A Nuclear Encoded and Mitochondrial Imported Dicistronic tRNA Precursor in Trypanosoma brucei *

The mitochondrial tRNAs of Trypanosoma brucei are nuclear encoded and imported into the mitochondrion. A heterogeneous population of RNAs having characteristics of precursor tRNAs have previously been identified within the mitochondrion ofT. brucei, suggesting that import occurs via a precursor molecule. In order to identify nuclear genes encoding tRNAs targeted to the mitochondrion, individual mitochondrial tRNAs were separated using two-dimensional gel electrophoresis and enzymatically sequenced. A 1.1-kilobase pair genomic DNA fragment was cloned containing three tRNA genes, tRNA1 Ser, tRNALeu, and tRNA2 Ser. Dicistronic precursors containing the tRNA1 Ser and tRNALeu transcripts with a 59-nucleotide intergenic sequence were identified by reverse transcriptase and polymerase chain reactions and the 5′ end of the precursors determined. The dicistronic precursor tRNA is present both in the cytosol and the mitochondrion supporting a model for tRNA import involving precursor tRNA transcripts.

The nuclear encoded tRNA genes in T. brucei have been categorized into three different classes: tRNAs specific to the cytoplasm, tRNAs specific to the mitochondria, and tRNAs that appear to be shared between both compartments (9). Sixteen tRNA genes have been identified in T. brucei (11)(12)(13)(14)(15). Two of these have been characterized as single copy genes and have been shown by hybridization studies to be present in both mitochondrial and cytoplasmic RNAs, suggesting that they are targeted to both compartments (14). The majority of the tRNA genes identified have been selected with a heterologous population of labeled tRNAs. Due to the ability of closely related tRNAs to cross-hybridize it is difficult to determine whether the other identified genes encode tRNAs targeted to the mitochondrion. The identification of nuclear genes encoding tRNAs targeted to the mitochondrion is crucial in identifying the substrate requirements and mechanism of mitochondrial RNA import.
Little is known about the mechanism by which a nuclear encoded RNA can be imported into the mitochondrion. It has been suggested that import of tRNAs may be in association with the cognate nuclear encoded aminoacyl tRNA synthetase of the tRNA (16). It has recently been shown that a tRNA Lys is imported into yeast mitochondria associated with the cognate synthetase (17). Transfection studies with both Trypanosoma and Leishmania have demonstrated mitochondrial import of endogenous, mutated, and exogenous tRNAs. These results suggest that the substrate requirement for import is contained within the mature tRNA structure, independent of the genomic 5Ј-and 3Ј-flanking sequences (18 -20). An in vitro import system has been described in Leishmania in which import was demonstrated by a ribonuclease protection assay (21). An antisense transcript from the 5Ј upstream region of ␤-tubulin appears to be imported, suggesting that import of small RNAs may be nonspecific in this system. More recent studies have suggested the presence of mitochondrial membrane proteins that serve as receptors for Leishmania tRNAs and the antisense tubulin transcript (22,23). We have previously reported the presence of a population of potential precursor tRNAs within T. brucei mitochondria. These RNAs were identified based on their ability to be posttranscriptionally labeled at their 3Ј end by tRNA nucleotidyl transferase and the ability of RNase P to cleave them to the same size and pattern as mature tRNAs (24). This suggested that for at least a subset of tRNAs imported into the mitochondria of T. brucei, the import substrate consists of a precursor tRNA. Attempts to characterize these precursors by Northern blot hybridization and primer extension analysis have failed, suggesting that the initial identification of precursor molecules may have been artifactual or that the precursors are present in very low abundance (25).
