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Originally published In Press as doi:10.1074/jbc.M007838200 on September 18, 2000

J. Biol. Chem., Vol. 275, Issue 48, 37907-37914, December 1, 2000
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End Processing Precedes Mitochondrial Importation and Editing of tRNAs in Leishmania tarentolae*

Stephen T. KapushocDagger §, Juan D. Alfonzo, Mary Anne T. Rubio||, and Larry Simpson**DaggerDagger

From the Departments of Dagger  Molecular, Cell, and Developmental Biology and  Microbiology, Immunology, and Molecular Genetics and the ** Howard Hughes Medical Institute, University of California, Los Angeles, California 90095

Received for publication, August 28, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

All mitochondrial tRNAs in Leishmania tarentolae are encoded in the nuclear genome and imported into the mitochondrion from the cytosol. One imported tRNA (tRNATrp) is edited by a C to U modification at the first position of the anticodon. To determine the in vivo substrates for mitochondrial tRNA importation as well as tRNA editing, we examined the subcellular localization and extent of 5'- and 3'-end maturation of tRNATrp(CCA), tRNAIle(UAU), tRNAGln(CUG), tRNALys(UUU), and tRNAVal(CAC). Nuclear, cytosolic, and mitochondrial fractions were obtained with little cross-contamination, as determined by Northern analysis of specific marker RNAs. tRNAGln was mainly cytosolic in localization; tRNAIle and tRNALys were mainly mitochondrial; and tRNATrp and tRNAVal were shared between the two compartments. 5'- and 3'-extended precursors of all five tRNAs were present only in the nuclear fraction, suggesting that the mature tRNAs represent the in vivo substrates for importation into the mitochondrion. Consistent with this model, T7-transcribed mature tRNAIle underwent importation in vitro into isolated mitochondria more efficiently than 5'-extended precursor tRNAIle. 5'-Extended precursor tRNATrp was found to be unedited, which is consistent with a mitochondrial localization of this editing reaction. T7-transcribed unedited tRNATrp was imported in vitro more efficiently than edited tRNATrp, suggesting the presence of importation determinants in the anticodon.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Targeting of one or more nucleus-encoded tRNAs to the mitochondrion occurs in a variety of organisms and allows mitochondrial protein synthesis to proceed in cells in which these tRNAs are not encoded in the mitochondrial genome. Importation of tRNAs into mitochondria has been studied in yeast (1), ciliates (2, 3), plants (4), and trypanosomatids (5-9) using both in vivo and in vitro techniques. In no case is the mechanism of importation of RNA into mitochondria fully understood, and there appear to be differences in the importation process in different organisms. The trypanosomatids Leishmania tarentolae and Trypanosoma brucei represent an extreme situation in which no tRNAs are encoded in the mitochondrial genome; and therefore, all tRNAs for mitochondrial translation must be encoded in the nucleus and imported from the cytosol (10, 11).

Targeting of specific tRNAs to the mitochondrion in trypanosomatids has been studied in vivo by transfection techniques (5) and in vitro by importation into isolated mitochondria (12-16). The specificity of targeting as well as the mechanism of transport are still obscure, but the specificity appears to involve, at least in part, the tertiary structure of the RNA molecule (16).

It was previously proposed, on the basis of the detection of precursor tRNAs in T. brucei mitochondria, that the substrate for importation is a 5'-precursor species that is processed by an RNase P-like activity within the organelle (17). However, Aphasizhev et al. (18) could not obtain any evidence for the existence of such mitochondrion-localized precursors in L. tarentolae or T. brucei. In addition, the presence of a genomic 5'-flanking sequence was not required for mitochondrial targeting of tRNAIle in L. tarentolae (5) or for mitochondrial targeting of tRNATyr in T. brucei (8). However, evidence was recently presented that a dicistronic tRNA transcript in T. brucei is the preferred substrate for importation into isolated mitochondria, but no evidence was presented for intramitochondrial processing of this transcript (19, 20).

To investigate the question of the nature of the in vivo substrate for tRNA importation into the mitochondrion, we have used RT-PCR1 to analyze the intracellular localization of 5'- and 3'-end processing and an in vitro assay to analyze the importation properties of several specific tRNAs in L. tarentolae. We have also investigated the subcellular localization of the C to U editing of the anticodon of the tRNATrp.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture, Cell Fractionation, and RNA Isolation-- L. tarentolae cells were grown at 27 °C in brain/heart infusion medium (Difco) supplemented with 10 µg/ml hemin (Calbiochem). Mitochondria were prepared from cultures at ~1 × 108 cells/ml by the hypotonic breakage method as described previously (21). The mitochondria were isolated in a 20-35% Renografin (Bracco) gradient. Isolated mitochondria were treated with micrococcal nuclease to remove any RNA bound to the outside of the vesicles.

