- Email: [email protected]
A role for RNAse MRP in mitochondrial RNA processing
Cell 16
Tam&s Kiss and Witold Filipowicz Friedrich Miescher-lnstitut PO Box 2543 4002 Base1 Switzerland
A Role for RNAase MRP in Mitochondrial RNA Processing
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The basic mode of mammalian mitochondrial DNA (mtDNA) replication has been established (Clayton, 1982), and the predominant control region for both replication and transcription has been located at one position on the circular genome. This site contains both major promoters, the origin of leading-strand mtDNA replication, and the signature feature of mammalian mtDNA, the displacement loop. Priming of leading-strand mtDNA synthesis begins by transcriptional initiation at a mtDNA promoter located 450 bp upstream of the most downstream nascent DNA 5’ termini that have been identified. There are multiple positions within this 450 bp zone at which RNA a*d DNA strand termini map. In most cases RNA 3’ends match DNA 5’ ends, and thus the former have been postulated to be primers for the latter. The number and relative positions of these RNA to DNA transition sites vary in complexity between different vertebrate species. In the case of mouse, in at least one instance, it was possible to demonstrate covalent linkage between an RNA whose 5’ end maps at the promoter transcriptional start site and DNA (Chang et al., 1985). The recognition that some mechanism must exist for the formation of these RNA termini in vivo prompted a search for any activity with such a capacity. These initial experiments led to the identification of a ribonucleoprotein endoribonuclease that cleaved at the most upstream RNA to DNA transition point in human and mouse mtDNAs; the activity was termed RNAase MRP for mitochondrial RNA processing (Chang and Clayton, 1987a, 1987b). Subsequent work has dealt with the sequence and properties of the RNAcomponent of RNAase MRP from several species (Chang and Clayton, 1989; Yuan et al., 1989; Topper and Clayton, 1990a, 1990b; Yuan et al., 1991) the nuclear gene for this small RNA from mouse (Chang and Clayton, 1989) and human (Topper and Clayton, 1990a; Yuan and Freddy, 1991) a possible relationship between RNAase MRP and the tRNA-processing enzyme RNAase P (Gold et al., 1989) and the determination of the critical regions of the mtRNA substrate that are required for cleavage (Bennett and Clayton, 1990). Kiss and Filipowicz (1992 [this issue of Cell]) have examined the abundance of a putative RNAase MRP RNA from a plant source and human RNAase MRP RNA from HeLa cells. By analyzing the hybridizable amount of the putative plant or known human full-length RNAase MRP RNA relative to other known RNA species at different stages of subcellular fractionation and purification, they conclude that the amount of detectable full-length RNAase MRP RNA is so low as to permit the deduction that the RNAase MRP ribonucleoprotein enzyme is not present in mitochondria in vivo and therefore presumably is only functional elsewhere in the cell, most likely in the nucleus. Results and Discusslon It is appropriate to be concerned about the high abundance of RNAase MRP RNA in the nucleus, and our as-
Matters 17
Arising
signment of a mitochondrial function for the enzyme itself has been based largely on its site-specific cleavage of mtRNAs. That only a very minor portion of the total cellular full-length RNAase MRP RNA is in mitochondrial fractions is seen in the original description of the RNA species in RNAase MRP: the amount of detectable full-length RNAase MRP RNA in the mitochondrial fraction is below 1% of that present in the nuclear fraction (Figure 76 in Chang and Clayton, 1987b). Concordantly, this same ratio can be reproduced when assaying the amount of RNAase MRP enzymatic activity from isolated subcellular fractions (Karwan et al., 1991). Reliable estimates on the relative amount of nucleus-localized RNAase MRP cleavage activity were not achieved in 1987, owing to substrate degradation in those crude extracts (see Discussion in Chang and Clayton, 1987b). Kiss and Filipowicz (1992 [this issue of CeIj) correctly note that we have not reported studies assessing the amounts of RNAase MRP RNA present following nuclease digestion of mitochondria or in submitochondrial mitoplasts. We have done such experiments as controls for other experiments, and a typical result is shown in Figure IA. The basic conclusions that can be drawn from these experiments are: first, most of the RNAase MRP and Ul RNAs associated with sucrose gradient-purified mitochondria are nuclease sensitive; second, a reproducible minor amount of RNAase MRP and Ul RNAs survives nuclease treatment and is rendered nuclease sensitive by solubilization of mitochondria with Triton X-l 00; and, third, preparation of mitoplasts by digitonin treatment shows a similar minor amount of RNAase MRP and Ul RNAs associated with mitoplasts. As independent controls, we tested for the presence of an mtDNA-encoded RNA and a small cytoplasmic RNA under the same conditions (Figure 16). The methionyl-tRNA known to be encoded by mtDNA and assumed to be localized entirely within the mitochondrial matrix is completely resistant to nuclease digestion until mitochondrial solubilization, whereas a nuclear-encoded seryl-tRNA is substantially removed by the incubation wash and can be entirely digested without mitochondrial solubilization. Because of our inability to digest Ul RNA completely, we did not pursue this approach further. Thus, less than 1% of the total full-length RNAase MRP RNA is present in purified