The pattern of transcription of the human mitochondrial rRNA genes reveals two overlapping transcription units

Cell, Vol. 34, 151-159.

August

1983, Copyright

Q 1983 by MT

0092-8674/83/080151-09

$02.00/O

The Pattern of Transcription of the Human Mitochondrial rRNA Genes Reveals Two Overlapping Transcription Units Julio Montoya,* George L. Gaines, and Giuseppe Attardi Division of Biology California Institute of Technology Pasadena, California 91125

Summary A detailed analysis of the mapping and kinetic properties of oligo(dT)-cellulose bound and unbound transcripts synthesized in HeLa cells has indicated that two distinct transcrfption events take place in the rDNA region. Of these, one appears to start -25 bp upstream of the tRNA- gene and to terminate at or near the 3’ end of the 16s rRNA gene, being responsible for the synthesis of the bulk of rRNA. The other transcription event starts near the 5’ end of the 12s tRNA gene, proceeds beyond the 3’ end of the 16s rFlNA gene, and results in the synthesis of a polycistronic molecule corresponding to almost the entire H-strand, which is destined to be processed to yield the mRNAs and most of the tRNAs encoded in the heavy strand. The existence of two overlapping transcription units with distinct promoters is probably the basis for the differential reg ulation of synthesis of the rftNAs and heavy-strandcoded mRNAs. Introduction

H-strand transcription with the synthesis of an oligo(dT)cellulose unbound RNA species (u4a), which spans the whole rDNA region and appears to be a precursor of the mature rRNA species. The downstream initiation site has been associated with the synthesis of an oligo(dT)-cellulose bound rDNA transcript (b4), and it is suggested that this RNA is a part of a polycistronic molecule that extends through almost the entirety of the H-strand and is processed to the downstream encoded mRNA and tRNA species.

Results Kinetics of Labeling of the Oligo(dT)-Cellulose Bound RNA 4 (b4) With the aim of determining which one of the two initiation sites for H-strand transcription detected in human mtDNA (Montoya et al., 1982) is utilized for rRNA synthesis, an investigation of the primary transcripts of the human rRNA genes was carried out. Among the oligo(dT)-cellulose bound transcripts encoded in HeLa cell mtDNA that had been identified by Amalric et al., (1978; these species will be designated in the text by their number with a prefix b to distinguish them from the corresponding oligo(dT)-cellulose unbound [u] species), RNA b4 seemed to be a good candidate for an rRNA precursor because of its mapping position corresponding to that of the 12s and 16s rRNA species (Ojala et al., 1980) and because of its relatively short half-life (40 min; Gelfand and Attardi, 1981). To obtain further evidence on the possible role of RNA b4, the flow of radioactivity into this RNA, as compared to the other mitochondrial RNA species, during short (5-3H) uridine pulses was investigated. HeLa cells were long-term labeled with 32P-orthophosphate under conditions of exponential growth and then exposed for very short periods to (5”H) uridine, always in the presence of 32P-orthophosphate. The most significant and surprising result of these experiments was that the extent of incorporation of (5-3H) uridine into RNA b4 at the time points analyzed was similar to the incorporation into most of the other RNA species whose coding sequences lie downstream of the rRNA genes, and it was, moreover, much lower than the labeling of the 12s rRNA (Figure 1). The appearance of a substantial amount of label in 12s rRNA after a 1 or 2 min pulse, when a negligible amount of radioactivity was associated with RNA b4, should particularly be noticed. Furthermore, a comparison of the ?-frJP ratios of the various components, which reflected their turnover rates, indicated that the turnover rate of RNA b4 is comparable to those of RNAs b5, b6, and bll (not shown). The above observations clearly excluded the possibility that RNA b4 is the kinetic precursor of the bulk of mitochondrial rRNAs.

The recent mapping and sequencing analysis of the transcripts of the heavy (H) strand of human mitochondrial DNA (mtDNA) (Ojala et al., 1980; Montoya et al., 1981; Ojala et al., 1981) and their alignment with the DNA sequence (Anderson et al., 1981) have led us to propose a model of transcription of the H-strand in the form of a single polycistronic molecule destined to be processed by endonucleolytic cleavages to mature or nearly mature products (Ojala et al., 1981). This model has raised the question of how a differential control of expression of the different genes encoded in the H-strand is achieved in this system; in particular, how is the higher rate of synthesis of the rRNAs relative to that of the individual mRNAs (10 to 30 fold), which is observed in mitochondria (Gelfand and Attardi, 1981) as in other systems, produced? Recently, two initiation sites for H-strand transcription have been identified in human mtDNA, one very close to the 5’ terminus of the 12s rRNA gene and the other 90 to 110 bp upstream of this site, i.e., 20 to 40 bp upstream of the tRNAPhe gene (Montoya et al., 1982). However, the functional significance of the two initiation sites could not be established. In the present work, a detailed analysis of the mapping and metabolic properties of the transcripts of the rDNA region of mtDNA synthesized in HeLa cells has made it possible to correlate the upstream initiation site for

Identification of Oligo(dT)-Cellulose Unbound rONA Transcripts

l Present address: Catedra de Bioquimica. ersidad de Zaragoza, Zaragoza, Spain.

