffith, I., Iynch, D., Merkel, Ch., Murpby, W. andy Ojala, D. (1976) in Genetics and Biogenesis of Mitochondria and Chloroplasts. Th.Bf9gher et al., eds, pp. 573-585, Biomed. Press, Amsterdam. 3. Upholl, W.B. and Dawid, I.B., ibid, pp. 587-592. 4. Avadbani, N.G., Lewis, F.S. and Rutman, R.I. (1975) Sub-CelL Biochem. 4, 93-145. 5. Spradling, A., Pardue, M.L. and Penman, S. (1977) J. Mol. Biol. 109, 559-587. 6. Lewis, F.S., Rutman, R.I. and Avadhani, N.G. (1976) Biochemistry 15, 3367-3372. 7. Coote, I.L. and Work, T.S. (1971) Eur. J. Biochem. 23, 564-
574.
8. Kuzela, S., Krempasky, V. and Kolarov, I. (1975) Eur. J. Biochem. 47, 165-171. 9. Schatz, G. and Mason, T. (1974) Ann. Rev. Biochem. 45, 51-87 4423
Nucleic Acids Research 10. Kisselev, O.I., Gaitskhoki, V.S., Klimov, N.A. (1975) Mol. Biol. Repts 2, 143-149. 11. Kisselev, O.I., Gaitskhoki, V.S. and Neifakh, S.A. (1977) Mol. Cell. Biochem. 14, 91-96. 12. Perry, R., LaTorre, D., Kelley, D. (1972) Biochim. Biophys. Acta 262, 220-226. 13. Palmiter, R.D. (1973) J. Biol. Chem. 248, 2095-2106. 14. Jelinek, W., Adesnik, M., Salditt, M., Sheiness, D. Wall, R., Molloy, G., Phillipson, L., Darnell, I.E. (19755 J. Mol. Biol. 75, 515-532. 15. Gaitskhoki, V.S., Kisselev, O.I. and Klimov, N.A. (1973) FEBS Letters 37, 260-263. 16. Peacock, A.C. and Dingman, C.W. (1967) Biochemistry 6, 18161827. 17. Gaitskhoki, V.S., Kisselev, 0.I., Klimov, N.A., Monakhov, N.K., Mukha, G.V., Schwrtzman, A.L. and Neifakh, S.A. (1974) fB Letters 43, 151-154. 18. Gaitskhoki, V.S., Golubkov, V.I., Grabovskaya, K.B., Kisselev, O.I., Totolyan, A.A. and Neifakh, S.A. (1974) Doklady Akademii Nauk SSSR (Russ.) 215, 1487-1490. 19. Sierra, M.F., Tzagoloff, A. (1973) Proc. Nat. Acad. Sci. USA 70, 3155-3159. 20. Lederman, M. and Attardi, G. (1973) J. Mol. Biol. 78, 275285. 21. Kuntzel, H. and Blossey, H.-Ch. (1974) Eur. J. Biochem. 47, 165-171. 22. Kadenbach,B. and Hadvary, P. (1973) Eur. J. Biochem. 32,
3435349-
23. Dianoux, A.C., Bof, M., Cesarini, R., Reboul, A. and Vignais, P.V. (1976) Eur. J. Biochem. 67, 61-67. 24. Blobel, G. and Dobberstein, B. (1975) J. Cell. Biol. 67, 855-851. 25. Rubio, V. and Grisolia, S. (1977) FEBS Letters 75, 281284.
