Expression of the mitochondrial genome in HeLa cells

J. Mol. Biol. (1972) 70, 375-381

LETTER TO TEE EDITOR

Expression of the Mitochondrial

Genome in HeLa Cells

XII. Relationship between Mitochondrial Fast-sedimenting RNA Components and Ribosomal and 4 s RNA About 40% of the mitochondrial RNA labeled in a five-minute [5-3H]uridine pulse in HeLa cells decays within one minute after blocking further RNA synthesis with ethidium bromide and actinomycm D. The decay involves a sharp decrease in the amount of fast-sedimenting pulse-labeled mitochondrial RNA molecules, which is only in minor part compensated by the accumulation of 16 s, 12 s and 4 s RNA. The data strongly suggest that these discrete species derive from larger precursors and, furthermore, that they are synthesized in a fairly

co-ordinate fashion. The occurrence in the mitochondrial fraction from HeLa cells of rapidly labeled heterogeneous RNA sedimenting between 4 s and more than 50 s, coded by mitochondrial-DNA, was previously reported from this laboratory (Attardi & Attardi, 1967,1968; Attardi et al., 1970). The relationship between this heterogeneous RNA and the discrete RNA species, with sedimentation coefficients of 16 s, 12 s and 4 s, which are also transcribed from m&DNA? is not known (Attardi et al., 1970; Penman et al., 1970) ; in particular, it is not known whether the heterogeneous RNA includes larger size precursors of the discrete species. The observation that in the newly synthesized RNA the fast-sedimenting components become labeled faster than the slower sedimenting ones and the evidence that the H strand of mit-DNA (which codes for the 16 S, 12 s and most of the 4 s RNA species (Aloni & Attardi, 1971a)) is completely or almost completely transcribed (Aloni & Attardi (1971b)), have suggested that this transcription might occur in the form of continuous long RNA chains, destined to be processed to smaller size functional molecules. The isolation, reported in the previous paper in this series (Aloni & Attardi, 1972), of transcription complexes of mit-DNA with growing RNA chains as long as expected for complete transcripts, is in agreement with this possibility. In this paper we present evidence indicating that a substantial portion of the rapidly labeled mitochondrial RNA has a very short half-life and strongly suggesting that the mitochondrial16 s and 12 s ribosomal RNA and 4 s RNA derive from precursors of larger size and are synthesized iu a fairly co-ordinate manner. The methods for preparation of the EDTA-washed and isopycnically-purified mitochondrial fraction, extraction and analysis of RNA and RNA/DNA hybridization with separated H or L m&DNA strands, have been described in detail in previous reports (Aloni & Attardi, 1971c, 1972; Attardi & Attardi, 1971). HeLa cells were labeled for 6 minutes with [5-3H]uridine, and then treated for different lengths of time with ethidium bromide at 1 pg/ml. and actinomycin D at 10 pg/ml. (a concentration previously shown to be adequate to block mitochondrial RNA synthesis (Attardi Q Attardi, 1967)). As shown in Figure 1, within one minute after addition of the drugs, a decrease of about 40% occurred in the amount of label present t Abbreviations

used: mit-DNA,

mitoehonckial 376

DNA:

H, heavy;

L, light.

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in mitochonclria-associated RNA, followed by a plateau or a slight increase in the next 14 minutes. That the labeled RNA found in the mitochondrial fraction after a 5minute [S3H]uridine pulse, or after a 5-minute pulse followed by a 15-minute ethidium bromide/actinomycin chase, is mainly coded by m&DNA, is indicated by the observation that about 90% (pulse) or 80% (chase) of its labeling is inhibited if the cells are treated with ethidium bromide rtt 1 pgfml. for 15 minutes before the [5-3H]uridine pulse (Pig. 2(a)). The observed decay of a substantial fraction of newly synthesized RNA reflects, at least in part, a metabolic instability of this RNA and not an effect of the drugs. This conclusion is based on the fact that mitochondrirtl RNA, in spite of its very high rate does not of synthesis (estimated to correspond to at least 4 x lo6 nucleotides/min/cellt) accumulate in the cytoplasm of HeLa cells, where it represents only 1 y. or less of the RNA. The incomplete decay of the mitochondrial fast-labeled RNA may in part be due to alterations in its processing induced by the drugs, as shown for the heterogeneous nuclear RNA (Penman, Vesco & Penman, 1968). Furthermore, the existence of s, slower decaying RNA fraction may be masked by the early arrival from the nucleus to the polysomes of the rough endoplasmic reticulum contaminating the mitochondrial fraction (Attardi, Cravioto t Attardi, 1969) of messenger RNA labeled during the 5minute [5-3H]uridine pulse, as suggested by the increase in the ethidium bromideresistant fraction 16 minutes after the chase (Pig. 2(a)). However, a portion of the apparently stable mitochondrial RNA is undoubtedly represented by the discrete mitochondrial 16 s, 12 s and 4 s RNA species, which accumulate during the chase (Fig. 2). Eth Br.* Act.0 i 100 -