The possible existence of primary transcripts of the rRNA genes in the oligo(dT)-cellulose unbound RNA fraction was

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with 32P-orthophosphate in medium containing 10e5 M phosphate in the presence of 0.1 pg/ml actinomycin D. The bound fraction shows the characteristic pattern with 18 discrete poly(A)-containing RNA components previously described (Amalric et al., 1978). The unbound fraction exhibits, aside from the prominent 16s and 12s rRNA bands, a series of minor bands, the majority of which align well with the bands of the poly(A)-containing RNA and which very probably represent RNA species differing from the latter by the absence or shorter length of the poly(A) tail. In particular, one can see in the pattern of the unbound RNA fraction a fairly pronounced band (~4) with the same apparent mobility as that of RNA b4; furthermore, there is a fainter, somewhat more slowly moving band (u4a), which is not well resolved from u4. The bands u4 and u4a are better resolved after a longer electrophoretic run (lane 4).

5’ End Mapping of rDNA Transcripts

23

Figure 1. Flow of 5.%-Uridine into Various Oligo(dT)-Cellulose Species and into 12s rRNA in Exponentially Growing Cells

Sound RNA

The total amount of ?-I radioactivity incorporated into each RNA species at the various time points, as determined by summing up the radioactivity in each peak after subtraction of an appropriate baseline, has been normalized to -4 x l@ cells and to a constant amount of 5-%-uriiine in the medium and corrected for variations in T-labeling of that particular species.

then investigated. Due to the difficulty of obtaining by the micrococcal nuclease procedure oligo(dT)-cellulose unbound mitochondrial RNA species completely free of contaminating cytoplasmic RNA components, we used cultures in which high molecular weight nuclear RNA synthesis had been inhibited by a low concentration of actinomycin v.

Figure 2A shows the autoradiogram, after electrophoresis through an agarose-CH,HgOH slab gel, of samples of the oligo(dT)-cellulose bound (lane 1) and unbound (lane 2) mitochondrial RNA fraction from cells labeled for 4 hr

In order to verify whether the two RNA species b4 and u4 were related to each other, as suggested above for all the oligo(dT)-cellulose bound and unbound RNA components with similar electrophoretic mobility, and in order to test whether RNA u4a mapped in the same mtDNA region as the other two components, Sl protection experiments were carried out to map the 5’ end of each of these RNA species. For this purpose, 32P-labeled RNAs b4, u4a, and u4 were eluted from an agarose-CHsHgOH gel, after a long electrophoretic run of the oligo(dT)-cellulose bound and unbound RNA, by using 2 mm slices serially cut from the wet gel to ensure complete recovery of the individual bands. The eluted RNA samples, as well as a sample of 3’P-labeled 12s rRNA, were hybridized with a molar excess of H-strands of unlabeled Hpa II fragment 8 (Hpa II-8H. Figure 3) and the mixtures were then digested with Sl nuclease. Figure 4A shows the Sl-resistant RNA-DNA hybrids analyzed under native conditions in a 5% polyacrylamide gel in TBM buffer. RNA b4 (lane 1) and RNA u4 (lane 3) both protected a DNA segment of -286 nucleotides, apparently identical in size to that protected by the 12s rRNA (lane 4). These results would place the 5’ end of both b4 and u4 very near to the 5’ terminus of the 12s rRNA gene. The RNA sample eluted from the slice of the gel of the bound RNA immediately above the RNA b4 band (corresponding in position to component u4a of the unbound RNA) did not protect any detectable fragments (not shown). On the contrary, the RNA eluted from the same region of the gel of the unbound RNA, which, therefore, contained RNA u4a, protected two fragments of Hpa II-8H strands, one of -380 nucleotides and the other of -286 nucleotides (lane 2). While the latter may be due to the presence of contaminating RNA u4 in the sample used, the 380 nucleotide fragment presumably results from hybridization of bona fide RNA u4a with Hpa II-8H. Figure 48 shows the results of an Sl protection experiment similar to that shown in Figure 4A, but carried out with unlabeled oligo(dT)-cellulose unbound RNA and 5’end-labeled Hpa II-8H strands. It is clear that the intensity of the bands corresponding to the protected DNA frag-

Two Overlapping 153

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Hours of pretreatment of Actinomycin

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(A) Oligo(dT)-cellulose bound (lanes 1 and 3) and unbound (lanes 2 and 4) RNA isolated frcm the micrococcal-nuclease-treated mitochondrial fraction of HeLa cells labeled for 4 hr with 32P-orthophosphate in the presence of 10” M unlabeled phosphate and 0.1 a/ml actinomycfn D were run through an agaroseCH&fgOH slab gel for 6 hr (lanes 1 and 2) or for 9 hr (lanes 3 and 4). The samples run in lanes 2, 3. and 4 derive from equivalent amounts of cells, the sample in lane 1 from 20 times that amount (lane 2 was exposed twice as long as lane 1). (B). (C) Effect of actlnomycin D pretreatment on the in viva labeling of mitochondrial RNA during a 3 hr exposure of the cells to “P-orthophosphate in the presence of the drug. Samples of 2.3 x l@ cells in 200 ml medium were labeled for 3 hr with gp-orthcphosphate (10 &i/ml) in the presence of 4 x IO” M unlabeled phosphate and in the absence of actinomycin D (lanes 5 and 9) or treated with 0.1 pg/ml of the drug for 30 min (lanes 6 and lo), 3 hr (lanes 7 and 1I), or 6 hr (lanes 8 and 12). and then labeled under the same conditions described above but in the presence of the drug at the same concentration, Portions of the oligo(dT)-cellulose bound or unbound RNA fractions from the various samples were run through agaroseCH&-fgOH slab gels. After exposure, the autcradicgrams were analyzed by densitometry. (B) Autoradiirams, after electrophoretii fractionation of equivalent portions (in terms of cell equivalents) of the oligo(dT)-cellulose unbound fractions of the control (lane 5). 30 min (lane 6) 3 hr (lane 7). and 6 hr pretreatment (lane 8) samples, and of portions with equal amount of radioactivity of the oligo(dT)-cellulose bound fractions of the control (lane 9). 30 min (lane lo), 3 hr (lane 1 l), and 6 hr pretreatment (lane 12) samples. (C) Relative labeling of 12s rRNA. 16s rRNA. and RNAs u4 + u4a (upper panel) and of total oligo(dT)-cellulose bound RNA and some individual poly(A)-contalning RNA species (lower panel) in the samples corresponding to different times of actinomycin D pretreatment of the cells, as determined from the densitometric tracings of approvriately exposed autoradiograms, after nonalization to the total amount in each sample. One hundred per cent represents the level of labeling in the absence~of actinomycin D.

