©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Human Mitochondrial tRNA Processing (*)

Walter Rossmanith (1), Apollonia Tullo (2), Thomas Potuschak (1)(§), Robert Karwan (1)(¶), Elisabetta Sbis (2)

From the (1) Institut für Tumorbiologie-Krebsforschung der Universität Wien, PG Genexpression, Borschkegasse 8a, 1090 Wien, Austria and the (2) Centro di Studio sui Mitocondri e Metabolismo Energetico, CNR Bari, Via Amendola 165/A, 70126 Bari, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

tRNA processing is a central event in mammalian mitochondrial gene expression. We have identified key enzymatic activities (ribonuclease P, precursor tRNA 3`-endonuclease, and ATP(CTP)-tRNA-specific nucleotidyltransferase) that are involved in HeLa cell mitochondrial tRNA maturation. Different mitochondrial tRNA precursors are cleaved precisely at the tRNA 5`- and 3`-ends in a homologous mitochondrial in vitro processing system. The cleavage at the 5`-end precedes that at the 3`-end, and the tRNAs are substrates for the specific CCA addition in the same in vitro system. Using a comparative enzymatic approach as well as biochemical and immunological techniques, we furthermore demonstrate that human cells contain two distinct enzymes that remove 5`-extensions from tRNA precursors, the previously characterized nuclear and the newly identified mitochondrial ribonuclease P. These two cellular isoenzymes have different substrate specificities that seem to be well adapted to their structurally disparate mitochondrial and nuclear tRNA substrates. This kind of approach may also help to understand the structural diversities and commonalities of tRNAs.


INTRODUCTION

Almost all RNA molecules are synthesized as immature precursors. Their conversion to functional species is a crucial step in gene expression. Contrasting with the diversity of RNA maturation processes of the various nuclear transcripts, RNA maturation in mammalian mitochondria is believed to involve only a handful of different steps (for recent review, see Refs. 1-3). The 11 mRNAs, 2 rRNAs, and 22 tRNAs encoded in the circular mitochondrial genome originate from polycistronic primary transcripts. These do not contain introns nor do most of the mRNAs have flanking untranslated regions. The tRNA sequences ``punctuate'' the transcripts since they are immediately contiguous to the rRNA and protein-coding (mRNA) sequences and almost regularly interspersed between them. This unique genetic arrangement led to a model which predicted that the endonucleolytic processing of mitochondrial transcripts is carried out mainly by precursor (pre-)tRNA() processing enzymes cleaving precisely at the 5`- and 3`-ends of the tRNAs. In other words, RNA processing of polycistronic primary transcripts would lead to tRNAs, mRNAs, and rRNAs as a natural consequence of tRNA processing (4, 5, 6, 7) . CCA addition, polyadenylation, and base modification of some nucleotides would then complete the maturation of the different RNA species. However, no mammalian mitochondrial tRNA processing enzyme (i.e. an enzyme from mitochondria capable of processing mitochondrial pre-tRNAs) has been found since the original proposal of this model in 1980/81 (discussed in Refs. 1, 8). Thus, testing the validity of the model by means of an in vitro processing system is a critical experiment and an indispensable need for the identification of mitochondrial tRNA processing enzymes.

This work represents the first direct demonstration of animal mitochondrial activities involved in the processing of mitochondrial tRNAs using a homologous in vitro processing system. We report the precise 5`- and 3`-endonucleolytic processing of human mitochondrial tRNA precursors in HeLa cell mitochondrial extracts and the 3`-terminal CCA addition to these processed mitochondrial tRNAs. Furthermore, we demonstrate that human cells contain two distinct activities that remove 5` leaders from pre-tRNAs (RNase P(s)). Their peculiar substrate specificities and the way they fractionate suggest strongly that the newly identified RNase P activity is mitochondrial whereas the other is nuclear, also indicating that previous mammalian RNase P isolations likely reflect the latter (9, 10, 11, 12, 13, 14) .


EXPERIMENTAL PROCEDURES

Precursor tRNA Substrates

phOL2, the template for (mt)pre-tRNA contains nucleotides 5577-5931 of the HeLa cell mitochondrial genome (numbering according to Ref. 6) cloned into the XbaI/EcoRI sites of the vector pGEM-1 (Promega). The plasmid was digested with TaqI prior to transcription with T7 RNA polymerase. phL, the template for (mt)pre-tRNA, contains nucleotides 3221-3608 of the human mitochondrial genome cloned into the XbaI/EcoRI sites of the vector pBS+ (Stratagene). phL was digested with RsaI prior to transcription with T3 RNA polymerase. pUC19pSer, the template for human (n)pre-tRNA, was digested with AvaI prior to transcription with T7 RNA polymerase. The sequence of the transcript is the following (tRNA in uppercase letters): gagguuguugaaggagguacGUAGUCGUGGCCGAGUGGUUAAGGCGAUGGACUUGAAAUCCAUUGGGGUCUCCCCGCGCAGGUUCGAAUCCUGCCGACUACGgcgugcuuuuuuuacucucgg. pUC19TyrT, the template for Escherichia coli, pre-tRNAsu, was digested with FokI prior to transcription with T7 RNA polymerase. The sequence of the transcript is identical to the sequence of the 131-nucleotide-long pre-tRNAsu isolated in vivo(15) .