mitochondrial fractions. Our earlier data, the data here, and the experiments of Kiss and Filipowicz (1992) are in agreement in this regard. Kiss and Filipowicz (1992) have extended this type of analysis by additional gradient procedures and nuclease treatment of isolated mitoplasts; we believe their experiments have been well executed. The central conclusion drawn by Kiss and Filipowicz (1992) is that at their ultimate levels of mitochondrial or mitoplast purification only a few molecules of full-length RNAase MRP RNA per cell are still associated with mitochondria (even less for mitoplasts) and that at these amounts it is not reasonable to assume a function for RNAase MRP in mitochondria. This argument, however, fails to take into consideration several important issues. The first is that the amount of detectable full-length RNAase MRP RNA may not be an accurate indicator of
the total level of active RNAase MRP. The most sensitive and unambiguous assay is RNA substrate cleavage. We do not regard this as a major point, but it is worth mentioning given the apparent ability of yeast mitochondrial RNAase P to be active under conditions in which the RNA component is not intact (Morales et al., 1989). A second matter is how much RNAase MRP must be in the mitochondrial fraction given its proposed role in mitochondrial primer RNA metabolism? Replication of mtDNA has been most extensively studied in the mouse L-cell system (Clayton, 1982), in which 4000 copies of mtDNA are synthesized per cell over a 20 hr period. This requires one leading-strand initiation event per minute per cell. In HeLa cells, the same calculation results in five initiations per minute per cell. In principle, this could be accomplished by one RNAase MRP enzyme in the cell. It should be noted that there is now a body of biochemical and cytological data that support the concept of a dynamic and largely continuous architecture for the mitochondrial mass in a living cell. However, nothing is known regarding the intramitochondrial mobility of RNAase MRP, the rate at which it could be delivered to the organelle and its exact intramitochondrial site of action, or its own half-life. It is worth noting that none of the control mtRNAs analyzed by Kiss and Filipowicz (1992), or by us, has any predictive value with regard to the expected abundance of RNAase MRP RNA. In addition, quantitating the absolute cellular amount of a given mtRNA has been notoriously difficult. A better comparison would be with an accepted enzymatic activity associated with mtDNA replication or to RNAase P (or its nuclear-encoded RNA component), which is a related ribonucleoprotein thought to be involved in mtRNA processing (Attardi and Schatz, 1988). The only such entity for which any type of quantitation is available is mtDNA polymerase. This enzyme has been historically hard to purify and characterize owing to its low cellular abundance. Systematic efforts to calculate the actual number of mtDNA polymerase molecules have rarely been attempted, but in one case (Xenopus embryogenesis; Zierler et al., 1985) avalue of roughly 0.5 to 1 .O polymerase molecule per mtDNA molecule was obtained. Given the slow polymerization rate in mitochondria, m5% of the total of ~1000 to 8000 mtDNA genomes per ceil (depending on cell type)are replicating in the steady state. Assuming both strands are being synthesized (Clayton, 1982) this would require an absolute minimum of 100 to 800 mtDNA polymerase molecules per cell. Therefore, mtDNA polymerase may be present in about 5- to lo-fold excess over the theoretical absolute minimum. To our knowledge, there is no evidence in any wild-type system that the amount of available DNA polymerase activity is rate limiting for DNA replication. Given that priming of mtDNA leading-strand synthesis begins by transcription at the most active promoter in mtDNA, it is unlikely that primer RNA synthesis per se is rate limiting. Therefore, some event intermediate between primer transcript synthesis and eventual elongation by mtDNA polymerase is likely a regulation point. In this regard, it is interesting that the process of doubling the number of mtDNA molecules results in some molecules replicating more than once and
Cdl
18
c
ml met-tRNA
f
cyto ser.tRNA
RNess MRP -t RNA
Ul RNA --)
Figure
1. Northern
Analysis
of RNAase
and Detergent-Treated
Mitochondria
to Examine
the Localization
of RNAase
MRP,
Ul,
and tRNAs
(A) Fractions of RNA isolated from mitochondria that had been treated with RNAase A or digitonin and subsequently blotted to nylon membranes and probed with the genes for human RNAase MRP RNA or Ul RNA. Mitochondria from 1 .O liter of human KB cells grown to mid-log phase were isolated by discontinuous sucrose gradients as described previously (Tapper et al., 1983). These were resuspended in 3.0 ml of buffer containing 0.25 M sucrose, 20 mM Tris-HCI (pli 7.4). 1 .O mM dithiothreitol, 10 mM KCI, 1.5 mM MgC12. 5 mM CaCI,, and 50 uglml Escherichia coli tRNA and brought to room temperature. This was aliquoted into three equal portions and designated mock reaction, Q NAase treated, and RNAase + Triton X-l 00, respectively. The fractions treated with RNA&e were supplemented with 1 .O mglml RNAase A and 400 U/ml micrococcal nuclease, from fresh stocks daily, and the Triton X-IOO-treated fraction was adjusted to 1% Triton X-100. The mock reactions had equivalent amounts of sterile water added. Individual fractions were then incubated for 30 min at room temperature. lodoacetamide was then added to a final concentration of 50 mM (to inactivate the RNAase A), and the incubation was continued for an additional 5 min. The mock reaction and RNAasetreated samples were then centrifuged in a microfuge for 10 min to collect the mitochondria, which were then resuspended in a buffer containing 0.25 M sucrose, 20 mM Tris-HCI (pH 8.0). 