4424
Nucleic Acids Research .M
Isolation and partial characterization of poly (A)-containing 7.5S messenger RNA from rat liver mitochondria
O.l.Kisselev, V.S.Gaitskhoki and S.A.Neifakh Laboratory of Biochemical Genetics, Institute of Experimental Medicine, 197022 Leningrad, USSR Received 3 November 1977
ABSTRACT
Poly(A)-containing low molecular weight (7.58) messenger RNA was isolated in a highly purified form from both polyribosomes and post-polysonal supernatant of rat liver sitochondria. Both mRNA's contain rather short pol(A) tracts (40-70 mononucleottdes) according to a profilo of their elution from poly(U)-Sepharose column with a gradient of formamide concentration. Both RNA's when added to a preincubated mitochondrial lysate prograed the synthesis of a hydrophobic polypeptide of a molecular weight about 9000 daltons which was soluble in the neutral chloroform-methanol mixture. INTRODUCTION A great number of data are available concern the primary products of the activity of mitochondrial (mt) genome within an animal cell (1-3). The major RNA classes synthesized within mitochondria were identified and the cistrons coding for rRNA and tRNA were localized along the complementary strands of mtDNA (1). Besides, the partial physical maps were constructed for certain regions of mtDNA from HeLa cells (1,2), Xenopus,
Drosophila and some higher animals (3). Messenger RNA is still one of the-poorly understood components of the mitochondrial system of protein synthesis (2,4,5). The analytical approach is the predominant one in the studies on mt mRNA because the preparative isolation and fractionation of this RNA is a very complicated problem. According to in vivo labelling data poly(A)-containing uRNA of animal mitochondria is represented by 8-18 components of different apparent molecular weights (2,5). Besides, several poly(A)lacking mRNA species were described which did not share common C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
4411
Nucleic Acids Research sequences with poly(A)-containing mRNA (6). Some poly(A)-containg mRNA species were identified as transcripts of mtDNA and the distribution of the corresponding coding sequences between complementary strands of mtDNA was established (2) . The number of polypeptides synthesized by mitochondrial translation machinery is from ten to twenty according to data obtained by different groups (7-9). It seems evident that a complete physical and functional mapping of a mt genome is hardly possible without preparative isolation of individual mt mRNA species concurrent with their functional identification in cell-free translation experiments. In this relation an attempt was undertaken in our laboratory to fractionate the total poly(A)-containing RNA from rat liver mitochondria. In this paper the isolation of a highly enriched low molecular weight mRNA species from both purified mt polysomes and postpolysomal supernatant is described. The product of the cell-free translation of these mRNA's in a homologous system was identified as a low molecular weight bydrophobic polypeptide component of mt membrane.
MATERIAI3 AND METHODS Isolation of mitochondria. 1iitochondria were isolated as described (10) and treated with digitonin to remove cytoplasmic contaminations (11). Isolation and purification of mt Polyribosomes. Mitochondrial pellet after digitonin treatment was washed and resuspended in 50 mM tris HCl, pH 7.6, 50 mM potassium chloride, 5 mM magnesium chloride, 5 mM 2-mercaptoethanol, 200 1g/ml heparin (polysomal buffer). Then 20% Triton X-100 was added up to a final concentration 2%. The lysate obtained after 20 min incubation at 00 was clarified (20000g, 10 min) and then layered on the cushion of 2.0M sucrose (10-12 ml). The polysomes were sedimented through a dense sucrose layer at 35000 rpm in a Spinco rotor No.42.I for 14-16 hours (temperature 40C). The supernatant was carefully decanted and served further as a source of post-polysomal RNA. The sucrose layer containing membrane aggregates was discarded and polysomal 4412
Nucleic Acids Research pellet was resuspended in the polJysomal buffer or in 0.IM acetate buffer, pH 6.0 containing 0.IM sodium chloride and 0.002M EDTA.
Sedimentation of polyribosomes Purified polysom. preparations were layered upon a linear (10-30%, w/w) gradient of sucrose concentration containing polysomal buffer and centrifuged in SW 27.1 bucket rotor of the Spinco L2-65B ultracentrifuge at 25000 rpm, 5 hours, 4°C'. The gradients were fractionated from the bottom and the UV profile was recorded at 260 nm using a flow cell adapted to Hitachi spectrophotometer. Aalytical ultracentrifugation. Polysomes dissociated with 0.68 potassium chloride-0.5 mM puromycin were centrifuged in a MSE analytical ultracentrifuge at 45000rpm and 200C.