Internally labeled substrate RNA was prepared by in vitro transcription with the appropriate RNA polymerase as described (16) . 10-µl reactions contained 500 µM of ATP, UTP, and CTP and 50 µM of GTP plus 22 µCi of [-P]GTP (3000 Ci/mmol). Transcripts were purified by denaturing polyacrylamide gel electrophoresis (16) . End-labeled substrate RNA was prepared by in vitro transcription with 1 unit/µl of the appropriate RNA polymerase in 80 mM HEPES-KOH (pH 7.6), 12 mM MgCl, 2 mM spermidine, 40 mM DTT, 1 mM each of ATP, CTP, GTP, and UTP, 1 unit/µl rRNasin (Promega), 5 units/ml yeast inorganic pyrophosphatase (Sigma), and 20-100 µg/ml template DNA (17) . Unlabeled transcripts were gel purified as described earlier (18) . Aliquots were 3`-end labeled with [P]pCp as described (19) or dephosphorylated and 5`-end labeled with [-P]ATP according to standard methods (20) . End-labeled substrate RNAs were purified as described above.

Preparation of Mitochondrial RNA Processing Activities

Exponential phase HeLa S3 cells were obtained as frozen pellets from the Computer Cell Culture Center (Mons, Belgium). Mitochondria were essentially isolated as previously described (21) . Gradient purified mitochondria (isolated from 5 10 cells) were resuspended in 4 ml of M3 (210 mM mannitol, 70 mM sucrose, 20 mM HEPES-KOH (pH 7.6), 10 mM KCl, 6 mM EGTA, 3 mM EDTA, 1 mM DTT). The protein concentration of this mitochondrial suspension was determined with the Bio-Rad protein assay (Bio-Rad) using bovine serum albumin as standard; 0.3-0.5 mg of digitonin (Fluka, Buchs, Switzerland) per mg of mitochondrial protein were added, and the whole mixture was brought to a final volume of 8 ml with M3 and incubated for 15 min on ice with intermittent vortexing. The suspension was further diluted with 12 ml of M3, and mitoplasts were isolated by centrifugation at 12,000 g. After an additional wash with 20 ml of M3, mitoplasts were resuspended in 4 ml of M4 (20 mM HEPES-KOH (pH 7.6), 10% glycerol, 3 mM EGTA, 0.5 mM EDTA, 1 mM DTT, proteinase inhibitor mix (1 mM phenylmethanesulfonyl fluoride, 1 mM benzamidine, 2 µM leupeptin, 1 µM pepstatin A) (all from Merck)). For extraction, 4 ml of M5 (M4 containing 300 mM KCl and 1% Triton X-100) were added, and the suspension was then vortexed and incubated on ice for 15 min. The extract was cleared by centrifugation at 20,000 g and stored frozen in aliquots at -70 °C. The average protein concentration of this mitoplast extract was 4-6 mg/ml (Bio-Rad protein assay, bovine serum albumin as standard).

Two such preparations were pooled, centrifuged at 33,000 rpm in a Beckman 50Ti rotor for 60 min, diluted with one-half volume of M0 (30 mM HEPES-KOH (pH 7.6), 10% glycerol, 1 mM EDTA, 1 mM DTT, proteinase inhibitor mix), concentrated by ultrafiltration (Centricon 30, Amicon), and applied at 2.5 ml/min to a 30-ml DEAE-Sepharose fast flow (Pharmacia LKB, Uppsala, Sweden) column equilibrated in M100 (M0 plus 100 mM KCl). Flow-through fractions active in 5`- and 3`-end processing were pooled, concentrated as before, and layered onto a linear 15-30% glycerol gradient (15-30% glycerol in 30 mM HEPES-KOH (pH 7.6), 150 mM KCl, 1 mM EDTA, 1 mM DTT, proteinase inhibitor mix). Centrifugation was at 41,000 rpm in a Beckman SW41 rotor for 25 h. Fractions were recovered from the bottom.

For experiments shown in Fig. 5, extracts were prepared from mitochondria/mitoplasts pretreated with micrococcal nuclease essentially as previously described (22) . Gradient purified mitochondria were resuspended in 4 ml of M2 (210 mM mannitol, 70 mM sucrose, 20 mM HEPES-KOH (pH 7.6), 10 mM KCl, 3 mM CaCl, 1 mM MgCl, 1 mM DTT) and either treated with digitonin as described above or with buffer only. Micrococcal nuclease at 800 units/ml was added, and after 15 min on ice, the mixture was diluted with M2 as described above and incubated for another 15 min at room temperature. Finally, EGTA and EDTA were added to 6 and 3 mM, respectively, and the washed mitochondria/mitoplasts were used for extract preparation as described above.