10 mM NaCI, 10 mM vanadyl-ribonucleoside complexes, 20 mM iodoacetamide, and 1 mM EGTA. The samples were then recentrifuged as above and resuspended in 0.25 M sucrose, 20 mM Tris-HCI (pH 8.0) IO mM NaCl, 0.3 mglml proteinase K, 0.1 mglml E. coli tRNA, 1 mM EGTA, 2 mM dithiothreitol, and 10 mM succinate and incubated for 30 min at 37OC. These steps served to expose the outside of the organelles to large amountsof RNAase and then eliminate the residual RNAase so as not to interfere with subsequent analysis of the remaining protected RNA. After this incubation, the mitochondria were lysed with 0.5% SDS, treated an additional 30 min with proteinase K, and the RNAs were isolated by the sodium perchlorate method as described (Fisher and Clayton, 1985). The sample that had received Triton X-100 in addition to the RNAase was treated with 25 mM dithiothreitol to inactivate the iodoacetamide prior to SDS-proteinase K extraction and sodium percholorate precipitation. For the digitonin treatment, the mitochondria were isolated as above and resuspended in the same buffer as above containing 50 ug/ ml E. coli tRNA. This was adjusted to 0.5% digitonin and agitated in the cold room for 15 min. The fraction was then diluted by 50% with buffer (minus digitonin) and homogenized five times with a Dounce glass homogenizer and a B pestle. The mitoplasts were recovered by centrifugation and washed once with the same buffer (minus digitonin); both the pellets and the pooled supernatants were treated with SDS and proteinase K, and RNA was isolated as above. Probes used for the Northern analyses consisted of restriction fragments of the human RNAase MRP RNA gene and the human Ul RNA gene that had been isolated and isotopically labeled by the method of random hexamers. (B) Control experiment utilizing probes for the mitochondrial methionyl-tRNA and a human cytoplasmic se@-RNA. In these experiments, the mitochondria were isolated and treated as described above with the following exception. An extra lane, labeled Untreated, is shown in this analysis. This corresponds to RNA isolated from mitochondria directly from the sucrose gradient and not subjected to any additional treatment. The mock incubation and subsequent isolation of mitochondria remove the bulk of the nuclear tRNAs. Probes: mitochondrial methionyl-tRNA was probed with the gene for this RNA inserted into an Ml3 vector and labeled by the reverse primer method as described previously (Chang and Clayton, 1989) and the seryl-tRNA species was probed with a restriction fragment of the gene (kindly provided by U. L. RajBhandary) inserted into an Ml3 vector and labeled by the method of reverse priming.
not at all (Flory and Vinograd, 1973; Bogenhagen and Clayton, 1977); this could be due to limited and localized availability of the required components for leadingstrand origin activation. Therefore, RNAase MRP, at low abundance and with RNA processing activity on mitochondrial primer RNA sequences, maintain8 two features consistent with its playing a potential regulatory and perhaps a rate-limiting role in mtDNA replication. Unfortunately, Other8
there is currently no available experimental system for studying mtDNA leading-strand origin function either in vivo or in vitro. Given the legitimate issue that is raised by the very low apparent abundance of full-length RNAase MRP RNA associated with mitochondrial fractions, it is important to examine carefully the current basis for assigning a mitochondrial function to it. Figure 2A presents the map positions
Matters 19
Mouse
Arising
S-U
A
A
G
A’G
G A’G
G
G G
G
”
-
G
G
G
G G
G
”
”
U G
-3
I I I I I I I I I I III I IIIII HumanS-CCAGAAGCGGGGGAGGGGGGGUUUG-3 I I 11111111111 I II Yeas,S-AAGUAAUAGGGGGAGGGGGUGGGUG-3 4
Figure
2. RNAase
ttt
4
MRP Cleavage
GC-cluster
C
of mtRNA
(A) The sites of primary cleavage by RNAase MRP on three mtRNA substrates are shown. Identical substrate nucleotides in pairwise comparison are shown by vertical lines. The dash in the mouse mtRNA sequence denotes the absence of a nucleotide at that position. The arrows denote the same five positions of cleavage of mouse, human, and yeast RNAase MRPs on their own and on each others substrate. The lengths of the arrows denote the relative frequency of cleavage at the respective positions in homologous cases of enzyme and substrate (downward arrows, mouse and human; upward arrows, yeast). (6) The table lists the current cases where RNAase MRP activities have been isolated and tested on mtRNA substrates from the same (homologous) or different (heterologous) species. The pattern of cleavage is intrinsic to the particular substrate; the only notable exception to date is the apparent inability of the yeast RNAase MRP to process RNA at multiple, separate sites (Karwan et al., 1991) in vitro. In the instances tested (plus sign) cleavage occurs in a similar manner in the heterologous as in the homologous combination ( f some differences in overall cleavage efficiency). Data on mouse and human heterologous cleavage are in Chang and Clayton (1987a). Topper et al. (1999) and Bennett and Clayton (1990). RNAase MRP assays with yeast mtRNA substrate are in Topper et al. (1988) and Stohl and Clayton (1992).Assayswith bovine RNAase MRPwith bovineandothermtRNA substrates were performed by D. J. Dairaghi of this laboratory (unpublished data). NT, not tested.