Isolation of RNA. Polysomal or post-polysomal RNA was isolated according to Perry et al. (12). Fractionation of RNA. Ethanol-precipitated RNA was washed with 3.0M lithium chloride and with 75% ethanol to remove the salts and then dissolved in bidistilled water. After short-time (1-2 hours) dialysis against 10 mM tris-HCl, pH 7.0. the RNA solution (no more then 750 ,gml) was adjusted to 1.5m with sodium chloride and allowed to stand overnight at 00. The precipitate formed was collected at 10000g, dissolved in bidistilled water and reprecipitated with ethanol. The supernatant containing low molecular weight RNA was dialysed shortly and then used for pdlr(U)-Sepharose fractionation. In some cases low molecular weight RNA fractions from several identical experiments were precipitated with ethanol and then pooled for further fractionation.
Poly(U )-Sepharose chromatograpby Poly(U)-Sbpharose chromatography was performed essentially as described earlier (11). RNA was dissolved in 20 mM tris-HCl 4413
Nucleic Acids Research buffer, pH 7.5 to adjust a final RNA concentration to 250-500 pg/ml. Then sodium chloride, EDTA and sodium dodecyl sulphate were added up to final concentrations 0.75M, 0.0021 and 0.2% respectively. All the solutions were treated with diethyl pyrocarbonate before use (13). Poly(U)-Sepharose columns (0.5x3.0 cm) were used for the fractionation. The retained RNA fraction was eluted with 0.2% sodium dodecyl sulphate. In some experiments the elution was made with a linear gradient of formamide concentration (0-80%, v/v) in 20 mM tris-HlC, pH 7.5 - 2 mM EDTA as described by Jelinek et al. (14) in order to estimate the length heterogeneity of poly(A) tracts. Poly(A)-containing RNA from cytoplasmic polysomes isolated as described earlier (15) or synthetic poly(A) were used as standards in these experiments. Eluate was concentrated with polyethylene glycol and RNA was reprecipitated with ethanol. For the cell-free translation experiments RNA was precipitated with ethanol directly from eluate using E.coli tRNA as a carrier. Sucrose gradient centrifugation of RNA RNA preparations were layered upon a linear (5-20%, w/w) sucrose concentration gradient prepared with 0.IM sodium acetate buffer, pH 6.0, 0.IM sodium chloride and 0.002U EDTA. The centrifugation was performed in a SW27.I bucket rotor at 25000 rpm and 40C. The duration of centrifugation is specified in the legends to figures.
Electrophoresis of RNA Electrophoresis of RNA was performed according to Peacock and Dingman (16) using 2.7% or 5% polyacrylamide gels. The gels were either scanned at 260 na or stained with methylene blue. Cell-free translation in a mitochondrial lysate RNA preparations were translated in a mitochondrial lysate from rat liver preincubated for 30 min to deplete the endogenous mlNA activity. The preparation of lysate and conditions for incubation were described earlier (11). The incubation was stopped with the addition of 80% methanol. Hydrophobic material of the precipitate was extracted with neutral chloroform-methanol (2:1, v/v). 4414
Nucleic Acids Research Electrophoresis of the product of cell-free translation
The electrophoresis of polypeptides synthesized in a cell-free system was performed in 7.5% polyacrylamide gels containing sodium dodecyl sulphate using tris-glycine buffer (11).
]RESULTS The sedimentation profile of mt polyribosomes is given in the figure IA. It is seen that mt polysomes sediment in a sucrose gradient rather slowly. Their sedimentation coefficients are in the range of 80-1908 corresponding to 2-9 monoribosomes per one mRNA molecule (17). On dissociation mt polysomes release 28S and 40S subparticles which were demonstrated with the aid of analytical ultracentrifugation (fig. IB). It is evident from this figure that there is no contaminating subunits of cytoplasmic ribosomes in the dissociated mt polysomal material. RNA extracted from mt polysomes is represented by two major components (11S and 16S) which were separated with sucrose gradient centrifugation (fig. 2). The 288 and 18S rRNA's of cytoplasmic origin were absent completely. Thus a conclusion could be drawn that polysomal preparations which served further as a starting material for mRNA isolation are in a highly purified state and practically free from cytoplasmic contaminations. Besides, it should be noted that neither mt lysate nor post-polysomal fraction contained ribonucleoprotein material with the properties of monosomes or ribosomal subunits. After 1.5M sodium chloride treatment about 20% of the total mt polysomal RNA remained in a supernatant. The sedimentation analysis showed that this fraction contained a bulk of 4S tRNA and a material sedimenting in a range of 5-8S (fig. 3A). A similar sedimentation behavior was also a characteristic of the RNA isolated from post-polysomal supernatant which contained a homogeneous 7.5S peak (fig. 3B). The electrophoresis in 2.7% polyacrylamide gel showed that both polysomal and post-polysomal low molecular weight RNA's contain a homogeneous 7.5S component differing significantly from rRNA in the electrophoretic mobility (Fig. 4).