Figure 5: Nuclear RNase P and mitochondrial tRNA processing? Extracts (mtE) were prepared from sucrose gradient-purified mitochondria either mock treated or treated with micrococcal nuclease (MN) or with digitonin and MN (dig./MN) as described under ``Experimental Procedures.'' Aliquots of the extract prepared from digitonin/MN-treated mitochondria were subjected to immunodepletions using anti-Th/To RNP sera, anti-Sm serum, or anti-RNP serum. Extracts, supernatants, and a fraction of DEAE/glycerol gradient-purified mitochondrial tRNA processing endonucleases (mtGG) were tested for their (mt)pre-tRNA processing capability (A) and analyzed by RNase protection for their content of H1 RNA and (mt)tRNA, respectively (B and C). A, processing of internally labeled (mt)pre-tRNA. Lane M, P-end-labeled DNA molecular weight marker VIII (Boehringer); lane 0, no enzyme added (mock); lanes 1-3, extracts prepared from mitochondria pretreated as indicated; lanes 4-7, supernatants of immunodepletions using the indicated antisera; lane 8, DEAE/glycerol gradient-purified mitochondrial tRNA processing endonucleases. Nucleotide length of DNA molecular weight standards are indicated on the left. The (mt)pre-tRNA processing products are schematically drawn on the right. B and C, RNase protection analysis using riboprobes complementary to H1 RNA (B) or (mt)tRNA (C). RNA was extracted from the indicated mitochondrial extracts (lanes 1-3), from immunodepletion supernatants (lanes 4-7), or from the DEAE/glycerol gradient fraction (lane 8). Lane M, marker as described in A. The RNase protection products corresponding to H1 RNA and (mt)tRNA are indicated on the right. In B as compared to C, the 10-fold amount of RNA was analyzed, the specific activity of the riboprobe was 10 times higher, and exposure time was 5 times longer.



Preparation of Nuclear RNase P

Nuclei recovered from the first differential centrifugation (21) were used for the preparation of nuclear RNase P. Nuclei were washed with RSB (10 mM Tris-Cl (pH 7.5), 10 mM KCl, 1.5 mM MgCl) and resuspended in 5 ml (per 10 cell equivalents) of N (20 mM HEPES-KOH (pH 7.6), 150 mM KCl, 10% glycerol, 1.5 mM MgCl, 1 mM DTT, proteinase inhibitor mix). Triton X-100 was added to 1% final concentration, and the suspension was vortexed vigorously and incubated on ice for 15 min. The extract was cleared by centrifugation at 16,000 g. 4 ml of this nuclear extract (approximately 12 mg of protein) were concentrated and applied to a 15-30% glycerol gradient as described above. Centrifugation was at 34,000 rpm in a Beckman SW41 rotor for 23 h. Fractions were recovered from the bottom.

Preparation of E. coli RNase P

A preparation of E. coli RNase P recently described was used in this study (18).

Enzyme Assays

Processing reactions were carried out in a 20-µl reaction mixture containing 30 mM HEPES-KOH (pH 7.6), 6 mM MgCl, 30 mM KCl, 2 mM DTT, 25 µg/ml bovine serum albumin, 1 unit/µl rRNasin, and 5000-50,000 cpm of gel-purified substrate RNA. 0.5-1 µl of mitoplast extracts, 3 µl of mitochondrial or nuclear glycerol gradient fraction, or E. coli RNase P were used per assay. For the assay of the mitochondrial ATP(CTP)-tRNA-specific nucleotidyltransferase, CTP and ATP at a concentration of 100 µM and 1 mM, respectively, were included in the reaction mixture. Reactions were incubated for 30 min at room temperature or at 37 °C. The reactions were stopped by guanidinium/phenol extraction, precipitated with ethanol in the presence of glycogen (Boehringer Mannheim), and subjected to denaturing polyacrylamide gel electrophoresis (6 or 8% polyacrylamide, 7 M urea) and autoradiography (20).

Citrate synthase activity was determined essentially as described (23) .

Analysis of Cleavage Products

The enzymatic and alkaline sequence ladders of end-labeled RNA substrates used to map the in vitro cleavage sites were generated as described (24) . Periodate oxidation and -elimination of RNA was done exactly as described (25) . Treatment with CIP was carried out according to standard methods (20) . To determine whether the in vitro processed tRNAs have a 5`-terminal phosphate, tRNAs were isolated from a gel, one aliquot was treated with CIP (see above), and the CIP-treated and untreated tRNA samples were incubated in 5 µl of 1 N NaOH at room temperature. After 60 h, 1 µl of 50% acetic acid was added and the mixtures were subjected to two-dimensional cellulose thin-layer chromatography as previously detailed (26) .

Immunodepletion

Mitoplast extracts were depleted of Th/To RNPs as has been described for nuclear RNase P/RNaseMRP preparations (27, 28) . The supernatants were used for pre-tRNA processing assays as well as RNA isolation. RNA was extracted with guanidinium/phenol and precipitated with ethanol using glycogen as carrier.