Nuclei were prepared from cultures at ~5 × 107 cells/ml. Cells were washed in 0.15 M NaCl and 0.02 M NaPO4 (pH 7.4); resuspended in 0.5 M hexylene glycol (Sigma), 1 mM PIPES (pH 7.4), and 1 mM CaCl2 (22); and broken using a Stansted Fluid Power apparatus at 60 p.s.i. A portion of this cell lysate was cleared by two successive centrifugations at 15,000 × g for 25 min each to obtain the cytosolic cell fraction. Nuclei were isolated from the cell lysate in a density gradient created by centrifugation of 35% Percoll (Amersham Pharmacia Biotech) at 60,000 × g for 35 min. Each step of the cell fractionation was monitored by phase and fluorescence microscopy. RNA was isolated from whole cells and from each cell fraction using the guanidinium thiocyanate/phenol/chloroform extraction method (23).

Northern Analysis-- RNA (1.5 µg) from each cell fraction was separated on an 8 M urea and 6% acrylamide gel. The gel was stained with ethidium bromide for RNA visualization and photography, and the RNA was transferred to Zeta-Probe membranes (Bio-Rad) according to the manufacturer's directions. The membranes were hybridized with the appropriate oligonucleotides, which were 5'-end-labeled with [gamma -32P]ATP (PerkinElmer Life Sciences) using T4 polynucleotide kinase (Life Technologies, Inc.).

Two-dimensional Gel Electrophoresis-- 7 µg of mitochondrial and cytosolic tRNAs were resuspended in 20 µl of 4 M urea and electrophoresed in the first dimension on a 4 M urea and 15% polyacrylamide gel (200 × 350 × 0.75 mm) at 750 V for 24 h at room temperature. The gel was stained with ethidium bromide, and the tRNA portion of the gel was excised and layered on top of a second dimension 8 M urea and 20% polyacrylamide gel (180 × 250 × 0.75 mm). The second dimension was electrophoresed at 400 V for 48 h at room temperature. The separated tRNAs were visualized by ethidium bromide staining for photography and transferred to a Zeta-Probe membrane for Northern analysis as described above.

Clamped Homogeneous Electric Field Electrophoresis-- L. tarentolae genomic DNA blocks were prepared as describe previously (24, 25) and separated on 0.5× Tris borate/EDTA-containing 1.5% agarose using the CHEF-DR II apparatus (Bio-Rad). The chromosome bands were visualized by ethidium bromide staining, and the gel was photographed using a C-80 UV Darkroom system (Ultra-Violet Products). The DNA was transferred to Nytran Plus membranes (Schleicher & Schüll), which were hybridized with the different tRNA PCR products labeled with [alpha -32P]dATP using the Prime-It II random primer labeling kit (Stratagene).

Oligonucleotides-- The following oligonucleotides were used for Northern analysis of RNA. S-3434 (5'-TTATCGGATTCAGAGTCCGAGGTG-3') was used to detect tRNAGln(CUG); S-3442 (5'-CCTGGATTTGGAATCCAATGCT-3') was used to detect tRNATrp(CCA); S-3444 (5'-TCCGGTTCATAAGACCAGCGTC-3') was used to detect tRNAIle(UAU); S-3443 (5'-ACGAGGTTAAAAGCCACGCGCT-3') was used to detect tRNALys(UUU); S-3437 (5'-ATCTCTCGCGTGTGAGGCGAATGTC-3') was used to detect tRNAVal(CAC); S-3313 (5'-CAACGTCCATCTGCGACGGCTTTA-3') was used to detect the 92-nucleotide small nucleolar RNA; U6-1 (5'-TCTTCACTGTTGAATTTCC-3') was used to detect the U6 snRNA; S-3315 (5'-GTTCCGGAAGTTTCGCATAC-3') was used to detect the spliced leader RNA; and S-3316 (5'-GTCTTCCTCTGAATGCGTAAGCG-3') was used to detect the RPS12-I guide RNA.

The following oligonucleotides (based on GenBankTM/EBI accession number L20948 (33)) were used for RT-PCR of mature and precursor tRNATrp. S-3213 (5'-ATCGAAAATGCGCTGTGACTTGTGG-3') was used for amplification of the tRNATrp 5'-leader; S-2820 (5'-AGCTCAGTGGTAGAGCATTGG-3') was used for amplification of the mature tRNATrp 5'-end; S-3215 (5'-CCAAAAAGGGCCCCCAGCGCGAAAACC-3') was used for amplification of the tRNATrp 3'-trailer; and S-2819 (5'-TGAGAGCTGCAGGGATTGAAC-3') was used for amplification of the mature tRNATrp 3'-end.