ments is much greater than in the previously discussed experiment, due to the high specific activity of the DNA. However, the striking result is that, in the experiment utilizing RNA u4a (lane 8) the band corresponding to the 286 nucleotide protected DNA fragment is much more intense than the 380 nucleotide band, rather than being of equal intensity as in the experiment utilizing labeled RNA and unlabeled DNA. Furthermore, this band is found with about the same intensity in the Sl-resistant samples obtained with RNA eluted from four adjacent slices, i.e., from the slice above the u4a region (lane 7) from the slice corresponding to u4a (lane 8) from the slice corresponding to u4 (lane 9) and from the slice immediately below (lane IO). This result suggested the presence in the oligo(dT)cellulose unbound RNA preparation of a large number of nascent chains having their 5’ end at or very near the 5’ terminus of the 12s rRNA gene (Montoya et al., 1982) with a substantial fraction being longer than RNA u4a, and therefore presumably extending beyond the 3’ end of the 16s rRNA gene (see below). Furthermore, the great difference in the relative intensities of the 286 and 380 nucleotide bands obtained with unlabeled (Figure 4A) and labeled DNA (Figure 48) indicated that, in the former case, these nascent chains were for the most part unlabeled in their

protected portion, and therefore represented preexisting chains not completed during the labeling period in the presence of actinomycin D. Lane 11 shows the Sl protection pattern obtained with an unlabeled nascent RNA chain preparation from HeLa cell mitochondria, exhibiting the two bands previously observed (Montoya et al., 1982)-one corresponding to the 380 nucleotide fragment, and the other, much more intense, corresponding to the 286 nucleotide protected fragment. Lane 6 shows the Sl protection pattern obtained with a total oligo(dT)-cellulose bound unlabeled RNA preparation; the expected presence of the 286 nucleotide protected fragment and the absence of the 380 nucleotide fragment should be noted here. 3’ End Mapping of rDNA Transcripts Sl protection experiments similar to those described above were carried out to map the 3’ ends of the rDNA transcripts. In particular, in order to map the 3’ end of the RNA b4, an unlabeled RNA sample eluted from the corresponding region of an agarose-CHsHgOH gel was hybridized under high formamide conditions (Casey and Davidson, 1977) with 3’-end-labeled Hpa II fragment 18 (isolated with the closely migrating fragment 19; Ojala and Attardi, 1977) and treated with Sl nuclease. Samples of 16s rRNA

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Figure 3. Portion of the HeLa cell mtDNA (H-Strand) Genetic and Transcription Map Illustrating the rRNA Gene Region and the Adjacent Regtons The diagram shows the precise mapping positions of the large rDNA transcripts identified in previous (Ama+r+c et a+., 1978) and the present work. The Hpa II and Mbo I fragments utiked in the Sl mapping experiments are shown A

12

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and RNA b10, which represents a small polyadenylated fraction (l %-2%) of the 16s rRNA (Amalric et al., 1978; Ojala et al., 1980) were similarlyhybridized with 3’-endlabeled Hpa II-18 and Sl-treated for the purpose of comparison. Figure 5A shows the Sl -resistant DNA fragments separated on a 10% polyacrylamide/7 M urea sequencing gel. As previously shown (Dubin et al., 1982) 16s rRNA protected two major, closely migrating fragments (lane 3) with sizes of 151 and 152 nucleotides (thus including the sequence up to the two nucleotides immediately preceding the tRNAL”gene; Figure 3). The same doublet of protected fragments was produced by RNA b10 (lane 4) and, in considerably lower amount, by RNA b4 (lane 5). The strong band aligning with Hpa II-18H and the weaker band aligning with Hpa II-18L, 19H, in lane 5, and the bands at the same positions in lanes 3 and 4 represent DNA strands completely protected, presumably, by contaminating nascent

A I23456

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Figure 4. Nuclease Sl Mapping of the 5’ Ends of the O+++o(dT)-Ce+lulose Bound and Unbound Large rDNA Transcripts (A) Samples of RNAs b4 (estimated amount before elution -20 ng, lane l), u4a (-15 ng. lane 2) and u4 (-25 ng, lane 3) from cells labeled for 4 hr with YP-orthophosphate in the presence of IO-’ M unlabeled phosphate and 0.1 pg/ml actinomycin D were isolated by elution from sequentially cut slices of agarose-CH&+gOH slab gels, and hybridized with a 4 to 8 molar excess of unlabeled Hpa IISH strands (-35 ng). A sample of 12s rRNA (-70 ng. lane 4) from the same RNA preparation was hybridized with -35 ng of Hpa II&-i strands. (B) Samples of oligo(dT)cellu+ose unbound unlabeled RNA species eluted from sequentially cut slices of an agarose-C+-bl-+gDH slab gel (lanes 7-10) a sample of unlabekd RNA from mtDNA transcription complexes (lane 1 l), and a sample of total digo(dT)-cellulose bound unlabeled RNA (lane 8) were hybridized with 5’end-labeled Hpa 11-8+-lstrands. The RNA utilized in the experiments shown in lanes 7-10 was eluted from a gel slice above the band of RNA u4a (lane 7) from the RNA u4a region (lane 8) and from two adjacent slices below RNA u4a (lanes 9 and 10). The mtDNA transcnption complexes were ikolated as previously described (Montoya et al.. 1982); lane 13: “-RNA” control. Lanes 5 and 12: marker represented by an Hpa II digest of HeLa cell mtDNA 3’end-labeled with E. coli DNA polymerase I and a-=P-dGTP and dCTP. Analysis of the protected DNA segments was carried out under native conditions in 5% polyacrykmide slab gels in TBM buffer.