FIG. 1. Decay of mitcchondrial RNA labeled during a 6-min [S-3Hluridine pulse after blooking further RNA synthesis with ethidium bromide (1 &ml.) and aatinomyoin D (10 &ml.). lo* HeLe oells, after SO-min treetment with 0.04 pg eatinomyoin D/ml. (to blook eeleotively oytoplssmio rRNA synthesis), were exposed for 6 min to [6-3H]uridine, and then immedintely treated with 1 H ethidium bromide/ml. and 10 H eotinomyoin D/ml. At the indioated times, portiona of the call suepension were removed from the spinner and poured onto frozen salt solution (0.13 M-NaCl/O.O26 AS-KCl/O*OOl M-M&~,). The aoid-preaipitable radioactive material present in the mitoohondriel fieotion from each aample (a), and that esaooiated with the RNA extmoted by the sodium dodeoyl sulf&e/oold phenol method (0) or the sodium dodeoyl sulf&e/pronae-a/ph~nol method (A) (normalized for the amount of 28 8 rRNA present in eaoh preparetion, as determmed after sediment&ion of e portion through 8 16 to 30% (w/w) ~uerose gradient) am plotted aa peroentages of the b-min v&16. t Estimate derived from the relative rates of lebeling with [6-3H]~dine of free polyaome 28 B RNA and mitoohondrie-eeeooid RNA (Attardi et d., 1969) and from the finding that the equilibration with exogenoue [6-aH]uridine of the intro-mitoohondrial UTP pool is slower than that of the extra-mitaohondrial pool (Pioa-bttiooie UCAttardi, 1971).

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Figure 2(a) shows the sedimentation profiles of labeled RNA extracted with sodium dodecyl sulfate/cold phenol from the mitoohondrial fraction of HeLa cells after a 5minute pulse, or a 5-minute pulse followed by a 15-minute ethidium bromide/actinomycin chase. The radioactivity profile of the 5-minute pulse RNA shows a 4 s peak, a broad peak in the region 12 to 22 s and heavier heterogeneous RNA components sedimeriting up to more than 50 s, with an appreciable proportion of the labeled RNA (approximately 20% in fractions 1 to 10) being prevented from pelleting by the dense sucrose cushion at the bottom of the tube. There is a hint of the discrete components sedimenting at about 33 s which have been reproducibly observed, usually more pronounced than here, in pulse-labeled mitochondrial RNA (Attardi, Aloni et al., 1969,197O; Pica-Mattoccia t Attardi, 1971). The radioactivity profile of the RNA extracted from the cells after the chase shows, in comparison with the B-minute pulse prolile, a sharp decrease in the amount of labeled components sedimenting to the dense cushion at the bottom of the tube, no change in the amount of label in the “33 s” region and an increase in the labeled RNA sedimenting in the 12 to 16 s region and in the 4 s RNA species. No increase could be seen in the radioactivity associated with the 4 s components in the “chase” RNA as compared with the “pulse” RNA in the samples pretreated with ethidium bromide, indicating that the accumulation of labeled 4 s RNA during the chase is not due to the -CCA turnover in these molecules. Extraction of pulse-labeled mitochondrial RNA with sodium dodecyl sulfate/cold phenol does not give a quantitative recovery of the RNA, the yield being 50 to 60% (Aloni & Attardi, 197lc). In particular, the faster sedimenting components are preferentially lost by this extraction procedure (Aloni $ Attardi, 1972). An almost complete recovery of the pulse-labeled mitoohondrial RNA can, on the contrary, be obtained by pronase treatment of the mitochondrial fraction in the presence of 0.5% sodium dodeoyl sulfate, followed by cold phenol extraction (Aloni & Attardi, 1971c). Figure 2(b) (3H pulse) shows the sedimentation profile of labeled mitochondrial RNA extraded by the above-mentioned procedure from cells exposed to [5-3H]uridine for 5 minutes. In comparison with the pattern obtained with RNA extracted without prior pronase digestion, a higher proportion of the labeled RNA is found near or in the dense sucrose cushion (approximately 50% in fractions 1 to 11). After a H&minute chase, there is here too a marked decrease, as compared to the pulse RNA, in the amount of labeled RNA sedimenting near or iu the dense sucrose cushion, while there is only a moderate increase in the labeled RNA sedimenting in the 12 to 16 s region (with partially resolved peaks) and at 4 s. A better resolution of the discrete RNA components sedimenting in the region of 4 to 16 s is achieved by a longer centrifugation on a sucrose gradient (Fig. B(c)). It is clear from Figure 2(b) that the increase in the amount of labeled RNA in the region corresponding to less than 20 s (equivalent to about 5% of the total pulse 3H ots/min) is considerably less than the decrease in the region of more than 20 s (amounting to about 37% of the total original cts/min), the difference (32%) being in good agreement with the data discussed above of decay of pulse-labeled mitochondrial RNA (Fig. 1). The absolute increase, after chase, in the amount of label associated with each of the 16 S, 12 s and 4 s RNA classes appears to be fairly similar. It is interesting to notice that the total DNA stretches coding for the three RNA classes are also rather similar in length, differing from each other by at most a factor of 2 (in fad, they correspond to about 11, 7 and 5.5% of the length of the m&-DNA molecule for 16 s, 12 s and 4 s RNA, respectively (Aloni & Attardi, 1971a; Robberson, Aloni, Attardi Q Davidson,