In this report, we describe the cloning of a nuclear gene encoding a mitochondrial tRNA Leu . The tRNA Leu gene is immediately downstream of a tRNA Ser gene. This unusual genomic organization has been seen for other tRNA genes of T. brucei (13)(14)(15). Using a sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) 1 assay a larger transcript containing tRNA Leu was identified within the mitochondrion. The 5Ј end of this transcript was determined by 5Ј RACE. This low abundance tRNA Leu precursor contains the tRNA Ser , a 59-nucleotide intergenic sequence and the tRNA Leu . This demonstrates the existence of an imported dicistronic precursor tRNA for a specific mitochondrial tRNA in T. brucei. * This work was supported by funds from the University of Alabama at Birmingham M.D./PhD. Program (to A. J. L.) and by National Institutes of Health Grant A121401. 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 indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM

MATERIALS AND METHODS
Trypanosoma Growth and Isolation of RNAs-The procyclic form of T. brucei (TREU 667) was grown at 27°C in a semi-defined medium (26) containing 10% heat-inactivated fetal bovine serum (Whittaker) and 20 g/ml gentamicin sulfate. Mitochondria were isolated from cells at a density of 1-2 ϫ 10 7 cells/ml as described previously (27). Briefly, cells were suspended in a hypo-osmotic buffer and then lysed by passage through a 26 gauge needle. The cytosolic RNAs were isolated from the supernatant after the mitochondria and membranous material had been pelleted at 15,800 ϫ g. Mitochondrial vesicles were isolated from a 20 -35% Percoll gradient. Mitochondrial vesicles from 2 ϫ 10 10 cells were solubilized in 300 l of buffer containing 100 mM NaCl, 10 mM EDTA, 10 mM Tris-HCl, pH 8.0, 0.5% sodium dodecyl sulfate. The RNAs from both mitochondria and cytosol were extracted with hot phenol (ϫ2), phenol/chloroform (1:1), and chloroform and then precipitated with sodium acetate and ethanol.
Metabolic Labeling of Mitochondrial tRNAs-Mitochondria isolated from 2.5 ϫ 10 10 cells were resuspended in 5 mM Hepes, pH 7.6, 3 mM potassium phosphate, pH 7.7, 125 mM sucrose, 10 mM magnesium acetate, 1 mM EDTA, pH 8.0, 5 mM 2-mercaptoethanol, 0.5 mM ATP, 0.5 mM UTP, and 1 mCi of [␣-32 P]CTP (NEN Life Science Products) in a volume of 1 ml. These mitochondria are deficient in transcription due to a reduction in the mitochondrial GTP pool resulting from the preincubation in the absence of NTPs and the omission of GTP in the reactions. The mitochondria were incubated at room temperature for 30 min followed by centrifugation in a microcentrifuge for 30 s at room temperature. Mitochondria were solubilized and RNAs were isolated as described above. Isolated RNAs were loaded in a denaturing gel-loading buffer and separated on a 8% polyacrylamide/4 M urea gel at 200 V for 6 h. The region of the gel containing the tRNAs and precursor tRNAs was visualized by ethidium staining and excised. RNAs were either eluted for use in the RNase P assays or separated in the second dimension, a 17.6% polyacrylamide/8 M urea gel.
[␣-32 P]CTP (NEN Life Science Products)-labeled precursor size RNAs (10,000 cpm) were incubated in the presence of increasing concentrations of an in vitro synthesized RNase P RNA from Escherichia coli, a gift from Dr. Norman Pace and Dr. Drew Smith. After a 60-min incubation the RNAs were isolated following phenol/chloroform extraction and ethanol precipitation. The RNAs were separated on a 10% polyacrylamide/8 M urea gel and visualized by autoradiography. As a control, a precursor tRNA Asp from Bacillus subtilis, also a gift from Dr. Norman Pace and Dr. Drew Smith, was digested under the same conditions. Two-dimensional Gel Electrophoresis-30 g of mitochondria and cytosolic RNAs were electrophoresed in the first dimension on a 7% polyacrylamide/4 M urea gel. Samples were loaded in a denaturing gel-loading buffer and run at room temperature at 200 V for 6 h. The region of the gel containing the tRNAs was visualized by ethidium staining, excised, and loaded in the second dimension, a 17.6% polyacrylamide/8 M urea gel. Electrophoresis was performed at 300 V at room temperature for 36 h. tRNAs were visualized by ethidium staining. Individual tRNAs were excised from the gel and electroeluted in a Isco electroeluter in 0.01ϫ TAE for 1 h at 3 watts.