4415
Nucleic Acids Research
I.
Fig. 1. Sedimentation of mitochondrial polyribosomes in sucrose density gradient and analytical ultracentrifugation of the dissociated polysomes (insert)
0,5
) Fig. 2. Sedimentation of mitochondrial polysomal RNA ( and cytoplasmic RNA ( - - -) in a sucrose concentration gradient. Centrifugation time - 18 hours. 4416
Nucleic Acids Research
F r a c t I ns
Fig. 3. Gradient centrifugation of a low molecular weight RNA isIoated from mitochondrial polysomes (A) and from post-polysomal supernatant (B). Centrifugation time - 21 .5 hours. 21 s
14.S l o,
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Electrophoresis of mitochondrial RNA preparations in 27W 6lyacrylamide gel. A. Total po lysomal RNA ( - -- ) and ). B. Total post-polysoits 1.51M NaCl-soluble fraction ( mal RNA.
Fig. 4
4417
Nucleic Acids Research In an attempt to fractionate further a low molecular weight mt RNA preparations the poly(U)-Sepharose chromatography was used. A certa±n poly(A)-containing fraction was found which was retained on the columns and eluted with 15-30% formamide (Fig. 5). In this respect mt poly(A)-containing RNA differed from cytoplasmic one which was resolved into three fractions eluted with higher formamide concentrations (30-40%) (Fig. 6). The synthetic poly(A) (average chain length about 300 nucleotides) was eluted from the poly(U)-Sepharose columns only with very high formamide concentrations (60-70%). The length of a poly(A)- tract in mt mRNA was calculated to be as short as 40-70 AMP residues, while a cytoplasmic mRNA is represented by three subfractions having poly(A)-tracts of different average length (50, 100 and 150-200 mononucleotides). As is shown in Fig. 7, poly(U)-chromatography resulted in a complete removal of tRNA from the retained fractions which sedimented in a sucrose gradient as 7.5S components. Poat-polysomal 7.5S RNA was more homogeneous than 7.5S RNS from mt polysomes. Gradient fractions containing 7.5S RNA were pooled and RNA was precipitated with ethanol using E.coli tRNA as a carrier and then added to a preincubated mt lysate. It was shown that polysomal 7.5S poly(A)-containing mRNA produced 4-6-fold stimulation of 14C-amino acid incorporation into acidE260
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supernatant (B).
4418
Nucleic Acids Research -insoluble material ( fig. 8A). Post-polysomal poly(A)-containing 7.5S mRNA produced 3-4-fold stimulation (fi,g. 8B). In both cases about 70-80% of the product of translation could be extracted from the incubation medium with a neutral chloroform-me-
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Nucleic Acids Research
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Fig;. 8. The template activity of 7.5S mPRNA from polysomes (A) and from post-polysomal supernatant (B) in the preincubated mitochondrial lysate. I - total acid-insoluble radioactivity; 2 - radioactivity of chloroform-methanol extracted polypeptides; 3 - the same without RNA &dded.
thanol mixture. The moderate stimulating effect of the RNA's added could be due to a high activity of exogeneous nucleases in the mt lysate (18). The gel electrophoresis under denaturing conditions revealed that chloroform-metbanol-extractable translation product was similar to a hydrophobic polypeptide extracted from liver mitochondria with chloroform-methanol according to Sierra and Tzagoloff (19). The molecular weigth of this polypeptide estimated in several independent experiments was in a range of 8000-9000 daltons (fig. 9).