378

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G. ATTARDI

6

4

60

(cl

18s 1 16.5

^ .^

- 0.05

5

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15

Volume (ml)

FIG. 2. Sedimentation patkerns of RNA from the mitochondrial fraotion of HeLe cells exposed to a 6-h [6-3H]uridine pulse, or to a [5-3H]uridine pulse followed by a 16-min ethidium bromide/ acrtinomycin D chase. (a) Two samples of lOa HeLtl cells, after 30-min treatment with 0.04 pg actinomycin D/ml., were exposed for 6 min to [6-3H]uridine in the absence (-@-a-) or in the presenoe (-A-A-) of 1 H ethidium bromide/ml. (added 16 min before the labeling). Two other equal samples. after a

LETTER

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1971)). This observation suggests that the synthesis of these discrete RNA species is fairly co-ordinate, in agreement with their kinetics of labeling (Attardi & Attardi, 1971). In view of the almost instantaneous block of further RNA synthesis under the chase conditions used here, the accumulation in substantial amounts with respect to the preexisting levels of labeled 16, 12 and 4 s RNA strongly supports the idea that these discrete species derive from processing of larger precursors. It should be noticed in this connection, that the increase in radioactivity associated with the mitochondrial ribosomal and 4 s RNA species is roughly what would be expected from processing of larger molecules, taking into consideration the amount of fast-sedimenting 3Hlabeled mitochondrial RNA which has decayed in the 15minute chase and the proportion of the total sequences of the H and L mit-DNA strands represented by the genes for these discrete species (about 11%) (Al oni t Attardi, 1971a). (On the basis of previously reported evidence (Aloni & Attardi, 1971c), it is assumed here that the 5minute pulse-labeled mitochondrial RNA contains equal amounts of transcripts of the two n&-DNA strands and that both strands are completely or almost completely transcribed.) That, in the case of 4 s RNA, the presumptive precursors must be as large as or larger than 12 s, is suggested by the observation that the increase in labeled 4 s RNA, which cannot be accounted for by terminal CCA turnover, is appreciably greater than the amount of labeled RNA components sedimentmg between 4 s and 12 s before the chase. In view of the observation that the 4 s RNA sites on the heavy strand are scattered at fairly regular intervals along the heavy mit-DNA strand (Wu, Davidson, Attardi & Aloni, 1972), the evidence suggesting that the 4 s RNA derives from precursors larger than 12 s and that the synthesis of the 16 s and 12 s RNA and of the bulk of 4 s RNA (presumably transcribed from the heavy strand, which contains the majority of 4 s genes (Aloni & Attardi, 1971a)) is co-ordinate, would be in agreement with the idea of a transcription of the heavy strand in the form of a continuous RNA chain. Direct evidence, however, is needed to establish this point conclusively. After the l&minute ethidium bromide/actinomycin chase, there is still some fastsedimenting labeled material (Fig. 2(a) and (b)). This fast-sedimenting material in the chase RNA extracted by sodium dodecyl sulfate/cold phenol is mainly ethidium bromide-sensitive (Fig. 2(a)), suggesting that it is represented by relatively stable RNA components. As discussed above, the apparent stability of these components may be due to secondary effects of the drug treatment. In order to obtain information on the nature of the labeled components sedimenting to the sucrose cushion in the chase RNA extracted with sodium dodecyl sulfate/pronase/phenol, their RNase resistance and homology to the separated n&DNA strands were investigated and compared

6-min [6-3H]uridine pulse in the 8bsence (-- O-- 0 --) or the presence (--A --A --) of the drug, were treated for 15 min with 1 H ethidium bromide/ml. and 10 a sctinomycin D/ml. The mitochondrial f&&ion w8a isolated from each ample, and its RNA extracted by the sodium dodeoyl sulfate/oold phenol method and run through 8 16 to 30% (w/w) suoroae gradient in sodium dodeoyl sulfate buffer (over 8 cushion of 1.5 ml. 64% sucrose in the same but%) in the Spinco SW27 rotor (small buckets) at 26,000 rev./min for 12 hr at 20°C. (b) Experimental conditions 88 in (a), except that the buoyant density oentrifugation in a sucrose gradient of the mitochondrial fraction w8a omitted, and the RNA was extracted by the sodium dodecyl sulfate/pronase/oold phenol method. (c) Experimental conditions 88 in (b), except that the centrifugation time was 25 hr.