The following oligonucleotides (based on GenBankTM/EBI accession number L20948 (33)) were used for RT-PCR of mature and precursor tRNAIle: S-3478 (5'-CCTACATCCATATTCGCAGTATGT-3') was used for amplification of the tRNAIle 5'-leader; S-3479 (5'-GCTCCCGTGGTCTAGTTGGTTAGG-3') was used for amplification of the mature tRNAIle 5'-end; S-3480 (5'-TTGGGGATTTTGGGGGCGGAAAAG-3') was used for amplification of the tRNAIle 3'-trailer; and S-3385 (5'-GGCTCGAACCCGCGACATCCGGTTC-3') was used for amplification of the mature tRNAIle 3'-end.

The following oligonucleotides (based on GenBankTM/EBI accession number X68207 (34)) were used for RT-PCR of mature and precursor tRNAGln. S-3475 (5'-TCACCTTCATCATCCTTGTATGAT-3') was used for amplification of the tRNAGln 5'-leader; S-3476 (5'-GCTCCTATA-GTGTAGCGGTTATCA-3') was used for amplification of the mature tRNAGln 5'-end; S-3477 (5'-GCCGCTTTTTGGTGGGGGGGTAAA-3') was used for amplification of the tRNAGln 3'-trailer; and S-3387 (5'-GGACTCGAACCAGGGTTATCGGATT-3') was used for amplification of the mature tRNAGln 3'-end.

The following oligonucleotides (based on based on GenBankTM/EBI accession number L20948 (33)) were used for RT-PCR of mature and precursor tRNALys. S-3864 (5'-TGGAGTAGCAATACGAATTCC-3') was used for amplification of the tRNALys 5'-leader; S-3865 (5'-GCACTTCTAGCTCAGTTGGTAG-3') was used for amplification of the mature tRNALys 5'-end; and S-3866 (5'-CGCACTCCGTGGGGCTCGAAC-3') was used for amplification of the mature tRNALys 3'-end.

The following oligonucleotides (based on GenBankTM/EBI accession number AF016249 (37)) were used for RT-PCR of mature and precursor tRNAVal. S-3861 (5'-TTGTAGCTCACATCCAGTAGC-3') was used for amplification of the tRNAVal 5'-leader; S-3862 (5'-GCGATGGTCGTCTAGTGGTTAG-3') was used for amplification of the mature tRNAVal 5'-end; and S-3863 (5'-TACGACGGGCGGGGATTGAAC-3') was used for amplification of the mature tRNAVal 3'-end.

The following oligonucleotides were used for 3'-RACE of tRNATrp. S-3122 (5'-TTGAATTCGCATTGAGCACCTGCTTTTTTTTTTTTTTTTTT-3') was used for reverse transcription of polyadenylated RNA, and S-3123 (5'-TTGAATTCGCATTGAGCACCTGC-3') was used for PCR amplification of the resulting cDNA.

RT-PCR, Cloning, and Sequencing-- For reverse transcription reactions, 40 pmol of the appropriate oligonucleotide primer were combined with 2 µg of each RNA in a 20-µl reaction using 200 units of Superscript II reverse transcriptase (Life Technologies, Inc.) following the directions of the manufacturer. Identical reactions were performed without the addition of Superscript II for the no-reverse transcriptase control. 1 µl of each reverse transcription reaction was used as a template for PCR amplification. Each 50 µl of PCR contained 40 pmol of each of the appropriate oligonucleotide primers and 1× PCR buffer (Promega) with 2 mM MgCl2, 500 µM each dNTP, and 5 units of Taq DNA polymerase. Thermal cycling was performed with an initial denaturation at 95 °C for 4 min, followed by 30 cycles of PCR (95 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s) and a final extension at 72 °C for 10 min. The PCR products were analyzed on a 2.5% Metaphor-agarose gel (FMC Corp. BioProducts). PCR products were cloned using the pCR-2.1-TOPO cloning kit (Invitrogen) and sequenced using Sequenase Version 2.0 (U. S. Biochemical Corp.).

HinfI Digestion of RT-PCR Products-- Precursor tRNATrp RT-PCR products were purified from a 2.5% Metaphor-agarose gel to separate the PCR product from unincorporated primers and nucleotides. The PCR products were digested with HinfI. After digestion, the reactions were extracted with phenol/chloroform, ethanol-precipitated, and analyzed on a 2.5% Metaphor-agarose gel.