The control experiments were performed in which the preincubated mt lysate was incubated further with 14C-amino acids and the newly formed polypeptides extractable with chloroformmethanol were analysed electrophoretically. It was shown that only a minor fraction of endogeneous product co-migrated with the marker hydrophobic polypeptide.
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Nucleic Acids Research
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Fig 9. Gel electrophoresis of a marker hydrophobic polypep), of endogeneous translation tide from mitochondria (A, the products of translation of 7.5S product (A,. ).of mRNA from potYsomes (B) and from post-polysomal supernatant (C). DISCUSSION We did not find aay data on the preparative fractionation of mt mRNA and isolation of its individual species in the literature available. Principally, two approaches could be used in the studies of such a kind: i) the immunochemical fractionation of at polysomes with the subsequent isolation of the mRNA concerned (17) and ii) the direct fractionation of the total mRNA into individ,ual species and their identification in the cell-free tranalation experiments. It should be noted that immunoprecipitation of at polysomes is significantly hampered by the presence of contaminatin hydrophobic proteins causing a non-specific aggregation of polysomes. Hence, the second approach was chosen in our experiments. 7.5S poly(A)-containing mRNA was isolated from both mt lolysomes and post-polysomal fraction. Polysoma 7.5S mRNA contained additional minor RNA components while post-polysomal 7.5S mRNA was-highly homogeneous according to sedimentation data. Tlhe preparations of 7.5S mRNA from both sources programmed the synthesis of a hydrophobic polypeptide with a molecular 4421
Nucleic Acids Research weight about 9000 daltons when added to a preincubated mitochondrial lysate. A similar mRNA fraction was found by Attardi et al. (2) in the preparations of in vivo labelled total mt poly(A)-containing RNA from HeLa cells. A peculiar polypeptide fraction of the apparent molecular weight about 8000-10000 daltons was identified as a product of both in vivo and in vitro mitochondrial translation (20-23). At least a part of the polypeptide components of this fraction could be extracted with neutral chloroform-methanol (22). The functions of this polypeptide in mitochondria remain to be understood. It is unknown also whether this polypeptide is a part of any oligomeric enzymatic complex of the inner mt membrane. With respect' to both molecular size and marked hydrophobicity it is similar to the subunit IX of the ATPase complex which was described in detail in the studies on the composition of ATPase and subcellular localization of the synthesis of its subunits in an yeast cell (19). In a number of works it was shown that polypeptide fraction synthesized within mitochondria and extractable with neutral chloroform-methanol is composed of at least two components (23). Hence, the identification of the product coded for by 7.5S mRNA from mitochondria could be hardly unequivocal. The product of the cell-free translation of 7.5S mRNA could be well different from the corresponding in vivo synthesized polypeptide. Firstly, the newly formed polypeptide could contain some additional amino acid sequences, in particular "anchor" NH2-terminal extensions which are known to be extremely hydrophobic (24). Such sequences could alter markedly the solubility of this polypeptide in both aqueous and organic solvents. Secondly, the product of in vitro translation of 7.5S mRNA could be well a fragment of a native polypeptide formed as a result of either proteolytic attack (25) or a premature termination which seems to be less probable in a homologous cell-free system. Of significant interest is the comparison of the molecular size of the poly(A)-containing 7.5S mRNA described here with that of the encoded polypeptide. From the molecular weight of the translation product it could be calculated that it is 4422
Nucleic Acids Research composed of 80-90 amino acid residues. The expected length of the coding part of the corresponding mRNA is 240-370 nucleotides. The apparent molecular weight of 7.58 mRNA calculated from its sedimentation and electrophoretic behaviour is about 1.1-1.2x105 daltons corresponding to a chain length about 360 nucleotides. This value could well accomodate a coding sequence of 240-270 nucleotides leaving 100 nucleotides in excess for poly(A) and other untranslated sequences. It has been shown in our earlier work (10) that the dissociation of mt polyribosomes with potassium chloride - puromycin resulted in the release of mRIA-containing ribonucleoprotein (mRNP); IOS mRNP being a major component. The size of this predominant mRNP corresponds well to the size of 7.5S mRNA. It seems possible that 7.5S mRNA coding for a hydrophobic polypeptide is a major mRNA species both in mt polysomes and in the non-polysomal RNP containing untranslated mRNA. Now we are studying a homology relationships between polysomal and post-polysomal poly(A)-containing 7.5S mRNA's using complementary DNA probe synthesized with reverse transcriptase. Some properties of post-polysomal mRNP from mitochondria are under study as well. The work was supported by grant No. 03/181/38 from the World Health Organization.