Figure 5. Sl Mapping of the 3’ Ends of the Oligo(dT)Ce+lulose Unbound Large rDNA Transcripts

Bound and

(A) Samples of unlabeled 16s rRNA (lane 3). RNA b10 (lane 4) and RNA b4 (lane 5) were hybridized under high fcrmamide conditions with 3’endlabeled Hpa II fragments 18 and 19 of HeLa cell mtDNA. After diitton with Si nuclease. the protected segments were analyzed by electrophoresis on a 10% po+yacry+amide/7 M urea sequencing gel. Lane 2: “-RNA” controi; lanes 1 and 6: a 3’-end-labeled Hpa II digest of HeLa cell mtDNA and a B’end-labeled Hha I digest of pBR322 DNA. The pBR322 Hha I fragments are designated by their original sizes (SutcBfe, 1978). Most of the restriction fragments were resolved into their complementary strands. (B) Samples of 16s rRNA (lane 8) RNA bl0 (lane 9) RNA b4 (lane IO), and RNA eluted from a region of the gel corresponding to specks u4a and u4 (lane 11) were hybridized with S’end-labeled Mbo I-1 1H strands of HeLa cell mtDNA. The hybrids were digested with 2.1 nuclease (250 U at 37°C for 30 min), and the protected fragments were analyzed by electrophoresis on a sequencing gel as described in (A). Lane 7: 3’end-labeled mtDNA Hpa II digest marker; lane 12: “-RNA’ control.

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RNA chains extending through Hpa II fragments 18 and 19. Figure 58 shows the results of Sl protection experiments utilizing samples of unlabeled RNA b4, u4 + u4a, 16s rRNA, or RNA b10 and 3’-end-labeled H-strands of Mbo I fragment II (corresponding to Mbo I fragment 9 of Drouin et al., 1979; Figure 3). Two closely migrating protected fragments, with an estimated size of approximately 166-167 nucleotides (with their 3’ ends corresponding in the mtDNA sequence to the two nucleotides immediately preceding the tRNAL” gene), were obtained with both 16s rRNA and RNA bl0 (lanes 8 and 9, respectively). RNA b4 also produced the doublet of protected fragments, although of considerably weaker intensity (lane 10); the same doublet was observed with a sample containing both RNA u4 and u4a (lane 11). The results of the Sl protection experiments discussed in this and the previous section thus showed that the 5’ ends of RNA b4 and RNA u4 map at the same or very similar positions, close to or at the 5’ terminus of the 12s rRNA gene, while the 5’ end of RNA u4a maps about 95 nucleotides upstream of that position. It is significant that these two sites correspond precisely to the two H-strand transcription initiation sites recently identified (Montoya et al., 1982). The results further showed that, at the 3’ end, RNA b4 and at least one of two oligo(dT)-cellulose unbound RNA species u4 and u4a map in correspondence

with the two nucleotides immediately preceding the tRNALW gene, exactly as do the mature 16s rRNA and RNA b10.

Kinetics of Labeling of Oligo(dT)-Cellulose Unbound rDNA Transcripts The results described above clearly pointed to the rDNA transcripts u4a and u4 as possible rRNA precursors, prompting an analysis of their metabolic properties. For this purpose, HeLa cells, labeled for 4 hr with =P-orthophosphate in the presence of 10m3 M phosphate and 0.1 rg/ml actinomycin D, were exposed for very short periods to (5-3H) uridine. Figure 6 shows the labeling pattern after a 10 min (5-3H) uridine pulse of the oligo(dT)-cellulose unbound (A) and bound (B) rDNA transcripts and of other mitochondrial RNA species migrating in the adjacent regions of the gel tracks, as determined by serial gel slicing and counting after long electrophoretic runs. The RNA species u4 and u4a are both labeled with (5”H) uridine to a much greater extent than RNA b4. Furthermore, the 3H/ %P ratio in the RNAs u4 and u4a is more than four times higher than in RNA b4, and five times as high as in 12s rRNA. Figure 6C represents the labeling of the various rDNA transcripts after cell exposure to different pulses of the precursor. One sees that there is no hint of saturation of 3H radioactivity in RNAs u4 and u4a, as would be expected if these species were obligatory kinetic precurFigure 6. Flow of 5?-I-uridine into Oligc+fT)-Cellulose Bound and Unbound rDNA Transcripts and Other H-Strand Transcripts in Cells Treated with 0.1 pgg/ml Actinomycin D

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(A-6) Analysis by serial gel slicing of the labeling of various digc@T)-cellulose unbound (A) and bound (6) RNA species separated on agaroseCH$-fgOH slab gels. RNA was isolated from cells labefed for 4 hr with %orthophosphate in the presence of IO” M unlabeled phosphate and 0.1 rg/ml actinomycin D, then labeled for 10 min with 5-?-l-uriiine in the presence of the antibiotic. The oligo(dT)cellulose bound RNA sample run on gel derived from about four times the amount of cells as the unbound RNA sample. (C) Kinetics of labeling of the RNA species in experiments illustrated in (A) and (B) and in other experiments rnvolving a 2 or 5 min 5-?-f-undine pulse. The total amount of radioactiiity incorpo rated into each RNA species at various time points has been nonalized as detailed in Figure 3.