RNase Protection

RNase protection assays with riboprobes complementary to H1 and (mt)tRNA were performed as recently described (27) . phN6-E-Cb, the template for the (mt)tRNA riboprobe, contains nucleotides 14625-14836 of the HeLa cell mitochondrial genome cloned in the XbaI/EcoRI sites of the vector pGEM-1.


RESULTS

Processing of Mitochondrial tRNA Precursors in Vitro

To identify the enzymatic activities for human mitochondrial pre-tRNA processing, we employed human mitochondrial tRNA precursors from the light (tRNA) and heavy strand (tRNA) produced by in vitro transcription with phage RNA polymerases. Fig. 1displays schematic structures of the (mt)pre-tRNAs used in our studies. Both substrate RNAs start with a short stretch of polylinker sequence at their 5`-end, followed by a few nucleotides of mitochondrial RNA flanking the respective tRNA at the 5`-end. The (mt)pre-tRNA substrates are extended at their 3`-end by approximately 35 nucleotides of mitochondrial sequence.


Figure 1: Schematic representation of mitochondrial pre-tRNA substrates. A, In vitro transcribed (mt)pre-tRNA is 157 nucleotides in length. The substrate RNA has 15 nucleotides of polylinker sequence at its 5`-end, followed by 31 nucleotides complementary to the 5`-end of the cytochrome oxidase subunit 1 mRNA (COX 1) and further 9 nucleotides of non-coding mitochondrial RNA. The pre-tRNA substrate is extended at its 3`-end by 36 nucleotides of tRNA sequence and terminates at a TaqI restriction site. Arrows mark the exact cleavage position mapped in this study (see Fig. 2, A and B). B, In vitro transcribed (mt)pre-tRNA is 156 nucleotides in length. The substrate RNA starts with 35 nucleotides of polylinker sequence, followed by the 12 3`-terminal nucleotides of 16 S rRNA, the sequence of tRNA, and terminates at an RsaI restriction site 34 nucleotides downstream of the 3`-end of tRNA in the NADH dehydrogenase subunit 1 mRNA (ND 1). Arrows mark the exact cleavage position mapped in this study (see Fig. 2, C and D). Structure drawings were prepared using the program loopDloop.



HeLa cell mitochondrial extracts were assayed for the presence of specific endonucleases capable of processing the (mt)pre-tRNAs. To minimize contamination with cytoplasmic enzymes, sucrose gradient-purified mitochondria were treated with digitonin. The resulting mitoplasts were washed, and a salt-detergent lysate was prepared. The mitoplast extract used for tRNA maturation assays is an S20 prepared from this lysate.

End-labeled (mt)pre-tRNA and (mt)pre-tRNA were incubated with mitoplast extract, and the reaction products were separated by denaturing gel electrophoresis alongside with enzymatic sequence ladders of the same substrate RNAs (Fig. 2). This allows to determine the exact cleavage site directly from the size of the reaction products. Two processing products are obtained using the 3`-end-labeled substrate RNAs; the longer corresponds to the (mt)pre-tRNA processed at the 5`-end of the tRNA but not at the 3`-end, whereas the smaller one is the removed 3`-trailer (see Fig. 2, A and C). The observed cleavage positions are precisely at the predicted 5`- and 3`-ends of the tRNAs (6) . Only one cleavage product is found using the 5`-end-labeled (mt)pre-tRNA substrates (see Fig. 2, B and D). It corresponds to the 5`-leader removed from the (mt)pre-tRNA. The lack of the 3`-phosphate in this reaction product (see below) results in a slower electrophoretic migration as compared with the reaction products of enzymatic and alkaline sequence ladders (mimicking approximately one nucleotide more). When internally labeled substrate RNA is incubated with mitoplast extract, a cleavage product corresponding to the tRNA is found in addition to the processing products obtained when using end-labeled substrate RNA (see Fig. 3A and Fig. 4, A and B). No processing intermediate corresponding to a (mt)pre-tRNA processed at the tRNA 3`-end but not at the 5`-end is found. The complete in vitro processing system requires MgCl at an optimal concentration of 4-8 mM and is stimulated by low concentrations (up to 80 mM) of KCl (data not shown). ()


Figure 2: Precise endonucleolytic processing of mitochondrial pre-tRNAs. End-labeled (mt)pre-tRNA substrates were incubated with mitoplast extract (mtE) or without (mock), and the cleavage products were separated by denaturing gel electrophoresis alongside with enzymatic and alkaline (OH) sequence ladders of the same substrate RNAs. The RNases used to generate the sequence ladders are as follows: RNase T1 (T1) cleaving after G; Bacillus cereus RNase (B. cereus) cleaving after C and U; RNase U2 (U2) cleaving after A. The sequence around the cleavage sites is shown on the left of each autoradiography with the cleavage site indicated by an arrow. The (mt)pre-tRNA substrate and the processing products are schematically drawn on the right with the label indicated by an asterisk. A, in vitro processing of 3`-end-labeled (mt)pre-tRNA. B, in vitro processing of 5`-end-labeled (mt)pre-tRNA. C, in vitro processing of 3`-end-labeled (mt)pre-tRNA. D, in vitro processing of 5`-end-labeled (mt)pre-tRNA.