Enzymatic Sequencing-Mitochondrial tRNAs were radiolabeled at the 3Ј end with [␣-32 P]pCp (NEN Life Science Products) using T4 RNA ligase (Life Technologies, Inc.) according to the manufacturer's recommendations. Mitochondrial tRNAs were 5Ј end-labeled with [␥-32 p]ATP (NEN Life Science Products) using T4 polynucleotide kinase (Life Technologies, Inc.). The tRNAs that were either 5Ј or 3Ј end-labeled were purified on a 8% polyacrylamide/8 M urea gel. The labeled tRNAs were visualized by autoradiography, and gel slices were cut out and eluted overnight at 37°C in 0.5 M ammonium acetate, 0.1% SDS, 1.0 mM EDTA, pH 8.0. The 5Ј and 3Ј end-labeled gel-purified mitochondrial RNA was sequenced by the enzymatic method using an Amersham Pharmacia Biotech RNA sequencing kit. RNase T1 was used for Gspecific cleavage, RNase U2 for A, RNase Phy M for AϩU, RNase B. cereus for CϩU, and RNase CL3 for C. Alkaline hydrolysis was performed in 0.15 M NaHCO 3 , 0.15 M Na 2 CO 3 , and 1 mM EDTA at pH 9.2 at 90°C for 5 min.
Northern Blot Analysis-For one-dimensional Northerns, mitochondrial RNA (15 g) and cytosolic RNA (15 g) were electrophoresed in a 8% polyacrylamide/8 M urea gel then electrotransferred onto Gene-Screen Plus (NEN Life Science Products) in 40 mM Tris, 20 mM acetic acid, 1 mM EDTA (1ϫ TAE) overnight at 15 V. For two-dimensional Northern blots, 20 g of mitochondrial and cytosolic tRNAs were separated as described previously then electrotransferred onto GeneScreen Plus (NEN Life Science Products). Following transfer, the RNAs were UV cross-linked to the membrane using the automatic cross-link mode of a Stratagene Stratalinker. Prehybridization was in 1 M NaCl, 10% dextran sulfate, 1% SDS at 42°C. The blot was hybridized at 42°C. Oligonucleotide probes used in the hybridizations were 5Ј end-labeled with [␥-32 P]ATP (NEN Life Science Products) using T4 polynucleotide kinase (Life Technologies, Inc.). A PCR product specific for tRNA Leu was labeled with a Random Primers DNA labeling system (Life Technologies, Inc.). Northerns were washed to low stringency in 1ϫ SSC and 1% SDS at 55°C. High stringency washes were performed in 0.5ϫ SSC and 1% SDS at 80°C (28).
Oligonucleotides 7, 20, and 002 are deoxyoligonucleotide primers used in the 5Ј RACE experiments described in Fig. 5. Oligonucleotides 20 (5Ј-TAGCTGGCGGTGTTGTC-3Ј) and 7 (5Ј-GGTGTTGTCTAAACA-AAATCTGTG-3Ј) were used, respectively, to prime either the RT or the amplification steps of the 5Ј RACE reactions. These oligonucleotides are complementary to sequences within the intergenic region of the tRNA 1 Ser /tRNA Leu dicistronic RNA precursor. Oligonucleotide 002 (5Ј-GCATGGCGCTAATACGACTCACTATAG-3Ј) is complementary to the ribo-oligonucleotide primer ligated onto the 5Ј end of the mitochondrial RNAs in the 5Ј RACE analysis. Synthetic ribo-oligonucleotide (5Ј-CGUACCGCGATTATGCUGAGUGATAUC-3Ј) was ligated onto the 5Ј ends of mitochondrial RNAs for the 5Ј RACE analysis.
RT-PCR Analysis-Primer extension analysis of mitochondrial RNA (40 g) and cytosolic RNA (40 g) was performed essentially as described previously (25). Briefly, 100 pM of D1 oligonucleotide and 100 pM of GS-2 oligonucleotide were hybridized with each RNA. A control reaction without RNA was also included. The RNAs and oligonucleotides were hybridized in 40 mM Pipes, pH 6.4, 0.4 M NaCl, and 1 mM EDTA at 92°C for 5 min and then allowed to slow cool overnight to 42°C. The extension reactions were performed at 40°C for 1 h and then ramped to 50°C over 1 h in reverse transcriptase buffer using 10 units of AMV reverse transcriptase (Promega). Mock reactions were performed in the absence of reverse transcriptase. After primer extension, the reactions were treated with 2 l of 10 mg/ml RNase A for 10 min at 37°C followed by incubation with 5 l of 10 mg/ml proteinase K for 10 min at 37°C. The reactions were phenol/chloroform extracted (1:1), ethanol precipitated, and resuspended in 20 l of distilled H 2 0. PCR reactions were performed with the tRNA Leu or cytochrome c-specific oligonucleotides. 2 l of the primer extension reactions or 2 l of a 1:200 dilution of the primer extension reactions were amplified using Taq Polymerase (Perkin-Elmer) for 30 cycles at an annealing temperature of 53°C, an extension temperature of 72°C, and a denaturing temperature of 92°C. 20 l of the 100-l PCR reaction was electrophoresed in a 2.5% Nusieve agarose (FMC) gel. PCR bands were visualized by ethidium staining. Quantification of the PCR products was performed by the addition of 1.0 Ci of [␣-32 P]dCTP (NEN Life Science Products) to the PCR reactions. The Nusieve gel was dried down under vacuum in the absence of heat, and the appropriate bands were quantitated using a Molecular Dynamics model 400E PhosphorImager.