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sors of the rRNAs. However, it should be noted that the rapid increase in specific activity of the intramitochondrial UTP and CTP pools during the (EL3H) uridine pulses (Gelfand and Attardi, 1981) contributes to a considerable extent to the observed kinetics of labeling of the various RNA species and would mask a saturation of the putative precursor rRNA pools. Furthermore, the rapid appearance of label in 12s rRNA (Figure 6C) already observed in exponentially growing cells (Figure 1) suggests that a part of the 12s rRNA may be directly processed from nascent chains (see Discussion). In any case, the rapid flow of (5 3H) uridine through RNAs u4 and u4a is clearly consistent with a role of rRNA precursors for these species, in sharp contrast to the labeling behavior of RNA b4. In the gel fractionation pattern of the oligo(dT)-cellulose unbound RNA shown in Figure 6A, one can recognize bands that correspond in migration to the bands formed by the polyadenylated RNAs b5, b6, b7, and b9 (Figure 6B), and very probably represent, as mentioned above, their nonadenylated or oligoadenylated counterparts. One can notice that these putative non- or oligoadenylated components, though represented in equivalent or somewhat lower amounts than the corresponding polyadenylated species, are labeled with (5-3H) uridine to a 5 to 15 fold greater extent (Figure 6C); this observation suggests that the non- or oligoadenylated species may be the kinetic precursors of the polyadenylated RNAs. In the gel fractionation pattern of the oligo(dT)-cellulose bound RNA (Figure 6B) one can recognize a well resolved peak corresponding to RNA bl0. This RNA appears to be labeled with (5-3H) uridine, relative to 32P-labeling, to a similar extent as the mRNA or mRNA precursors b5, b6, b7, and b9, and considerably less than 16s rRNA (about one-fourth as much): The data shown in Figure 6 clearly indicated the distinct kinetic behavior of RNA b4 as compared to RNAs u4a and ~4, strongly suggesting its derivation from a different transcription event. This interpretation was supported by an analysis of the effects of pretreatment of cells with actinomycin D at 0.1 rg/ml for different times on the labeling of mitochondrial RNA species during a 3 hr exposure to 32P-orthophosphate in the presence of the drug. As shown in Figures 28 and 2C, in this experiment the labeling of the two rRNA species was reduced to 60%-70% relative to the level of labeling in the absence of the drug after a 3 hr pretreatment, and to 20%-30% after a 6 hr pretreatment, with a parallel decrease in the labeling of RNAs u4a and u4 (Figure 2C). The labeling of the oligo(dT)-cellulose bound RNA fraction showed a roughly similar course. However, the labeling of RNA b10 and that of RNA b4 for the first 3 hr of drug pretreatment did not decrease, but actually increased during the drug treatment applied (Figure 2C).

human mtDNA. These transcription events have been correlated with the two initiation sites for H-strand transcription previously identified in this genome (Montoya et al., 1982) and it has thus been possible to make a functional assignment for these two sites. One transcription event, relatively frequent, appears to start at a site (IT2) -25 bp upstream of the tRNAh gene, under the control of the putative promoter PHR(Figure 7) and is responsible for the synthesis of the bulk of rRNA. The other transcription event, much less frequent, starts at a site (ITI) near the 5’ end of the 12s rRNA gene under the control of the putative promoter PHTand proceeds beyond the 3’ end of the 16s rRNA gene, resulting in the synthesis of a polycistronic molecule that corresponds to almost the entire H-strand and is destined to be processed to mature tRNAs and mRNAs. The functional assignment proposed for the two H-strand initiation sites is justified by two main results reported here: 1) the kinetic evidence indicating that the previously identified polyadenylated transcript originating at the downstream initiation site (RNA b4) is not the precursor of the rRNA, and is synthesized at a rate comparable to the rate of synthesis of the H-strand-encoded mRNAs, and 2) the discovery of a discrete rDNA transcript that maps with its 5’ end at the uptstream initiation site (RNA u4a) and is synthesized at a rate compatible with its being a precursor of the rRNA. RNA u4, which is also synthesized at a high rate, very probably represents a processing intermediate produced by the excision’ from RNA u4a of the tRNAPhe and of the 5’ leader that precedes it. Further support for a functional role in rRNA synthesis of the upstream initiation site comes from the results of mtDNA transcription experiments utilizing isolated mitochondria (Gaines and Attardi, unpublished data). Transcription of mtDNA in isolated organelles results in the formation of a set of transcripts very similar to that of the in vivo

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Discussion

Figure 7. Diagram Representing the Proposed Model of Transcription RNA Processing in the rDNA Region of Human mtDNA

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The main conclusion of this study is that two overlapping transcription events take place in the rDNA region of

IT: site of initiation of transcription. Pw~: putative promoter for the transcrip tion of the total H-strand; Pnn: putative promoter for rRNA synthesis. T: termination.