Y. ALONI

380

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G. ATTARDI

DNA (pgl FIG. 3. Homology to separated mit-DNA strands of labeled mitoohondria-essooiated RNA components sedimenting to the sucrose on&ion from cells exposed to a 6min [6-3H]nridine pnlso, or to e pnlsc followed by a l&mm ethidinm bromide/actinomycin D ohase. Samples oontaining about 300 ots/min of the [sH]RNA from the pooled fraotions indicsted in Fig. 2 were ctnnealed with increasing amounts of L or H mit-DNA strands. The hybridization values hove been corrected for the background obtained without DNA (< 1% of input). -O-O-, -O-O-, pulse RNA; --a--@--, --O--O--, chase RNA.

with those of the corresponding components of the pulse RNA. No difference in the kinetics of RNA degradation by pancreatic RNase between the RNA components sedimenting to the dense sucrose cushion in the pulse and in the chase experiment was observed. On the other hand, an appreciably larger amount of labeled RNA hybridizable with the L m&-DNA strand, relative to that hybridizable with the H strand, was found in the fast-sedimenting components of the pulse RNA (L/H N 2.0) as compared to those of the chase RNA (L/H -1.6). This points to a more rapid turnover of the L transcripts, in agreement with previous observations (Aloni & Attardi, 1971c). 50% to 60% of the fast-sediment&g RNA, both in the pulse and in the chase experiment, hybridized with the L or H m&DNA strands. However, this has to be considered a minimum estimate of the fraction of this fast-sedimenting RNA coded by m&DNA; in fact, under the conditions of hybridization used here, a certain portion of complementary sequences of RNA was expected to form RN&se-resistant duplex structures, presumably destined to be in most part lost in the Sltration through nitrocellulose membranes. These observations argue, therefore, against a substantial contemirmtion of the fast-sedimenting mitochondrial RNA components by RNA of non-mitoohondrial 0IigiI-l.

This work was supported by a research grant from the U.S. Public Health Service (GM-11726) to one of us (G. A.) and by a Dernham Fellowship of the Amerioau Cancer Society to the other (Y. A.) We thank Mm L. Wenzel and Mrs B. Keeley for their excellent assistance. Division of Biology California Institute of Technology Pasadena, Calif. 91109, U.S.A. Received

10 January

Aloni, Y. & Attardi, Aloni, Y. &Attardi, Aloni, Y. & Attardi,

1972, and in revised form 4 April

Y.

&ON1

G. ATURDI

1972.

REFERENCES G. (1971a). J. Mol. Biol. 65, 271. G. (1971b). J. Mol. Bid. 65, 261. G. (1971~). Proc. Nat. Acad. Sci., Wmh. 68, 1767.

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Aloni, Y. & Attardi, G. (1972). J. Mol. Bid. 70, 363. Attardi, B. & Attardi, G. (1967). Proc. Nat. Acad. Sk., Wash. 58, 1061. Attardi, B. & Attardi, G. (1971). J. Mol. Bid. 55, 231. Attardi, B., Cravioto, B. & Attardi, G. (1909). J. Mol. Bid. 44, 47. Attardi, G. & Attardi, B. (1968). Proc. Nat. Ad. Sci., Wash. 61, 261. Attardi, G., Aloni, Y., Attardi, B., Lederman, M., Ojala, L., Pies-Mattoccie, L. & Storrie, Sywupos. on Autmwmy and Biogenesis of Mitochondria and B. (1969). In Intern&. ChZoro@z&, p. 293. Canberra, Amsterdam: North Holland Publ. Co. Attardi, G., Aloni, Y., Attardi, B., Ojala, D., Pica-Mattoocia, L., Robberson, D. & Storrie, B. (1970). Cold Spr. Harb. Syq. Quant. Bid. 35, 599. Ojala, D. t Attardi, G. (1972). J. Mol. Bid. 65, 273. Penman, S., Fan, H., Perlman, S., Rosbash, M., Weinberg, R. & Zylber, E. (1970). CoZd Spr. Harb. Syrup. Quant. Bid. 85, 561. Penman, S., Vesco, C. t Penman, M. (1968). J. Mol. Bid. 84, 49. Pica-Mattoccia, L. & Attardi, G. (1971). J. Mol. Bid. 57, 616. Robberson, D., Aloni, Y., Attardi, G. & Davidson, N. (1971). J. Mol. BioZ. 60, 473. Wu, M., Davidson, N., Attardi, G. & Aloni Y. (1972). J. Mol. Boil. in the press.