Figure 3: Characterization of cleavage products and their further maturation. A, Internally labeled (mt)pre-tRNA (lanes 1-8) or precleaved, gel-purified (mt)tRNA (lanes 9 and 10) were incubated with mitoplast extract. ATP and/or CTP were included in some reactions as indicated. Reaction products were resolved by denaturing gel electrophoresis after treatment with CIP or after periodate -elimination (JO/-elim.) where indicated. The (mt)pre-tRNA substrate and the processing products are schematically drawn on the right. Nucleotide length of DNA molecular weight standards are indicated on the left. Lane M is a P-end-labeled MspI digest of pBR322. Lanes 1, 3, and 5 show untreated standard cleavage reaction products (mock). Products in lane 2 were treated with CIP. Products in lane 4 were subjected to periodate -elimination. Note that since -elimination generates 3`-phosphate termini, thereby increasing the net negative charge of the respective cleavage products, the electrophoretic mobility of those is altered by more than one nucleotide. In lane 6 CTP, in lane 7 ATP, and in lanes 8 and 9 CTP and ATP were included in the reaction. B and C, in vitro processed (mt)tRNA was isolated and divided into two equal portions, which were incubated under the same conditions either with (C) or without CIP (B). Thereafter, samples were hydrolyzed, and the nucleotide composition was determined by two-dimensional thin layer chromatography. Autoradiographies are shown. The positions of individual nucleotides (nucleoside 2`(3`)-phosphates, Gp, Ap, and Up; guanosine 2`(3`),5`-diphosphate, pGp) and free phosphate (P) are indicated. Arrows show the direction of chromatography from the origin (marked with a point) in the first and second dimension. Note that Cp is not labeled because no C residue precedes a G in the tRNA sequence. We have not determined the identity of the weak spot labeled X.




Figure 4: Comparison of the enzymatic capabilities of mitochondrial and nuclear RNase P. (mt)pre-tRNAs, (n)pre-tRNA and E. coli pre-tRNAsu, were incubated without enzyme (mock, lanes 1), with mitoplast extract (mtE, lanes 2), with DEAE/glycerol gradient-purified mitochondrial tRNA processing endonucleases (mtGG, lanes 3), or with glycerol gradient-purified nuclear RNase P (nGG, lanes 4) in parallel under identical conditions. Processing products were resolved in parallel by denaturing gel electrophoresis. Lane M is a P-end-labeled MspI digest of pBR322. Nucleotide length of DNA molecular weight standards are indicated. The position of the specific pre-tRNA processing products is indicated. A, uniformly labeled (mt)pre-tRNA. B, uniformly labeled (mt)pre-tRNA. C, 3`-end-labeled (n)pre-tRNA. D, 3`-end-labeled pre-tRNAsu. A processing reaction with E. coli RNase P was included as reference (lane 5).



Characterization of Cleavage Products and Their Further Maturation

From the results obtained with end-labeled or internally labeled substrates, the endonucleolytic nature of mammalian mitochondrial tRNA processing is evident. The tRNA processing endonucleases, RNase P and pre-tRNA 3`-endonuclease, generally produce tRNAs with 5`-phosphate and 3`-hydroxyls. We determined the fate of the phosphate group at the cleavage site to test whether the identified endonucleases are analogous to known tRNA processing enzymes in this respect (Fig. 3; identical results were obtained for both (mt)pre-tRNA substrates used, but only data for (mt)pre-tRNA are shown). The cleavage products of an in vitro processing reaction were treated with CIP (Fig. 3A, lane 2) and resolved by gel electrophoresis along with untreated processing products (Fig. 3A, lanes 1 and 3). A slight mobility shift of all cleavage products treated with CIP is observed. The slower migration is consistent with the removal of charge by dephosphorylation. To directly determine whether the in vitro processed tRNAs contain a 5`-phosphate, (mt)pre-tRNA was uniformly labeled with [-P]GTP, the tRNA cleavage product isolated and subjected to total alkaline hydrolysis. The nucleotide composition was analyzed by two-dimensional thin layer chromatography and subsequent autoradiography (Fig. 3B). A guanosine 2`(3`),5`-diphosphate is present in the hydrolysate of the tRNA along with the expected nucleoside 2`(3`)-phosphates. We have not determined the identity of an additional spot, labeled X and possibly representing a modified nucleotide. The guanosine 2`(3`),5`-diphosphate spot is not observed if the tRNA has been treated with CIP prior to alkaline hydrolysis (Fig. 3C). The presence of 3`-hydroxyls in the processing products was substantiated by oxidation with periodate and removal of the 3`-terminal base by the -elimination reaction (Fig. 3, compare lane 4 with lanes 3 and 5). Taken together, these results demonstrate that the human mitochondrial RNase P and pre-tRNA 3`-endonuclease activities leave 5`-phosphates and 3`-hydroxyls on their cleavage products.