5Ј RACE of Mitochondrial RNA-A 30-nucleotide synthesized RNA was ligated to mitochondrial RNA by combining 80 pmol of the synthetic RNA and 10 g of mitochondrial RNA. The mixture was heated to 95°C for 2 min, and the volume was then brought to 100 l in a buffer containing a final concentration of 50 mM Hepes, pH 7.9, 20 mM MgCl 2 , 5 mM dithiothreitol, 10% Me 2 SO, 10 g/ml bovine serum albumin, 1 unit/ml RNasin, and 240 pmol ATP. T4 RNA ligase (100 units) was added, and the mixture was incubated at 16°C for 2 h. The RNAs were extracted with phenol and phenol/chloroform (1:1) and precipitated with sodium acetate and ethanol. For reverse transcription, 2 g of the ligation reaction was added to oligonucleotide 20, incubated at 85°C for 5 min, and placed on ice. Then a mixture containing 500 M of each deoxynucleotide, 1ϫ Enhanced AMV buffer (Sigma), 1 unit/l Enhanced AMV-RT (Sigma), and 1 unit/l RNasin (Sigma) was added. The reactions were incubated at 55°C for 1 h and then 95°C for 5 min. A PCR reaction containing 5 l of the RT reaction, 200 M deoxynucleotides, 1ϫ PCR buffer (Perkin-Elmer), 1 pmol 5Ј oligonucleotide 002, and 1 pmol 3Ј oligonucleotide 7 was amplified using Taq Polymerase (Perkin-Elmer) for 35 cycles at a denaturing temperature of 94°C, an annealing temperature of 55°C, and an extension temperature of 72°C. The PCR amplified cDNAs were collected by phenol/chloroform (1:1) extraction and ethanol precipitated with sodium acetate. The cDNAs were then ligated into the TA vector (Invitrogen) using the 5-min ligation kit (Roche Molecular Biochemicals). Briefly, 1:100 of the PCR reaction was added to 50 ng of TA vector, 1ϫ DNA dilution buffer, 1ϫ T4 ligation buffer, T4 DNA ligase and incubated at room temperature for 5 min. One-tenth of the ligation was then transformed into MAX efficiency DH5␣ competent cells (Life Technologies, Inc.), and the 5Ј ends of the resulting clones were determined by dideoxynucleotide sequencing using the 3Ј oligonucleotide 7.
Nucleotide Sequence Accession Number-The sequence identified in this paper has been assigned GenBank TM accession number U63718.