:bv; Overlapping

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rDNA

synthesized RNAs, including the species u4a, u4, and b4. Particularly significant in the present context is the detection in these in vitro experiments of a large fraction of incompletely processed 12s rRNA carrying at its 5’ end the tRNAP”” sequence and the 5’ leader, and of a minor processing intermediate lacking this leader. This finding is consistent with the proposed functional assignment for the upstream initiation site. Figure 7 summarizes in a schematic form our interpretation of the data concerning the transcription and RNA processing patterns of the rDNA region of human mtDNA. For simplicity, only one possible processing pathway from u4a to mature rRNAs is represented. In this diagram, the transcription event producing the oligo(dT)-cellulose unbound rDNA transcripts u4a and u4 is shown as terminating at the 3’ end of the 16s rRNA gene. The evidence for this termination is based mostly on the observation that the rate of labeling with (5-3H) uridine of the oligo(dT)cellulose bound and unbound RNA species encoded in the segments downstream of the rRNA genes is much lower than the rate of labeling of the rRNA species. It is interesting that the 3’terminal region of the 16s rRNA gene of several mammalian mtDNAs, including human, can be folded in a structure resembling, in a rudimentary form, the hairpin-oligo(U) signal postulated for bacterial termination-attenuation (Attardi et al., 1982; Dubin et al., 1982). Further support for a termination of transcription near the 3’ end of the 16s rRNA gene comes from the discovery that the 3’ ends of mammalian 16s rRNA are ragged (Dubin et al., 1982)-an observation that points to a certain imprecision of the process leading to their formation, in contrast to the absolute precision of the processing event leading to the release of the 3’ ends of the mRNAs (Ojala et al., 1981). A testable implication of the model shown in Figure 7 is that the tRNAPhe and tRNAVa’ genes are transcribed at a much higher rate than most of the other H-strand tRNA genes, leading to a larger accumulation or turnover of the corresponding tRNA species. Preliminary experiments have indeed indicated that the steady state amounts of the tRNAPhe and tRNA”” in‘exponentially growing cells are two to four times higher than those of several other Hstrand-coded tRNAs (including the tRNA &‘L encoded on the 3’ end side of the 16s rRNA gene) without bearing any relationship to the expected codon usage for mitochondrial translation (King and Attardi, unpublished data). Although the transcribed moieties of RNA b4 and RNA u4 appear to have identically or similarly mapping 5’ and 3’ termini, these two RNA species have clearly distinct kinetic properties, and their synthesis shows different behavior in vivo in the presence of actinomycin D and in isolated mitochondria under appropriate experimental conditions. RNA b4 may have no physiological role other than that of joining the promoter near the origin of replication to the main portion of the transcription unit lying downstream with respect to the rRNA genes. This RNA has a poly(A) tail long enough to cause it to be retained on an oligo(dT)cellulose column, like all the H-strand-coded mRNAs or

mRNA precursors (Amalric et al., 1978; Ojala et al., 1980) and in contrast to the rRNA species, which only have short stretches of A’s at their 3’ ends (Dubin et al., 1982). This fact points to a similarity of RNA processing events for RNA b4 and the H-strand-coded mRNAs, and is again consistent with the interpretation proposed above. The idea that RNA b4 derives from processing of longer transcripts extending beyond the 3’ end of the 16s rRNA gene is also supported by the evidence in the present work indicating that a substantial portion of the nascent rDNA transcripts mapping with their 5’ end at or very near to the downstream initiation site extends beyond the 3’ end of the 16s rRNA gene. With regard to RNA b10, the estimates of RNA synthesis rate (Gelfand and Attardi, 1981) clearly exclude its being a precursor of the mature 16s rRNA. The results of the (53H) uridine pulse labeling experiments in the presence of actinomycin D and the long-term effects of treatment with this drug fully support this conclusion. It seems likely that RNA b10 derives from processing of RNA b4. In exponentially growing cells, mature 12s rRNA is already labeled to a substantial extent after a 1 min [5-3H] uridine pulse, suggesting that most of the processing of the rRNA precursor to mature species under normal conditions occurs while this precursor is being synthesized, and that the discrete species u4a and u4 detected here result from the slowing down of the processing under the conditions of actinomycinD treatment or in vitro incubation of isolated organelles (Gaines and Attardi, unpublished data). The situation may be analogous to that occurring in E. coli, where the large rRNA precursor (30s) is not normally observed in vivo due to the rapid cleavage by RNAase Ill of the sensitive sites in the nascent chains, but does accumulate in RNAase Ill-deficient strains (Dunn and Studier, 1973; Nikolaev et al., 1973). A difference in the efficiency of initiation at the two promoters is very probably the main factor controlling the different rates of rRNA and mRNA synthesis in human mitochondria. The existence of multiple transcripts of the same gene with different 5’ ends and under separate regulation has been previously observed in procaryotic (Reichardt and Kaiser, 1971) and in eucaryotic systems (Young et al., 1981; Carlson and Botstein, 1982). However, an unusual feature of the mitochondrial system discussed here is that, apart from the control of the rate of transcription, the fate of each of the two kinds of transcripts of the rDNA region is determined by its having started at a specific site. This difference concerns primarily the termination or continuation of transcription at or near the 3’ end of the 16s rRNA gene, but may also determine the extent of addition of A residues at the 3’ end of the 16s rRNA sequence, as well as the capacity of the transcript to undergo processing at the 3’ end of the 12s rRNA sequence. The involvement of two different polymerases and/or the extra nucleotide stretch at the 5’ end of the RNA u4a may be responsible for the difference in behavior mentioned above. The situation differs from the transcription attenuation described in other systems (Yanofsky,

cell 158

1981; Hay et al., 1982) where alternative secondary structures of the same nascent RNA molecule determine the termination or continuation of transcription. In those systems, the secondary structure of the 5’ leader is assumed to produce the formation of the termination signal in the region immediately downstream. In contrast, in the mitochondrial system discussed here the 5’ end of the rDNA transcript originating at the upstream promoter would be required to transmit the signal for transcription termination to a site -2675 nucleotides downstream. If processing of the mitochondrial rRNA precursor under normal conditions occurs while this is being synthesized, as the kinetic data presented here strongly suggests, the long-range effect of the 5’ end of this precursor must involve some form of polar transmission of the signal, as by sequential formation of secondary structures and sequential binding of ribosomal proteins. The elucidation of the factors controlling the initiation of transcription from each of the two H-strand promoters and the events occurring at the 3’ end of the 16s rRNA gene must await the development of suitable “open” in vitro systems. Experimantal