Mammalian mitochondrial tRNA genes do not encode the 3`-terminal CCA sequence found in mature tRNAs (6, 29) . A rat liver mitochondrial tRNA nucleotidyltransferase activity capable of incorporating [C]ATP into yeast tRNA was characterized some time ago (30). We analyzed if the in vitro processed mitochondrial tRNAs can act as a substrate for the specific addition of the CCA sequence (see Fig. 3A). Indeed, if the cleavage reaction is performed in the presence of CTP and ATP or if a precleaved tRNA is incubated with these nucleotides in mitoplast extract, a product that is 3 nucleotides longer than the tRNA-sized cleavage product is observed (Fig. 3A, lanes 8 and 9). Intermediates containing only one or two additional nucleotides are also observed. Incubation with CTP alone leads to a tRNA elongated by either one or two nucleotides, whereas the addition of ATP alone does not change the length of the tRNA (Fig. 3A, lanes 6 and 7). None of the other cleavage products is altered by the addition of CTP and/or ATP. Thus, our HeLa cell in vitro system for mitochondrial tRNA processing contains an ATP(CTP)-tRNA-specific nucleotidyltransferase activity that specifically adds the sequence CCA to 3`-end-processed mitochondrial tRNAs.

Human Cells Contain Two Distinct RNase P Enzymes

We were interested if the identified mitochondrial RNase P activity is identical to or shares components with the previously characterized nuclear RNase P activity (9, 10, 11, 12, 27) . We compared the enzymatic capability of both activities to cleave different pre-tRNA substrates at the 5`-end of the respective tRNA. For this purpose, we employed the two (mt)pre-tRNAs, a human nuclear (n)pre-tRNA and E. coli pre-tRNAsu, in processing reactions with mitoplast extract, partially purified mitochondrial RNase P, or partially purified nuclear RNase P (Fig. 4). While (mt)pre-tRNA is not detectably cleaved by nuclear RNase P at the tRNA 5`-end (Fig. 4B), (mt)pre-tRNA is cleaved by nuclear as well as mitochondrial RNase P (Fig. 4A). The involvement of nuclear RNase P in the in vitro cleavage reaction was confirmed by immunodepletion with a specific antiserum (data not shown). In the reverse experiment, i.e. employing (n)pre-tRNA in processing reactions with mitochondrial enzyme(s), we observed no 5`-processing of this nuclear pre-tRNA by our mitochondrial processing system (Fig. 4C); the cleavage site of nuclear RNase P is precisely at the expected 5`-end of the (n)tRNA (mapping data not shown). A substrate of special interest is E. coli pre-tRNAsu, which has been used to characterize human nuclear RNase P (9, 10, 11, 12) as well as RNase P activities purified from HeLa cell and rat liver mitochondria (13, 14) . The mitochondrial RNase P activity described in this paper, which specifically 5` matures (mt)pre-tRNAs, does not cleave pre-tRNAsu at the 5`-end of the mature tRNA employing the same reaction conditions; instead, cleavage events are observed 4 nucleotides upstream of the mature 5`-end (Fig. 4D). We do not know if these cleavage events are due to mitochondrial RNase P. Nuclear RNase P cleaves pre-tRNAsu exactly at the same site as E. coli RNase P (Fig. 4D). These results furthermore demonstrate that the used mitoplast extracts do not contain traceable nuclear RNase P activity (Fig. 4, C and D, lane 2).

To verify the copurification of the identified tRNA processing enzymes with the mitochondrial matrix fraction, we compared extracts prepared from untreated mitochondria and from mitoplasts (digitonin treated), respectively. The use of mitoplast rather than e.g. straight mitochondrial extract does not significantly affect the mitochondrial RNase P activity and the mitochondrial pre-tRNA 3`-endonuclease (Fig. 5A) or a mitochondrial marker enzyme (citrate synthase; data not shown). The digitonin pretreatment of mitochondria nevertheless leads to the almost complete removal of contaminating snRNAs, such as H1 RNA (an RNA molecule that copurifies with nuclear RNase P; Ref. 11), without affecting authentic mitochondrial RNAs (Fig. 5, B and C, lanes 1-3).

Since, using highly sensitive means, trace amounts of H1 RNA are still detectable in extracts prepared from highly purified mitoplasts (Fig. 5B, lane 3; see below for discussion), we employed autoimmune sera that specifically and quantitatively immunoprecipitate nuclear RNase P (anti-Th/To RNP sera; Refs. 10, 27). No significant difference was observed concerning the processing capability of these mitoplast extracts depleted of residual Th/To RNPs as compared with appropriate controls (Fig. 5A, compare lanes 4 and 5 with 6 and 7). Residual H1 RNA and fragments thereof, however, are completely removed by immunoprecipitation (Fig. 5B, lanes 4 and 5). Moreover, H1 RNA does not copurify with the partially purified mitochondrial RNase P activity (Fig. 5B, lane 8; compare with Fig. 4 , A and B, lane 3, and Fig. 5A, lane 8).