Identification of Precursor tRNAs within the Mitochondria of T. brucei-In vitro
labeling of a population of potential precursor tRNAs was accomplished as in previous studies (24). Isolated mitochondria were incubated in the presence of [␣-32 P]CTP in the absence of transcription. RNAs were isolated from these organelles and separated on a 7% polyacrylamide/4 M urea gel. The RNAs were visualized either by ethidium staining or by autoradiography (Fig. 1A). The entire lane was excised and separated on a 17.6% polyacrylamide/8 M urea gel and visualized by autoradiography (Fig. 1B). CTP labeling of two groups of RNA species is believed to be due to turnover of the 3Ј terminus CCA by the endogenous nucleotidyl transferase. Run-on mitochondrial transcription was inhibited by depletion of the mitochondrial GTP pool prior to labeling with CTP (24). Two-dimensional electrophoresis shows that both groups of RNAs are retarded in the second dimension, consist-ent with having significant secondary structure (Fig. 1B). The smaller RNAs ranging in size from 65 to 95 nucleotides have previously been identified as mature mitochondrial tRNAs (9). The larger RNAs, which range in size from 150 to 300 nucleotides, are believed to be precursor tRNAs having a mature 3Ј terminus formed by CCA addition. A significant portion of these slowly migrating RNAs are ligated circular tRNAs that are either formed in vivo or accumulated during the in vitro labeling reactions (Ref. 25 and data not shown). Consistant with this interpretation, only a portion of this RNA population, containing circular tRNAs and putative precursor tRNAs, are processed by RNase P from E. coli to mature tRNAs (Ref. 24 and Fig. 2). The RNAs that are substrates for processing by RNase P are likely to be tRNA with long 5Ј extensions. Fig. 2 also shows that the processing of mitochondrial precursor tRNAs is dependent on enzyme. In order to show that the 5Ј extensions of the precursor tRNAs identified are derived from continuous upstream genomic sequence, it is necessary to identify nuclear genes encoding tRNAs targeted to the mitochondrion.
Identification of Nuclear Genes Encoding Mitochondrial tRNAs-A number of nuclear encoded tRNA genes have been identified in T. brucei. Identification of these genes has been based on analysis of cloned genomic fragments (14) or by screening with a heterogeneous population of labeled tRNAs (11)(12)(13)(14)(15). In order to identify genes specifically encoding mitochondrial tRNAs of T. brucei, we have isolated and sequenced After a 60-min incubation at 60°C, the RNAs were isolated and separated on a 10% polyacrylamide/8 M urea gel and visualized by autoradiography. As a control, a precursor tRNA Asp from B. subtilis was digested under the same conditions. individual mitochondrial tRNAs. RNAs were isolated from purified preparations of T. brucei mitochondria (27). RNAs were also prepared from a cytosolic fraction following sedimentation of organelles at 12,000 ϫ g. The cross-contamination of the RNAs from the cytosol and mitochondria was established by hybridization with probes specific for mitochondrial and cytosolic RNAs (see Fig. 6, C and D). In order to isolate individual tRNAs, mitochondrial and cytosolic RNAs were separated on a 7% polyacrylamide/4 M urea gel (Fig. 3A). Gel slices corresponding in size to the mature tRNAs indicated by the bracket in Fig.  3A were excised and separated on a 17.6% polyacrylamide/8 M urea gel. The tRNAs were visualized by ethidium staining (Fig.  3B). Comparison of the pattern of tRNAs from the mitochondria versus the cytoplasm shows several differences. Most of the tRNAs appear to be shared between the two compartments (including RNAs 1, 3 and 4 indicated by small arrows), but several are compartment specific (large arrows).
Fifteen individual tRNAs were excised from the two-dimensional gel and eluted. The tRNAs were then 5Ј or 3Ј end-labeled and subjected to enzymatic sequencing (data not shown). Clear sequence was obtained from all of these RNA indicating the purity of the products from the two-dimensional gel purification. The sequence obtained from one 3Ј end-labeled tRNA (arrow 1 in Fig. 3B) was used to design a complementary oligonucleotide designated D1.
The specificity of oligonucleotide D1 for a single tRNA was confirmed by Northern blot hybridization of two-dimensional gels of mitochondrial and cytosolic tRNAs (Fig. 4). Oligonucleotide D1 hybridizes to the single tRNA, from which its sequence was derived, in both the cytoplasm and the mitochondrion. The second tRNA that appears to hybridize in the cytosolic tRNAs is a degradation product that occurred during the two-dimensional separation. Enzymatic sequencing of this tRNA species was identical to tRNA 1 except for the absence of a single nucleotide at the 3Ј terminus (data not shown).
Oligonucleotide D1 was used to screen a genomic library derived from T. brucei. A 1.1-kilobase pair genomic fragment was isolated that contained sequence corresponding to the tRNA hybridizing to oligonucleotide D1. The tRNA was identified based on its anticodon as a tRNA Leu . This genomic fragment also contains two closely related tRNA Ser genes that are shown diagramatically in Fig. 5. The tRNA 1 Ser and tRNA Leu are oriented in the same direction and are separated by 59 nucleotides. The tRNA 2 Ser is in the opposite orientation and is separated from tRNA Leu by 210 nucleotides. The sequence and secondary structure predictions for these tRNAs are shown in Fig. 5.