of the primary RNAase MRP cleavages on mtRNA substrates from three sources. The most extensively studied substrate for RNAase MRP is a mouse mtRNA sequence derived from the leading-strand origin region. The principal site of cleavage within this RNA sequence is located immediately adjacent to a highly conserved sequence block in vertebrate mtDNA, conserved sequence block II (CSB II) (Figure 2A, top line). Kiss and Filipowicz (1992) argue that this site of cleavage may be simply fortuitous, because it does not correspond exactly to an RNA to DNA transition site derived from nucleic acids isolated from mouse mitochondria. It is true that we have described the relationship between this cleavage site and the RNA to DNA transition zone in conservative terms given the precision of the mapping data of these very low abundance species. Based on strand-sizing data, the three major RNAase MRP cleavage points could be 8-8 nt upstream of the downstream boundary for DNA 5’ ends. In this regard, the most unequivocal data are those of Gillum and
Clayton (1979), where the actual 5’-end nucleotides of nascent strands were determined directly. Assuming that nascent strands are complementary to their template strand, the data of Figure 8 of that work show unambiguously that the 5’ ends of principally DNA strands contain rA and rG in a distribution that can only be rationalized by placing those 5’ ends exactly at the RNAase MRP cleavage site. We note that isolates of RNAase MRP that represent the bulk of the cellular activity are also capable of cleaving a mouse mtRNA substrate -50 nt downstream of the site shown in Figure 2A (Karwan et al., 1991). This second site is 2-4 nt upstream of the next downstream 5’-end map position of nascent mouse mtDNA strands (Gillum and Clayton, 1979; Chang et al., 1985). Nascent human mtDNA strands have not been as completely characterized as those for mouse, but the same pattern of cleavage at CSB II is observed, and human and mouse RNAase MRPs cleave their respective substrates at exactly the same location at CSB II. In addition, yeast mtDNA contains a sequence associated with putative origins of replication (termed GC-cluster C) that is very similar to CSB II (de Zamaroczy et al. 1984; Faugeron-Fonty et al., 1984). Yeast mtRNA that would be transcribed from known yeast promoters in these sequences is cleaved by a site-specific yeast ribonucleoprotein endoribonuclease at the positions shown in Figure 2A (Stohl and Clayton, 1992). This cleavage site is exactly the reported site of transition between RNA linked to DNA in two separate cases of in vivo mitochondrial nucleic acids analyzed (Baldacci et al., 1984). Because this yeast activity shares physical purification and cleavage-reaction features with mammalian RNAase MRPs, we believe it is the yeast RNAase MRP homolog (Stohl and Clayton, 1992). The yeast mtDNA replication origin is very simple compared with vertebrate mtDNAs in that only one RNA to DNA transition site has been reported. The yeast endonuclease cleaves precisely at the predicted 3’primer RNA positions. The physical structure of the mtRNA substrate that is required to enable cleavage by RNAase MRP is highly conserved between species (Figure 28). RNAase MRPs from yeast to humans not only cleave their own homologous substrates, but in every heterologous case tested a similar basic pattern of cleavage characteristic of the substrate is observed. In every case these cleavages are at or are at least very close to the reported positions of in vivo nascent nucleic acid termini. Furthermore, RNAase MRP does not cleave at any other sites in these substrates that would then require additional explanations. It is important to recognize that the required short sequences in these mtRNA substrates are highly conserved in a region of the mtDNA genome in which they are flanked by otherwise completely different sequences. The substrate requirement for the CSB II type of sequence, as shown in Figure 2A, has been established, and even single point mutations within this sequence can result in profound alterations in the ability of RNAase MRP to cleave normally (Bennett and Clayton, 1990). If, in fact, there were no RNAase MRP available for mtRNA processing in the cases cited above, then the following would have to be true: some other process, cur-
Cdl 20
rently unknown, exists in mitochondria and can effect at least a very similar type of RNA end formation as achieved by RNAase MRP in vitro; RNAase MRP functions only elsewhere in the cell, where it cleaves RNA of currently unknown origin; the conservation of the NRF-1 site (nuclear respiratory factor 1; Evans and Scarpulla, 1990) a target sequence for a transactivator protein important for efficient transcription of some nuclear genes encoding components of the mitochondrial electron transport chain, in the 5’ upstream region of both human and mouse RNAase MRP RNAgenes is fortuitous; and the only evolutionarily conserved sequences at currently identified mitochondrial origins are by chance mimicking the true RNAase MRP substrate to a degree that site-directed mutagenesis implicates their required intact presence in order for RNA cleavage to occur. Although these situations do not represent an impossibility, the fourth requires a degree of stringent coevolution of separate discrete processes from yeast to humans that seems highly implausible. Conclusions and Prospects In the first reports on the identification of RNAase MRP there was evidence to indicate that the major amount of the full-length RNA component was located in the nuclear compartment. All experiments since that time have supported that view and more recent data are consistent with the substantial majority of enzymatic activity also being nuclear in location (Topper and Clayton, 1990a; Karwan et al., 1991). Kiss and Filipowicz (1992) have assayed for the presence of the full-!ength RNAase MRP RNA component through a series of increasingly stringent mitochondrial and submitochondrial purification procedures. Their central conclusion is that very little, if any, full-length RNAase MRP RNA remains protected within the mitochondrial matrix in the final analysis. From this they deduce that RNAase MRP only functions in the nucleus of the cell, most likely in rRNA processing. There has never been any doubt that a nuclear role for RNAase MRP would almost certainly exist, and this point has been made repeatedly in all previous publications from our laboratory. As for a function in mitochondria, we believe, for the reasons stated, that an amount of full-length RNAase MRP RNA even at the level of detectability in the experiments of Kiss and Filipowicz (1992) would, in principle, be sufficient for its purported role based on all data available at this time. In this light, it is important to note that RNAase MRP enzymatic activity, not RNAase MRP RNA abundance, is the critical assay. It is important to confirm or deny a role for RNAase MRP in mitochondria in a definitive manner and to learn its role in the nucleus. To these ends, the discovery of an RNAase MRP type of endoribonuclease in yeast (Stohl and Clayton, 1992) should permit the identification of its respective component(s) and, in turn, its function in various cellular compartments. Genetic evidence from yeast will likely provide a definitive answer for or against a requirement for RNAase MRP in maintaining wild-type mtDNA. We thank Mark E. Schmitt for useful discussions regarding the manuscript. This work has been supported by the National Institute of General Medical Sciences (GM33088).