Taken together, our results demonstrate that (i) HeLa cells contain (at least) two distinct RNase P activities, (ii) these have different but partially overlapping substrate specificities, and (iii) neither nuclear RNase P nor its RNA or immunogenic protein components are involved in mitochondrial tRNA processing in our in vitro system. Our data furthermore strongly suggest that one RNase P activity is mitochondrial whereas the other is nuclear.


DISCUSSION

Using a homologous cell-free in vitro system, we have identified three enzymatic activities involved in the biosynthesis of mammalian mitochondrial tRNAs. Highly purified HeLa cell mitochondria/mitoplasts contain 5`- and 3`-endonucleases and an ATP(CTP) nucleotidyltransferase activity; these specifically process human mitochondrial tRNA precursors to mature CCA containing tRNAs. Our results provide direct support for the tRNA punctuation model of mitochondrial RNA processing (5) . Moreover, the development of this first homologous animal mitochondrial in vitro processing system provides the basis for a future biochemical characterization of the enzymes involved as well as for an analysis of the role of tRNA processing in certain human mitochondrial diseases (31, 32, 33) .

The endonucleolytic activity that cofractionates with mitochondria and specifically cleaves human mitochondrial tRNA precursors at the predicted 5`-end of the tRNA should be termed mitochondrial RNase P for this reason (34, 35) ; the enzyme is analogous to all other known RNase P activities, with regard to its magnesium ion requirement as well as the generation of 5`-phosphate and 3`-hydroxyl termini at the site of cleavage, suggesting a possibly similar cleavage mechanism. Apart from these similarities, our data demonstrate that the identified mitochondrial RNase P is distinct from all previously described mammalian RNase P activities (9, 10, 11, 12, 13, 14) . The mitochondrial RNase P described in this paper does, in particular, not correspond to RNase P activities previously purified from HeLa cell or rat liver mitochondria (13, 14), which were identified and characterized by their ability to cleave E. coli pre-tRNAsu at the same site as E. coli RNase P. In contrast to these previous reports, the mitochondrial activity described herein does not cleave E. coli pre-tRNAsu but cleaves authentic mammalian mitochondrial tRNA precursors and thereby fulfills the functional criterion for a mitochondrial RNase P not previously demonstrated for any other mammalian RNase P activity (for discussion, see Refs. 1, 8). Considering the cleavage potential of mitochondrial and nuclear RNase P described in this paper, those earlier preparations probably reflect the nuclear enzyme; indeed, this earlier RNase P activity was recently reported to copurify with an RNA identical to that of the nuclear RNase P (2) .

This discrepancy to previous reports and the minute traces of H1 RNA found in extracts of rigorously defined mitochondrial fractions raise the question of whether there is a second mitochondrial RNase P species identical to the nuclear enzyme. In evaluating this possibility, it has to be considered that (i) the amount of mitoplast-associated H1 RNA is rather low and corresponds to that reported for other snRNAs,()e.g. spliceosomal U snRNAs, estimated to be less than 1 molecule per 100 mitochondria (22) ; (ii) nuclear RNase P is not detectable as an activity in our mitochondrial in vitro system (probably due to its extremely low abundance, see (i)); (iii) all residual H1 RNA (and breakdown products thereof) is assembled as a Th/To RNP and thus most likely indeed represents nuclear RNase P. Considering that no bacterial, nucleocytoplasmic, or organellar genetic system has thus far been shown to contain two different RNase P enzymes, mammalian mitochondria would be the leading case of a partial RNase P redundancy.

The finding that human cells contain two distinct RNase P enzymes led us to a comparative study of the HeLa cell RNase P isoenzymes. In other words, we have asked: what is the conserved capacity, in terms of substrate recognition and cleavage, of nuclear or mitochondrial RNase P, or which are the conserved features of nuclear or mitochondrial encoded tRNAs that make them substrates for one or the other? Interestingly, (mt)pre-tRNA is a model substrate both for the nuclear as well as the mitochondrial enzyme. In contrast, (mt)pre-tRNA, which is efficiently cleaved by the mitochondrial enzyme, is not a substrate for nuclear RNase P. These results are consistent with the observation that (mt)tRNA is the most conventional mitochondrial tRNA in the sense that it retains all the invariant and semi-invariant nucleotides conserved in nonorganellar tRNAs (6, 36, 37) . Its tertiary structure may thus be assumed to be more similar to cytoplasmic or bacterial tRNAs than that of other mitochondrial tRNAs, e.g. (mt)tRNA, which lack one or more of these conserved features. In particular the D- and TC-loops of many mitochondrial tRNAs deviate in length and base composition. The inability of mitochondrial RNase P to cleave (n)pre-tRNA or E. coli pre-tRNAsu (both of which are substrates for the nuclear enzyme) poses the question of what differentiates these tRNAs from (mt)tRNA. All three tRNAs have all the conserved features, and the only apparent difference is the length of the variable arm. Considering what is known about RNase P substrate recognition (reviewed in Ref. 35), we cannot decide if the distinct but overlapping cleavage potential of mitochondrial and nuclear RNase P can be attributed to this structural variation. Nevertheless, our results make us confident that it is possible to study the structural diversities and commonalities of tRNAs as well as the coevolution of the two cellular RNase P enzymes with their in vivo substrates by a quantitative enzymatic study.