To confirm that the genomic sequence corresponds to the original enzymatically sequenced tRNA, a Northern blot of a two-dimensional gel of mitochondrial and cytosolic tRNAs was hybridized with a labeled PCR product containing the genomic tRNA Leu sequences (data not shown). High stringency washes identify the single tRNA encoded by the genomic sequence, corresponding to the original tRNA that was enzymatically sequenced. tRNA 1 Ser and tRNA 2 Ser hybridize to tRNAs identified as arrows 3 and 4, respectively in Fig. 3B in both the mitochondrion and cytosol. 2 These results indicate that the 1.1-kilobase pair genomic fragment encodes three tRNAs that are targeted to both the mitochondrion and the cytoplasm.
tRNA Leu Precursor Identification-In order to determine whether a precursor tRNA Leu was present in the mitochondrion and cytoplasm, we developed a sensitive RT-PCR method. The tRNA Leu D1 oligonucleotide complementary to the variable loop and T-stem region of mature tRNA Leu was annealed to mitochondrial or cytosolic RNAs and was extended using reverse transcriptase. The single-stranded DNA products were amplified with the D1 oligonucleotide and each of 4 oligonucleotides complementary to sequences upstream of the mature tRNA Leu genomic sequence. The position of the oligonucleotides used and the expected size products are shown in  chondria and cytosolic reactions as well as the predicted size D1, 1-14 product in the cytosolic reaction were confirmed using nested PCR (data not shown). Additional bands in the cytosolic D1, 1-15, D1, 1-14, and D1, 1-13 reactions have not been identified. The RT-PCR results show that a precursor tRNA is present both in the cytoplasm and the mitochondrion, extending a minimum of 60 nucleotides upstream of the mature 5Ј end of tRNA Leu (Fig. 6B). Furthermore a precursor extending over 50 nucleotides upstream of the mature tRNA 5Ј end can be identified within the cytoplasm as shown by the oligonucleotides D1, 1-15 product found in the cytosolic RNA. This indicates that the cytoplasmic precursor extends through the intergenic region.
The possibility that the precursor tRNA identified within the mitochondria by RT-PCR was due to cytosolic contamination of the mitochondrial RNA preparation was addressed by two ap-FIG. 5. Identification of a genomic fragment containing the mitochondrial tRNA identified by oligonucleotide D1. The genomic fragment isolated by screening a genomic library with oligonucleotide D1 is shown diagramatically. The predicted secondary structures of the tRNA Leu , identified by the D1 oligonucleotide, and two tRNA Ser contained in this genomic fragment are also shown.
FIG. 6. Identification of precursor tRNA Leu in the mitochondrion and cytoplasm. A and B, the D1 oligonucleotide was annealed to mitochondrial (Mito) or cytosolic (Cyto) RNAs and was extended using reverse transcriptase. The single-stranded DNA products were PCR amplified with the D1 oligonucleotide and each of 4 oligonucleotides complementary to sequences upstream of the mature tRNA Leu genomic sequence (shown diagramatically in A and experimentally in B). The bottom portion of the gel in panel B containing the primers and primer dimer products is not shown. C, to determine cytosolic contamination of mitochondrial RNA, mitochondrial and cytosolic RNAs were separated on polyacrylamide gels, Northern blotted, and probed with cytosolic and mitochondrial specific probes. D, in addition, control primer extension reactions were performed using an oligonucleotide complementary to cytosolic cytochrome c mRNA under the same conditions used for precursor tRNA Leu , except that the amount of RT product used in these reactions was reduced 200-fold.
proaches. Mitochondrial and cytosolic RNAs were separated on a polyacrylamide gel, Northern blotted, and probed with cytosolic and mitochondrial-specific probes (Fig. 6C). An oligonucleotide complementary to the 140-nucleotide spliced leader RNA of T. brucei hybridized preferentially to the cytosolic RNAs. An oligonucleotide complementary to the mitochondria enriched tRNA 5, identified by typical migration on two-dimensional gels (Fig. 3B) and analyzed by partial enzymatic sequencing (data not shown), preferentially hybridized to the mitochondrial tRNAs (Fig. 6C). Quantitation of the hybridization to these RNAs suggests that cytosolic contamination of the mitochondrial RNA was approximately 1%. Less than 5% of the cytosolic tRNA pool was a consequence of leakage of mitochondrial RNAs during mitochondrial purification.