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Tam&s Kiss and Witold Filipowicz Friedrich Miescher-lnstitut PO Box 2543 4002 Base1 Switzerland
A Role for RNAase MRP in Mitochondrial RNA Processing
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The basic mode of mammalian mitochondrial DNA (mtDNA) replication has been established (Clayton, 1982), and the predominant control region for both replication and transcription has been located at one position on the circular genome. This site contains both major promoters, the origin of leading-strand mtDNA replication, and the signature feature of mammalian mtDNA, the displacement loop. Priming of leading-strand mtDNA synthesis begins by transcriptional initiation at a mtDNA promoter located 450 bp upstream of the most downstream nascent DNA 5’ termini that have been identified. There are multiple positions within this 450 bp zone at which RNA a*d DNA strand termini map. In most cases RNA 3’ends match DNA 5’ ends, and thus the former have been postulated to be primers for the latter. The number and relative positions of these RNA to DNA transition sites vary in complexity between different vertebrate species. In the case of mouse, in at least one instance, it was possible to demonstrate covalent linkage between an RNA whose 5’ end maps at the promoter transcriptional start site and DNA (Chang et al., 1985). The recognition that some mechanism must exist for the formation of these RNA termini in vivo prompted a search for any activity with such a capacity. These initial experiments led to the identification of a ribonucleoprotein endoribonuclease that cleaved at the most upstream RNA to DNA transition point in human and mouse mtDNAs; the activity was termed RNAase MRP for mitochondrial RNA processing (Chang and Clayton, 1987a, 1987b). Subsequent work has dealt with the sequence and properties of the RNAcomponent of RNAase MRP from several species (Chang and Clayton, 1989; Yuan et al., 1989; Topper and Clayton, 1990a, 1990b; Yuan et al., 1991) the nuclear gene for this small RNA from mouse (Chang and Clayton, 1989) and human (Topper and Clayton, 1990a; Yuan and Freddy, 1991) a possible relationship between RNAase MRP and the tRNA-processing enzyme RNAase P (Gold et al., 1989) and the determination of the critical regions of the mtRNA substrate that are required for cleavage (Bennett and Clayton, 1990). Kiss and Filipowicz (1992 [this issue of Cell]) have examined the abundance of a putative RNAase MRP RNA from a plant source and human RNAase MRP RNA from HeLa cells. By analyzing the hybridizable amount of the putative plant or known human full-length RNAase MRP RNA relative to other known RNA species at different stages of subcellular fractionation and purification, they conclude that the amount of detectable full-length RNAase MRP RNA is so low as to permit the deduction that the RNAase MRP ribonucleoprotein enzyme is not present in mitochondria in vivo and therefore presumably is only functional elsewhere in the cell, most likely in the nucleus. Results and Discusslon It is appropriate to be concerned about the high abundance of RNAase MRP RNA in the nucleus, and our as-
Matters 17
Arising
signment of a mitochondrial function for the enzyme itself has been based largely on its site-specific cleavage of mtRNAs. That only a very minor portion of the total cellular full-length RNAase MRP RNA is in mitochondrial fractions is seen in the original description of the RNA species in RNAase MRP: the amount of detectable full-length RNAase MRP RNA in the mitochondrial fraction is below 1% of that present in the nuclear fraction (Figure 76 in Chang and Clayton, 1987b). Concordantly, this same ratio can be reproduced when assaying the amount of RNAase MRP enzymatic activity from isolated subcellular fractions (Karwan et al., 1991). Reliable estimates on the relative amount of nucleus-localized RNAase MRP cleavage activity were not achieved in 1987, owing to substrate degradation in those crude extracts (see Discussion in Chang and Clayton, 1987b). Kiss and Filipowicz (1992 [this issue of CeIj) correctly note that we have not reported studies assessing the amounts of RNAase MRP RNA present following nuclease digestion of mitochondria or in submitochondrial mitoplasts. We have done such experiments as controls for other experiments, and a typical result is shown in Figure IA. The basic conclusions that can be drawn from these experiments are: first, most of the RNAase MRP and Ul RNAs associated with sucrose gradient-purified mitochondria are nuclease sensitive; second, a reproducible minor amount of RNAase MRP and Ul RNAs survives nuclease treatment and is rendered nuclease sensitive by solubilization of mitochondria with Triton X-l 00; and, third, preparation of mitoplasts by digitonin treatment shows a similar minor amount of RNAase MRP and Ul RNAs associated with mitoplasts. As independent controls, we tested for the presence of an mtDNA-encoded RNA and a small cytoplasmic RNA under the same conditions (Figure 16). The methionyl-tRNA known to be encoded by mtDNA and assumed to be localized entirely within the mitochondrial matrix is completely resistant to nuclease digestion until mitochondrial solubilization, whereas a nuclear-encoded seryl-tRNA is substantially removed by the incubation wash and can be entirely digested without mitochondrial solubilization. Because of our inability to digest Ul RNA completely, we did not pursue this approach further. Thus, less than 1% of the total full-length RNAase MRP RNA is present in purified mitochondrial fractions. Our