Procadums

HeLa cells were grown in suspension in modified Eagle’s medium supplemented with 5% calf serum. Pulse labefing with 5?iuridine was carried out on cells that had been previously long-term labeled with “Porthophosphate either under condiiions of exponential growth or in the presence of a low concentration of actinomycin D. In the former case, cell cultures at an initial concentration of -2.0 x 105 cells/ml were exposed to 32porthophosphate (13 &i/ml) for 24 hr at 37% in modiied Eagle’s medium containing 20” M phosphate and supplemented with 5% dialyzed calf serum (Gelfand and Attardi, 1981). The cells were then sedimented at 37’C, resuspended at a concentration of 1.8 x I@ cells/ml in the same medium containing qcrthophosphate (13 &i/ml), and exposed to 5-?-iuridine (43 or 65 &i/ml) for different lengths of time (1, 2, 3.5, 5, and 10 min). After the pulse, the cells were rapidly harvested on crushed frozen isotonic salt solution (0.13 M NaCI, 0.005 M KCI, 0.001 M MgCI,). In the experiments in which actinomycin D was used to inhibit the synthesis of nuclear rRNA, cell cultures at -2.0 x 106 cells/ml were grown for 24 hr in modified Eagle’s medium containing 10” M phosphate. Actinomycin D (0.1 Ag/ml) was then added and, after 1 hr incubation, the cells were exposed to gP-o+thophosphate (13 &i/ml) for 4 hr. Pulse-labeling with 5 ?--uridine of these cells (65 &i/ml) was wried out as described above, except that actinomycin D (0.1 pg/ml) was also present in the medium. In some experiments, labeling of the RNA with gP-orthophosphate was carried out in the presence of actinomycin D at a high cell density in a low phosphatecontaining medium. For this purpose, cells from exponentially growing cultures were resuspended at a concentration of 1.5 x I@ cells/ ml in modified Eagle’s medium containing 10e5 M phosphate and incubated for 30 min in the presence of the drug (0.1 rg/ml); T-orthophosphate (75 &i/ml) was then added and incubation continued for 4 or 4.5 hr. To study the effect of actinomycin D pretreatment on mitochondrial RNA synthesis, cells were incubated in the presence of the drug for different times, under the conditions described above, and then labeled for 3 hr with 32porthophosphata Exbnction, Fractionstion, and Analysis of Mihondrial RNA lsolatiin of total mitochondrfai nucleic acids from HeLa cells by the micrococcal nuclease procedure, oligo(dT)-cellulose chromatography, electrophoresis through agarose-CH&tgCH slab gels, and isolation of the individual RNA species were carried out as previously described (Ojala et al., 1980). For the kinetic studies, the slab gels containing the fractionated mitochondrill RNA were dried, and after autoradiiraphy, whole tracks or portions thereof were sliced sequentially into 1 mm sections on a Micker gel slicer.

The radioactivity in the individual sections was eluted by heating them at 95’C for 30 min in 1 ml of low salt buffer (0.01 M Tris-HCI. pH 7.4; 0.001 M EDTA) (Gdfand and Attardi, 1981). Isafatkm, End-labeling, and Strand ¶tion of ResWtian Fmgn~~ts Human mtDNA Hpa II fragment 8 was isdated from the plasmid pEiHK2, which contains the middle-sized of the three Kpn I fragments of human mtDNA (Greenberg et al., 1963). 5’.end-labeling of this fragment (Crews and Attardi, 1980) and separation of its strands (Cantatcre and Attardi, 1960) were performed as described. Hpa II fragments 18 and 19 and Mbo I fragment 11 were isoiated from HeLa cell mtDNA and S’end-labeled with a-=P-dCTP and a-?-dGTP in the case of Hpa II-18 and Hpa 11-19,or with all four a-TdNTPs in the caSe of Mbo I-1 1, and E. cdi DNA polymerase I as previously detailed (Dubin et al., 1982). Separation of the strands of Mbo I-1 1 was carried under the conditions previously described for Hpa II. 8 (Cantatore and Attardi, 1980). Sl Profaction Analysis Hybridization of separated strands of restriction fragments or whde fragments, unlabeled or labeled (at the 5’ end or 3’ end), with unlabeled M labeled (in vivo or in vitro) RNA species. respectively, was carded out under high formamide condkions favoring RNA-DNA hybridization ,over DNADNA reassociation (Casey and Davidson, 1977) as previously described (Ojala et al., 1980). The St nuclease (Sigma) was used at 100-500 lJ in a 0.2 ml volume for 30 min at 45°C unless otherwise specified. Electrophoretic analysis of the protected DNA segments was performed either under native conditions in 5% polyacryfamide slab gels in Tris-borate-Mg++ buffer (TBM) (Maniatis et al., 1975) or under denaturing condiions in Tris-borate-EDTA buffer (TEE) (Maniatis et al., 1975) as described earlier (Ojaja et at., 19&l), or in 10% polyacrylamidefl M urea sequencing gels (Dubin et al., 1982). Acknowledgments These investigations were supported by National Institutes of Health grants GM-11726 and T32 GM-07616. We are very grateful to Bs. Masayasu Nomura and Anne Chomyn for their critical reading of the manuscript. The technical assistance of Ms. Arger Drew is gratefully acknowledged. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indite this fact. Received