In addition, a control primer extension reaction was performed using an oligonucleotide complementary to cytosolic cytochrome c mRNA. PCR reactions were carried out under the same conditions used for the RT-PCR of the precursor tRNAs (Fig. 6D) except that the amount of RT product used in these reactions was reduced 200-fold. Quantitation of the PCR product corresponding to the cytochrome c mRNA shows that the cytosolic contamination of the mitochondrial RNA preparation is approximately 0.4%. This demonstrates that the precursor tRNA Leu is present in the mitochondrion at a 25-60-fold higher amount than can be explained by simple cytosolic contamination of the mitochondrial RNAs.
The RT-PCR products seen in Fig. 6B were difficult to interpret because it was not possible to determine whether the lack of a PCR product indicated the 5Ј end of the precursor or a truncated product resulting from the inability of the reverse transcriptase to extend through the upstream tRNA. In addition, it is possible that multiple 5Ј ends exist within the mitochondrial RNA pool. These possibilities were addressed by 5Ј RACE of total mitochondrial RNA (Fig. 7). Total T. brucei mitochondrial RNA was ligated to a 30-nucleotide synthetic RNA and reverse transcribed with the oligonucleotide 20, which is complementary to the intergenic region immediately upstream of the tRNA Leu . To decrease premature termination because of RNA secondary structure, reactions were done at 55°C using the Enhanced AMV-RT (Sigma). The cDNAs were then amplified with oligonucleotide 002, complementary to the ligated RNA oligonucleotide, and oligonucleotide 7, complementary to the tRNA Ser /tRNA Leu intergenic sequence. PCR products were cloned into the TA vector and sequenced using oligonucleotide 7. Three independent clones containing intergenic sequence were identified. The sequence of each clone showed that the synthetic RNA was ligated to a dicistronic precursor 14 nucleotides upstream of the mature 5Ј end of the tRNA Ser . These results are consistent with the precursor transcript identified in these studies being a dicistronic tRNA Ser / tRNA Leu formed by polymerase III transcription read-through of the upstream tRNA Ser . DISCUSSION We have previously defined a population of mitochondrial RNAs with characteristics of precursor tRNAs (22). A subset of the precursor size molecules can be processed by an E. coli RNase P RNA to mature mitochondrial tRNAs. These findings suggest that import of at least a subset of the tRNAs may require sequences flanking the tRNAs. In this paper we have identified the nuclear genes encoding two tRNAs targeted to the mitochondrion and determined that a dicistronic precursor tRNA exists within the mitochondrion.
We have separated individual mitochondrial and cytosolic tRNAs using two-dimensional gel electrophoresis (Fig. 3B). A complementary oligonucleotide was designed based on the partial enzymatic sequence of one mitochondrial tRNA. This oligonucleotide was used to identify a genomic DNA fragment containing three tRNAs, tRNA 1 Ser , tRNA Leu , and tRNA 2 Ser (Fig. 5).