earlier data, the data here, and the experiments of Kiss and Filipowicz (1992) are in agreement in this regard. Kiss and Filipowicz (1992) have extended this type of analysis by additional gradient procedures and nuclease treatment of isolated mitoplasts; we believe their experiments have been well executed. The central conclusion drawn by Kiss and Filipowicz (1992) is that at their ultimate levels of mitochondrial or mitoplast purification only a few molecules of full-length RNAase MRP RNA per cell are still associated with mitochondria (even less for mitoplasts) and that at these amounts it is not reasonable to assume a function for RNAase MRP in mitochondria. This argument, however, fails to take into consideration several important issues. The first is that the amount of detectable full-length RNAase MRP RNA may not be an accurate indicator of
the total level of active RNAase MRP. The most sensitive and unambiguous assay is RNA substrate cleavage. We do not regard this as a major point, but it is worth mentioning given the apparent ability of yeast mitochondrial RNAase P to be active under conditions in which the RNA component is not intact (Morales et al., 1989). A second matter is how much RNAase MRP must be in the mitochondrial fraction given its proposed role in mitochondrial primer RNA metabolism? Replication of mtDNA has been most extensively studied in the mouse L-cell system (Clayton, 1982), in which 4000 copies of mtDNA are synthesized per cell over a 20 hr period. This requires one leading-strand initiation event per minute per cell. In HeLa cells, the same calculation results in five initiations per minute per cell. In principle, this could be accomplished by one RNAase MRP enzyme in the cell. It should be noted that there is now a body of biochemical and cytological data that support the concept of a dynamic and largely continuous architecture for the mitochondrial mass in a living cell. However, nothing is known regarding the intramitochondrial mobility of RNAase MRP, the rate at which it could be delivered to the organelle and its exact intramitochondrial site of action, or its own half-life. It is worth noting that none of the control mtRNAs analyzed by Kiss and Filipowicz (1992), or by us, has any predictive value with regard to the expected abundance of RNAase MRP RNA. In addition, quantitating the absolute cellular amount of a given mtRNA has been notoriously difficult. A better comparison would be with an accepted enzymatic activity associated with mtDNA replication or to RNAase P (or its nuclear-encoded RNA component), which is a related ribonucleoprotein thought to be involved in mtRNA processing (Attardi and Schatz, 1988). The only such entity for which any type of quantitation is available is mtDNA polymerase. This enzyme has been historically hard to purify and characterize owing to its low cellular abundance. Systematic efforts to calculate the actual number of mtDNA polymerase molecules have rarely been attempted, but in one case (Xenopus embryogenesis; Zierler et al., 1985) avalue of roughly 0.5 to 1 .O polymerase molecule per mtDNA molecule was obtained. Given the slow polymerization rate in mitochondria, m5% of the total of ~1000 to 8000 mtDNA genomes per ceil (depending on cell type)are replicating in the steady state. Assuming both strands are being synthesized (Clayton, 1982) this would require an absolute minimum of 100 to 800 mtDNA polymerase molecules per cell. Therefore, mtDNA polymerase may be present in about 5- to lo-fold excess over the theoretical absolute minimum. To our knowledge, there is no evidence in any wild-type system that the amount of available DNA polymerase activity is rate limiting for DNA replication. Given that priming of mtDNA leading-strand synthesis begins by transcription at the most active promoter in mtDNA, it is unlikely that primer RNA synthesis per se is rate limiting. Therefore, some event intermediate between primer transcript synthesis and eventual elongation by mtDNA polymerase is likely a regulation point. In this regard, it is interesting that the process of doubling the number of mtDNA molecules results in some molecules replicating more than once and
Cdl
18
c
ml met-tRNA
f
cyto ser.tRNA
RNess MRP -t RNA
Ul RNA --)
Figure
1. Northern
Analysis
of RNAase
and Detergent-Treated
Mitochondria
to Examine
the Localization
of RNAase
MRP,
Ul,
and tRNAs
(A) Fractions of RNA isolated from mitochondria that had been treated with RNAase A or digitonin and subsequently blotted to nylon membranes and probed with the genes for human RNAase MRP RNA or Ul RNA. Mitochondria from 1 .O liter of human KB cells grown to mid-log phase were isolated by discontinuous sucrose gradients as described previously (Tapper et al., 1983). These were resuspended in 3.0 ml of buffer containing 0.25 M sucrose, 20 mM Tris-HCI (pli 7.4). 1 .O mM dithiothreitol, 10 mM KCI, 1.5 mM MgC12. 5 mM CaCI,, and 50 uglml Escherichia coli tRNA and brought to room temperature. This was aliquoted into three equal portions and designated mock reaction, Q NAase treated, and RNAase + Triton X-l 00, respectively. The fractions treated with RNA&e were supplemented with 1 .O mglml RNAase A and 400 U/ml micrococcal nuclease, from fresh stocks daily, and the Triton X-IOO-treated fraction was adjusted to 1% Triton X-100. The mock reactions had equivalent amounts of sterile water added. Individual fractions were then incubated for 30 min at room temperature. lodoacetamide was then added to a final concentration of 50 mM (to inactivate the RNAase A), and the incubation was continued for an additional 5 min. The mock reaction and RNAasetreated samples were then centrifuged in a microfuge for 10 min to collect the mitochondria, which were then resuspended in a buffer containing 0.25 M sucrose, 20 mM Tris-HCI (pH 8.0). 