REFERENCES 1. Wu, M., Davidson, N., Attardi, G. and Aloni, J. (1972) J. Mol. Biol. 71, 81-93. 2. Attardi, G., Albring, M., AmalriO, F., Ge$and, R., Griffith, I., Iynch, D., Merkel, Ch., Murpby, W. andy Ojala, D. (1976) in Genetics and Biogenesis of Mitochondria and Chloroplasts. Th.Bf9gher et al., eds, pp. 573-585, Biomed. Press, Amsterdam. 3. Upholl, W.B. and Dawid, I.B., ibid, pp. 587-592. 4. Avadbani, N.G., Lewis, F.S. and Rutman, R.I. (1975) Sub-CelL Biochem. 4, 93-145. 5. Spradling, A., Pardue, M.L. and Penman, S. (1977) J. Mol. Biol. 109, 559-587. 6. Lewis, F.S., Rutman, R.I. and Avadhani, N.G. (1976) Biochemistry 15, 3367-3372. 7. Coote, I.L. and Work, T.S. (1971) Eur. J. Biochem. 23, 564-
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8. Kuzela, S., Krempasky, V. and Kolarov, I. (1975) Eur. J. Biochem. 47, 165-171. 9. Schatz, G. and Mason, T. (1974) Ann. Rev. Biochem. 45, 51-87 4423
Nucleic Acids Research 10. Kisselev, O.I., Gaitskhoki, V.S., Klimov, N.A. (1975) Mol. Biol. Repts 2, 143-149. 11. Kisselev, O.I., Gaitskhoki, V.S. and Neifakh, S.A. (1977) Mol. Cell. Biochem. 14, 91-96. 12. Perry, R., LaTorre, D., Kelley, D. (1972) Biochim. Biophys. Acta 262, 220-226. 13. Palmiter, R.D. (1973) J. Biol. Chem. 248, 2095-2106. 14. Jelinek, W., Adesnik, M., Salditt, M., Sheiness, D. Wall, R., Molloy, G., Phillipson, L., Darnell, I.E. (19755 J. Mol. Biol. 75, 515-532. 15. Gaitskhoki, V.S., Kisselev, O.I. and Klimov, N.A. (1973) FEBS Letters 37, 260-263. 16. Peacock, A.C. and Dingman, C.W. (1967) Biochemistry 6, 18161827. 17. Gaitskhoki, V.S., Kisselev, 0.I., Klimov, N.A., Monakhov, N.K., Mukha, G.V., Schwrtzman, A.L. and Neifakh, S.A. (1974) fB Letters 43, 151-154. 18. Gaitskhoki, V.S., Golubkov, V.I., Grabovskaya, K.B., Kisselev, O.I., Totolyan, A.A. and Neifakh, S.A. (1974) Doklady Akademii Nauk SSSR (Russ.) 215, 1487-1490. 19. Sierra, M.F., Tzagoloff, A. (1973) Proc. Nat. Acad. Sci. USA 70, 3155-3159. 20. Lederman, M. and Attardi, G. (1973) J. Mol. Biol. 78, 275285. 21. Kuntzel, H. and Blossey, H.-Ch. (1974) Eur. J. Biochem. 47, 165-171. 22. Kadenbach,B. and Hadvary, P. (1973) Eur. J. Biochem. 32,
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23. Dianoux, A.C., Bof, M., Cesarini, R., Reboul, A. and Vignais, P.V. (1976) Eur. J. Biochem. 67, 61-67. 24. Blobel, G. and Dobberstein, B. (1975) J. Cell. Biol. 67, 855-851. 25. Rubio, V. and Grisolia, S. (1977) FEBS Letters 75, 281284.
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