March 8, 1983; revised

May 27, 1963

References Amalric, F., Merkel, C., Gelfand. Ft., and Attardi, G. (1978). Fractionation of mitochondnal RNA from HeLA cells by high-resolution electrophcresis under strongly denaturing conditions. J. Mot. Bid. 7 18, 1-25. Anderson, S., Bankier, A. T., Etarrell, 6. G., de Bruijn, M. H. L., Co&on, A. R.. Drouin, J., Eperon. I. C., Niedich, D. P., Roe, 6. A., Sanger, F., Schreier, P. H., Smith. A. J. H., Staden, R.. and Young, I. G. (1981). Sequence and organization of the human mitochondrial genome. Nature 290, 457-465. Attardi, G., Cantatore, P., Chomyn, A.. Crews, S.. Gelfand. R.. Merkel, C., Montoya, J., and Ojata, D. (1982). A comprehensive view of mitochondrial gene expression in human cells. In Miicchondrial Genes, P. Sloninski, P. Borst, and G. Attardi, eds. (Cold Spring Harbor, New York: Cdd Spring Harbor Laboratory), pp. 51-71. Cantatore, P., and Attardi, G. (1980). Mapping of nascent light and heavy strand transcripts on the physical map of HsLa cell mitochondrfai DNA. Nucl. Acids Res. 8, 2605-2624. Carbon, M.. and Rotstein, D. (1982). Two differentially regulated mRNAs with different 5’ ends encode secreted and intracetlular forms of yeast invertase. Cell 28. 145-154. Casey, J.. and Davidson, N. (1977). Rates of formation and thermal stabilities of RNA:DNA and DNADNA duplexes at high concentrations of formamide. Nucl. Acids Res. 4. 1539-1552. Crews, S.. and Attardi, G. (1980). The sequences

of the small ribosomaj

;;;

Overlapping

Transcription

Units of Mtochondrial

rDNA

RNA gene and the phenylatanine tRNA gene are joined end to end in human mitochondrial DNA. Cell 19,775-784. Drouin, J. (1980). Cloning of human mitochondrial J. Mol. Biol. 140, 15-34.

DNA in Escheriihia

coli.

Dubin. D. T., Montoya, J., Timko, K. D., and Attardi, G. (1992). Sequence analysis and precise mapping of the 3’ ends of HeLa cell mitochondrtal ribosomal RNA?.. J. Mol. Biol. 157. l-19. Dunn, J. J., and Studier, F. W. (1973). T7 early RNAs and Eschertchia coli ribosomal RNAs are cut from large precursor RNAs in vivo by ribonuclease Ill. Proc. Nat. Acad. Sci. USA 70, 3296-3300. Geffand, R., and Attardi, G. (1961). Synthesis and turnover of mitochondrfal ribonucleic acid in HeLa cells: the mature ribosomal and messenger rtbonucleic acid species are metabofiily unstable. Mol. Cell. Biil. 1, 497511. Greenberg, B. D., Newbold, J. E., and Sugino, A. (1983). Intraspecific nucleotide sequence variability surrounding the origin of reptication in human mitochondrial DNA. Gene, 21, 33-49. Hay, N., Skolnik-David, H., and Aloni, Y. (1982). of SV40 gene expression. Cell 29, 183-193.

Attenuation

in the control

Maniatis, T., Jeffrey, A., and van de Sand@, H. (1975). Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel dectrophoresis. Biochemistry 74,3787-3794. Montoya. J., Ojafa. D.. and Attardi, G. (1981). Distinctive terminal sequences of the human mftochondriaf mRNAs. 470.

features of the 5’ Nature 290, 465

Montoya, J., Christianson, T.. Levens, D., Rabinowitz, M., and Attardi, G. (1982). Identification of initiation sties for heavy strand and light strand transcription in human mitochondrial DNA. Proc. Nat. Acad. Sci. USA 79, 7195-7199. Nikolaev, N., Silengo. L., and Schlessinger, precursor of ribosomal RNA in a mutant Acad. Sci. USA 70,3361-3365.

D. (1973). Synthesis of a large of Escherichia cofi. F’roc. Nat,

Ojala. D., and Attardi, G. (1977). A detailed physical map of HeLa cell mitochondrial DNA and its alignment with the positions of known genetic markers. Fiasmid 1, 78-105. Ojala, D., Merkel. C.. Gelfand. R., and Attardi, G. (1980). The tRNA genes punctuate the reading of genetic information in human mitochondrii DNA. Cell 22, 393-403. Ojala, D., Montoya, of RNA processing

J., and Attardi, G. (1981). The tRNA punctuation in human mitcchondria. Nature 299, 470-474.

Reichardt, L., and Kaiser, A. D. (1971). Control Proc. Nat. Acad. Sci. USA 68, 2185-2189.

of A repressor

model

synthesis,

Sutcliffe, J. G. (1978). pBR322 restriction map derived from the DNA sequence: accurate DNA size markers up to 4361 nucfeotide pairs long. Nucl. Acrds Res. 5, 2721-2728. Yanofsky, C. (1981). Attenuation operons. Nature 289, 751-758.

in the controi of expression

of bacterial

Young, R. A., Hagenbijchle. 0.. and Schibler, U. (1981). A single mouse a-amylase gene specifies two different tissue-specific mRNAs. Cell 23, 451-458.