10 mM NaCI, 10 mM vanadyl-ribonucleoside complexes, 20 mM iodoacetamide, and 1 mM EGTA. The samples were then recentrifuged as above and resuspended in 0.25 M sucrose, 20 mM Tris-HCI (pH 8.0) IO mM NaCl, 0.3 mglml proteinase K, 0.1 mglml E. coli tRNA, 1 mM EGTA, 2 mM dithiothreitol, and 10 mM succinate and incubated for 30 min at 37OC. These steps served to expose the outside of the organelles to large amountsof RNAase and then eliminate the residual RNAase so as not to interfere with subsequent analysis of the remaining protected RNA. After this incubation, the mitochondria were lysed with 0.5% SDS, treated an additional 30 min with proteinase K, and the RNAs were isolated by the sodium perchlorate method as described (Fisher and Clayton, 1985). The sample that had received Triton X-100 in addition to the RNAase was treated with 25 mM dithiothreitol to inactivate the iodoacetamide prior to SDS-proteinase K extraction and sodium percholorate precipitation. For the digitonin treatment, the mitochondria were isolated as above and resuspended in the same buffer as above containing 50 ug/ ml E. coli tRNA. This was adjusted to 0.5% digitonin and agitated in the cold room for 15 min. The fraction was then diluted by 50% with buffer (minus digitonin) and homogenized five times with a Dounce glass homogenizer and a B pestle. The mitoplasts were recovered by centrifugation and washed once with the same buffer (minus digitonin); both the pellets and the pooled supernatants were treated with SDS and proteinase K, and RNA was isolated as above. Probes used for the Northern analyses consisted of restriction fragments of the human RNAase MRP RNA gene and the human Ul RNA gene that had been isolated and isotopically labeled by the method of random hexamers. (B) Control experiment utilizing probes for the mitochondrial methionyl-tRNA and a human cytoplasmic se@-RNA. In these experiments, the mitochondria were isolated and treated as described above with the following exception. An extra lane, labeled Untreated, is shown in this analysis. This corresponds to RNA isolated from mitochondria directly from the sucrose gradient and not subjected to any additional treatment. The mock incubation and subsequent isolation of mitochondria remove the bulk of the nuclear tRNAs. Probes: mitochondrial methionyl-tRNA was probed with the gene for this RNA inserted into an Ml3 vector and labeled by the reverse primer method as described previously (Chang and Clayton, 1989) and the seryl-tRNA species was probed with a restriction fragment of the gene (kindly provided by U. L. RajBhandary) inserted into an Ml3 vector and labeled by the method of reverse priming.
not at all (Flory and Vinograd, 1973; Bogenhagen and Clayton, 1977); this could be due to limited and localized availability of the required components for leadingstrand origin activation. Therefore, RNAase MRP, at low abundance and with RNA processing activity on mitochondrial primer RNA sequences, maintain8 two features consistent with its playing a potential regulatory and perhaps a rate-limiting role in mtDNA replication. Unfortunately, Other8
there is currently no available experimental system for studying mtDNA leading-strand origin function either in vivo or in vitro. Given the legitimate issue that is raised by the very low apparent abundance of full-length RNAase MRP RNA associated with mitochondrial fractions, it is important to examine carefully the current basis for assigning a mitochondrial function to it. Figure 2A presents the map positions
Matters 19
Mouse
Arising
S-U
A
A
G
A’G
G A’G
G
G G
G
”
-
G
G
G
G G
G
”
”
U G
-3
I I I I I I I I I I III I IIIII HumanS-CCAGAAGCGGGGGAGGGGGGGUUUG-3 I I 11111111111 I II Yeas,S-AAGUAAUAGGGGGAGGGGGUGGGUG-3 4
Figure
2. RNAase
ttt
4
MRP Cleavage
GC-cluster
C
of mtRNA
(A) The sites of primary cleavage by RNAase MRP on three mtRNA substrates are shown. Identical substrate nucleotides in pairwise comparison are shown by vertical lines. The dash in the mouse mtRNA sequence denotes the absence of a nucleotide at that position. The arrows denote the same five positions of cleavage of mouse, human, and yeast RNAase MRPs on their own and on each others substrate. The lengths of the arrows denote the relative frequency of cleavage at the respective positions in homologous cases of enzyme and substrate (downward arrows, mouse and human; upward arrows, yeast). (6) The table lists the current cases where RNAase MRP activities have been isolated and tested on mtRNA substrates from the same (homologous) or different (heterologous) species. The pattern of cleavage is intrinsic to the particular substrate; the only notable exception to date is the apparent inability of the yeast RNAase MRP to process RNA at multiple, separate sites (Karwan et al., 1991) in vitro. In the instances tested (plus sign) cleavage occurs in a similar manner in the heterologous as in the homologous combination ( f some differences in overall cleavage efficiency). Data on mouse and human heterologous cleavage are in Chang and Clayton (1987a). Topper et al. (1999) and Bennett and Clayton (1990). RNAase MRP assays with yeast mtRNA substrate are in Topper et al. (1988) and Stohl and Clayton (1992).Assayswith bovine RNAase MRPwith bovineandothermtRNA substrates were performed by D. J. Dairaghi of this laboratory (unpublished data). NT, not tested.