The genomic organization of the tRNA genes of T. brucei is unusual (11)(12)(13)(14)(15). tRNA 1 Ser and tRNA Leu are closely linked, separated by only 59 nucleotides. This genomic arrangement may play a role in distinguishing between tRNAs targeted to the mitochondrion or targeted to the cytoplasm. We have shown that the tRNA 1 Ser and tRNA Leu genes are transcribed to give a dicistronic precursor transcript. Normally RNA polymerase III initiates tRNA transcription 10 -20 nucleotides upstream of the mature 5Ј tRNA sequence (reviewed in (29)). The 5Ј terminus of the dicistronic precursor tRNA was determined to be 14 nucleotides upstream of the tRNA 1 Ser . This suggests that the precursor tRNA 1 Ser /tRNA Leu is formed by the inability of polymerase III to efficiently terminate the transcription of the upstream tRNA 1 Ser . The presence of the tRNA 1 Ser /tRNA Leu dicistronic molecule within the mitochondrion implies that these precursor tRNAs must somehow escape the tRNA processing events that normally occur within the nucleus. A tRNA precursor consisting of two linked tRNAs may fold into an unusual structure (30), which could allow the precursor to escape nuclear processing. Alternatively, the precursor molecule may be recognized by specific RNA binding proteins that participate in the export of the unprocessed RNA from the nucleus and its subsequent import into the mitochondrion. In mammalian cells it has recently been demonstrated that processing of precursor tRNAs by RNase P is modulated by the La antigen phosphoprotein (31). A similar pathway for protein-mediated protection FIG. 7. Identification of the 5 end of the precursor tRNA Leu transcript in mitochondria. A 30-nucleotide synthesized RNA (hatched box) was ligated to mitochondrial RNA. The ligated RNA was then reverse transcribed with oligonucleotide 20. The resulting cDNAs were then PCR amplified using both the 5Ј oligonucleotide 002, which is complimentary to the 30-nucleotide ligated RNA, and the nested 3Ј oligonucleotide 7. The products from the PCR reaction were ligated into the TA vector and sequenced using oligonucleotide 7. The 5Ј end of the precursor tRNA Leu transcript was mapped to be 14 nucleotides upstream (closed triangle) of the predicted mature 5Ј end of the tRNA Ser (open triangle).
of precursor tRNA Leu may allow it to be exported from the nucleus in an unprocessed form.
Some processing events may occur prior to, during, or shortly after mitochondrial import. Processing or modifications of the precursor tRNA Ser /tRNA Leu once inside the mitochondria may allow these transcripts to be recognized and processed into mature functional tRNAs by the mitochondrial RNase P and nucleotidyl transferase (24). In support of this, a mitochondrial specific modification at position 32 of tRNA Leu was identified as 2Ј-O-methylcytidine and occurs following mitochondrial tRNA import (20). 2 Import of tRNAs does not appear to be dependent on this modification, and the importance of this modification in forming functional mitochondrial tRNAs is not known (20). We are currently investigating modifications of imported tRNAs and the roles these modifications may play in correctly processing precursor tRNAs and forming functional mature tRNAs.
In previous studies (22), oligonucleotides complementary to conserved sequences in tRNAs (D-loop and stem) were used in both Northern hybridization and primer extension reactions to identify putative precursor tRNAs. When similar experiments were performed using an oligonucleotide complementary to specific sequences immediately upstream of the mature tRNA Leu 5Ј terminus, no products were observed in the mitochondrial or the cytosolic fractions. 2 This is consistent with a recent study indicating that precursor molecules with 5Ј extended sequences were not detected either by primer extension analysis or Northern blot hybridization (25). In this report we have used a highly sensitive RT-PCR assay to identify precursor tRNA Leu molecules within the mitochondrion and cytosol, suggesting that precursor tRNA Ser /tRNA Leu is present at very low abundance in T. brucei. We now believe that the majority of higher molecular weight RNAs observed to incorporate radiolabeled CTP in a metabolic labeling experiment (Fig. 1A) are actually artificial ligation products that are formed during our mitochondrial isolation procedure. We have observed during metabolic labeling experiments with isolated mitochondria that over time there is an increase in both CTP incorporation and intensity of ethidium staining in precursor size RNA molecules with a corresponding decrease in mature tRNA molecules. 2 This would explain why only a small percentage of the metabolically labeled precursor tRNAs are cleaved in our RNase P assay (Ref. 24 and Fig. 2).
Transfection studies have been used to investigate the determinants of tRNA import into trypanosome mitochondria (18,19,21). The results from these studies suggest that the mature tRNA structure alone can direct import of several tRNAs into the mitochondria, irrespective of their genomic context or genetic origin. These findings are an apparent contradiction to our findings. Although we do not have an explanation for these differences, it is possible that the high levels of tRNAs expressed from the episomal vectors used in the transfection studies may have influenced the distribution of tRNAs. The studies reported here do not directly address whether the dicistronic precursor or mature tRNAs are preferentially imported into trypanosome mitochondria. However, the data demonstrate the presence of a nuclear encoded, dicistronic, tRNA 1 Ser /tRNA Leu precursor within the mitochondria. Recently, we have investigated the import of RNAs into the mitochondria of T. brucei. The dicistronic tRNA 1 Ser /tRNA Leu molecules are efficiently imported in this in vitro system, whereas mature tRNA 1 Ser and tRNA Leu are poor import substrates. 3