The tRNA punctuation model of mitochondrial RNA processing (5) implies that the tRNA 3`-ends and concomitantly the 5`-ends of rRNAs and mRNAs are formed by single endonucleolytic cuts. This contrasts with the best characterized mechanism of tRNA 3`-end formation in eubacteria, where exo- and endonucleases are required (38) . Human mitochondrial tRNA precursors are cleaved precisely and endonucleolytically at the predicted 3`-end of the tRNA in our in vitro system. The 3`-CCA sequence is then added stepwise by an ATP(CTP)-tRNA-specific nucleotidyltransferase activity to the 3`-hydroxyl terminus. The endonucleolytic mode of mammalian mitochondrial tRNA 3`-end formation and the posttranscriptional CCA addition resemble the tRNA maturation mechanisms reported for organellar tRNAs of other organisms (25, 39, 40, 41) . We have not yet attempted to separate the pre-tRNA 5`- and 3`-endonucleolytic activities found in HeLa cell mitochondria. However, considering that different enzymes act on the 5`- and 3`-ends of pre-tRNAs in other genetic systems, it is likely that also in human mitochondria 5`- and 3`-cleavages are carried out by distinct enzymes.

The lack of a detectable processing intermediate corresponding to a (mt)pre-tRNA with processed tRNA 3`-end but unprocessed 5`-end suggests an ordered pathway of processing of mitochondrial tRNAs in vitro, with 5`-cleavage preceding 3`-cleavage. Biochemical separation of the mitochondrial pre-tRNA 3`-endonuclease activity from RNase P will be necessary to determine if the 3`-cleavage strictly depends on a 5`-processed substrate. Even this would not necessarily imply an obligatory order of cleavages in vivo, and in fact a tRNA processed at its 3`-end but not at the 5`-end, most likely representing a processing intermediate, has been observed in rat mitochondria (42) . Similar observations have been made for yeast mitochondrial tRNA processing; while in vitro the action of a 3`-endonuclease appears to depend on a 5`-matured pre-tRNA substrate (25), petite mutants of Saccharomyces cerevisiae containing mitochondrial tRNA genes but lacking the mitochondrial RNase P RNA locus accumulate mitochondrial tRNA precursors with 5`-extensions and mature 3` termini (43, 44, 45) . Taken together, these observations suggest that the cellular route to mature mitochondrial tRNAs is not rigidly fixed.

While resecting tRNAs from primary transcripts obviously accounts for the majority of mammalian mitochondrial RNA processing, some mitochondrial RNA termini cannot be explained by tRNA processing; these are the 5`-ends of the cytochrome oxidase subunit 1 and 3 mRNAs and of the cytochrome b mRNA, respectively, the 3`-ends of the ATPase subunit 6 mRNA and the NADH dehydrogenase subunit 5 mRNA (4, 5, 7, 46) , as well as the different displacement loop RNA species believed to arise from RNA processing of light strand transcripts in this region (reviewed in Ref. 1). It is tempting to speculate that one of the activities described here processes these potential substrates, in analogy to E. coli RNase P, which cleaves the precursor to 4.5 S RNA in addition to pre-tRNAs (47) . Careful in vitro analysis will be necessary to answer these questions.


FOOTNOTES

*
This work was supported in part by the Austrian Science Foundation and Austrian Ministry of Foreign Affairs, European Molecular Biology Organization (short-term fellowship ASTF 7159 (to A. T.)), Progetto Finalizzato Ingegneria Genetica (CNR Italy), and MURST (Italy). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Institut für Botanik der Universität Wien, Abteilung für Zytologie und Genetik, Rennweg 14, 1030 Wien, Austria.

To whom correspondence should be addressed. Tel.: 43-1-40154-240; Fax: 43-1-4060790.

The abbreviations used are: pre-tRNA, precursor tRNA; RNase P, ribonuclease P; (mt), mitochondrial; (n), nuclear; pre-tRNAsu, precursor suppressor III tRNA; DTT, dithiothreitol; CIP, calf intestinal alkaline phosphatase; RNP(s), ribonucleoprotein(s); RNaseMRP, ribonuclease mitochondrial RNA processing; snRNA, small nuclear RNA.

W. Rossmanith and R. Karwan, unpublished data.

LoopDloop (Gilbert, D. G. (1992)), a Macintosh program for visualizing RNA secondary structure, is published electronically on the Internet and available via anonymous ftp to ftp.bio.indiana.edu.


ACKNOWLEDGEMENTS

We thank P. Breit for help with the figure preparation, D. A. Clayton for pUC19pSer, R. Lührmann for antisera, N. C. Martin for helpful advice, M. Nardelli for help in subcloning phL, C. Saccone for helpful advice, W. J. van Venrooij for antisera, V. Wintersberger for helpful advice, and the Dept. of Molecular Genetics (this institute) for instrumental equipment and support.


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