of the primary RNAase MRP cleavages on mtRNA substrates from three sources. The most extensively studied substrate for RNAase MRP is a mouse mtRNA sequence derived from the leading-strand origin region. The principal site of cleavage within this RNA sequence is located immediately adjacent to a highly conserved sequence block in vertebrate mtDNA, conserved sequence block II (CSB II) (Figure 2A, top line). Kiss and Filipowicz (1992) argue that this site of cleavage may be simply fortuitous, because it does not correspond exactly to an RNA to DNA transition site derived from nucleic acids isolated from mouse mitochondria. It is true that we have described the relationship between this cleavage site and the RNA to DNA transition zone in conservative terms given the precision of the mapping data of these very low abundance species. Based on strand-sizing data, the three major RNAase MRP cleavage points could be 8-8 nt upstream of the downstream boundary for DNA 5’ ends. In this regard, the most unequivocal data are those of Gillum and
Clayton (1979), where the actual 5’-end nucleotides of nascent strands were determined directly. Assuming that nascent strands are complementary to their template strand, the data of Figure 8 of that work show unambiguously that the 5’ ends of principally DNA strands contain rA and rG in a distribution that can only be rationalized by placing those 5’ ends exactly at the RNAase MRP cleavage site. We note that isolates of RNAase MRP that represent the bulk of the cellular activity are also capable of cleaving a mouse mtRNA substrate -50 nt downstream of the site shown in Figure 2A (Karwan et al., 1991). This second site is 2-4 nt upstream of the next downstream 5’-end map position of nascent mouse mtDNA strands (Gillum and Clayton, 1979; Chang et al., 1985). Nascent human mtDNA strands have not been as completely characterized as those for mouse, but the same pattern of cleavage at CSB II is observed, and human and mouse RNAase MRPs cleave their respective substrates at exactly the same location at CSB II. In addition, yeast mtDNA contains a sequence associated with putative origins of replication (termed GC-cluster C) that is very similar to CSB II (de Zamaroczy et al. 1984; Faugeron-Fonty et al., 1984). Yeast mtRNA that would be transcribed from known yeast promoters in these sequences is cleaved by a site-specific yeast ribonucleoprotein endoribonuclease at the positions shown in Figure 2A (Stohl and Clayton, 1992). This cleavage site is exactly the reported site of transition between RNA linked to DNA in two separate cases of in vivo mitochondrial nucleic acids analyzed (Baldacci et al., 1984). Because this yeast activity shares physical purification and cleavage-reaction features with mammalian RNAase MRPs, we believe it is the yeast RNAase MRP homolog (Stohl and Clayton, 1992). The yeast mtDNA replication origin is very simple compared with vertebrate mtDNAs in that only one RNA to DNA transition site has been reported. The yeast endonuclease cleaves precisely at the predicted 3’primer RNA positions. The physical structure of the mtRNA substrate that is required to enable cleavage by RNAase MRP is highly conserved between species (Figure 28). RNAase MRPs from yeast to humans not only cleave their own homologous substrates, but in every heterologous case tested a similar basic pattern of cleavage characteristic of the substrate is observed. In every case these cleavages are at or are at least very close to the reported positions of in vivo nascent nucleic acid termini. Furthermore, RNAase MRP does not cleave at any other sites in these substrates that would then require additional explanations. It is important to recognize that the required short sequences in these mtRNA substrates are highly conserved in a region of the mtDNA genome in which they are flanked by otherwise completely different sequences. The substrate requirement for the CSB II type of sequence, as shown in Figure 2A, has been established, and even single point mutations within this sequence can result in profound alterations in the ability of RNAase MRP to cleave normally (Bennett and Clayton, 1990). If, in fact, there were no RNAase MRP available for mtRNA processing in the cases cited above, then the following would have to be true: some other process, cur-
Cdl 20
rently unknown, exists in mitochondria and can effect at least a very similar type of RNA end formation as achieved by RNAase MRP in vitro; RNAase MRP functions only elsewhere in the cell, where it cleaves RNA of currently unknown origin; the conservation of the NRF-1 site (nuclear respiratory factor 1; Evans and Scarpulla, 1990) a target sequence for a transactivator protein important for efficient transcription of some nuclear genes encoding components of the mitochondrial electron transport chain, in the 5’ upstream region of both human and mouse RNAase MRP RNAgenes is fortuitous; and the only evolutionarily conserved sequences at currently identified mitochondrial origins are by chance mimicking the true RNAase MRP substrate to a degree that site-directed mutagenesis implicates their required intact presence in order for RNA cleavage to occur. Although these situations do not represent an impossibility, the fourth requires a degree of stringent coevolution of separate discrete processes from yeast to humans that seems highly implausible. Conclusions and Prospects In the first reports on the identification of RNAase MRP there was evidence to indicate that the major amount of the full-length RNA component was located in the nuclear compartment. All experiments since that time have supported that view and more recent data are consistent with the substantial majority of enzymatic activity also being nuclear in location (Topper and Clayton, 1990a; Karwan et al., 1991). Kiss and Filipowicz (1992) have assayed for the presence of the full-!ength RNAase MRP RNA component through a series of increasingly stringent mitochondrial and submitochondrial purification procedures. Their central conclusion is that very little, if any, full-length RNAase MRP RNA remains protected within the mitochondrial matrix in the final analysis. From this they deduce that RNAase MRP only functions in the nucleus of the cell, most likely in rRNA processing. There has never been any doubt that a nuclear role for RNAase MRP would almost certainly exist, and this point has been made repeatedly in all previous publications from our laboratory. As for a function in mitochondria, we believe, for the reasons stated, that an amount of full-length RNAase MRP RNA even at the level of detectability in the experiments of Kiss and Filipowicz (1992) would, in principle, be sufficient for its purported role based on all data available at this time. In this light, it is important to note that RNAase MRP enzymatic activity, not RNAase MRP RNA abundance, is the critical assay. It is important to confirm or deny a role for RNAase MRP in mitochondria in a definitive manner and to learn its role in the nucleus. To these ends, the discovery of an RNAase MRP type of endoribonuclease in yeast (Stohl and Clayton, 1992) should permit the identification of its respective component(s) and, in turn, its function in various cellular compartments. Genetic evidence from yeast will likely provide a definitive answer for or against a requirement for RNAase MRP in maintaining wild-type mtDNA. We thank Mark E. Schmitt for useful discussions regarding the manuscript. This work has been supported by the National Institute of General Medical Sciences (GM33088).
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