Assay of in Vitro Importation of RNA into Isolated Mitochondria-- The in vitro RNA importation assay (16) was performed in a 20-µl reaction volume containing 50,000 cpm radioactively labeled RNA, 1 mg of mitochondria (~40 µg of protein), 1 mM ATP, 2 mM dithiothreitol, 10 mM MgCl2, 0.63 mM creatine phosphate, and 22.5 mg/ml creatine phosphokinase (5, 12, 14, 15). After incubation at 27 °C for 5-30 min, 100 units of micrococcal nuclease (Roche Molecular Biochemicals) and 5 mM CaCl2 were added to digest the RNAs that were not imported into the mitochondria. Micrococcal nuclease was then inhibited by the addition of 10 mM EGTA (pH 8). To isolate imported RNAs, the mitochondria were pelleted and suspended in 150 µl of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 0.1% SDS and extracted with phenol; and the RNA was precipitated with ethanol. The radioactively labeled RNAs were separated by electrophoresis on a 7 M urea and 8% acrylamide gel. After electrophoresis, the nuclease-protected radioactively labeled RNAs were visualized and quantitated using a Storm PhosphorImager and ImageQuant software (Molecular Dynamics, Inc.).

3'-RACE Analysis of tRNATrp-- To map the 3'-end of tRNATrp by RT-PCR, 15 µg of RNA from each cell fraction were polyadenylated using a combination of yeast poly(A) polymerase (Amersham Pharmacia Biotech) and Escherichia coli poly(A) polymerase (Life Technologies, Inc.) according to previously established protocols (26). The resulting polyadenylated RNA was used as a template for cDNA synthesis in a 20-µl reaction using 200 units of Superscript II and primer S-3122. A 5-µl portion of the cDNA reaction was used as a template in PCR using primers S-2820 and S-3123.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Subcellular Fractionation of L. tarentolae-- Several methods exist for the isolation of kinetoplast-mitochondrial fractions from L. tarentolae cells with <5% contamination with cytosolic rRNA (5). Cells can be ruptured in hypotonic (21) or isotonic (27) media, and mitochondria can be isolated by isopycnic sedimentation in Renografin (21) or Percoll (28) gradients. The hypotonic rupture/Renografin method yields mitochondrial fractions of the highest purity and yield (5).

These methods cannot, however, be used to obtain nuclear fractions due to the presence of chelating agents, which cause the nuclei to rupture during isolation. We have modified the method of Shapiro and Doxsey (22), which was developed for T. brucei, by disrupting cells in the Stansted Fluid Power cell disruption apparatus in the presence of 0.5 M hexylene glycol and CaCl2 and banding nuclei in a Percoll gradient. As shown in the micrograph in Fig. 1A, this method produces a high yield of morphologically intact nuclei with essentially no intact cells or contaminating debris visible at the level of light microscopy. Furthermore, since the kinetoplast-mitochondrion remains intact and associated with the flagellum and cell ghost after cell rupture, clarification of the lysate yields a cytosolic cell fraction with little apparent mitochondrial or nuclear contamination.


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  		Fig. 1.   Separation of L. tarentolae into nuclear, cytosolic, and mitochondrial fractions. A, microphotography of isolated nuclei. The purified nuclei were photographed at 1000× under both phase-contrast and 4,6-diamidino-2-phenylindole (DAPI)-stained (with ultraviolet light) conditions (as indicated). B, Northern analysis of RNA isolated from different cell fractions. 1.5 µg of RNA isolated from whole cells (total), the cytosolic fraction (cyto), purified nuclei (nuc), or purified mitochondria (mito) were separated on an 8 M urea and 6% acrylamide gel. The ethidium bromide-stained gel is shown with the results of Northern hybridizations using oligonucleotide probes specific for several marker RNAs (as indicated). U6 snRNA, U6 small nuclear RNA; SL RNA, spliced leader RNA; gRNA RPS12-I, RPS12 block I guide RNA.

To ascertain the purity of the subcellular fractions, equal amounts of RNA isolated from each cell fraction were analyzed by gel electrophoresis as shown in Fig. 1B. The cytosolic and nuclear fractions show, in addition to tRNA, the predominant five small rRNAs characteristic of trypanosomatids (29), and the mitochondrial fraction shows only the 9 S and 12 S mitochondrial rRNAs (30) and tRNA. In addition, Northern analysis was performed using hybridization probes for marker RNAs specific for the nucleolus (92-nucleotide small nucleolar RNA) (31), the nucleus (U6 snRNA), and the mitochondrion (RPS12-I guide RNA) (32). The probe for the 92-nucleotide small nucleolar RNA showed specific hybridization to the nuclear fraction. The probe for the U6 snRNA showed a large enrichment in the nuclear fraction with some hybridization to the cytosolic fraction; it is unclear whether the latter represents leakage from the nucleus during cell fractionation or the presence of transient U6 snRNA traversing the cytosol during normal RNA maturation and small nucleolar ribonucleoprotein assembly. The guide RNA probe showed strong hybridization to the mitochondrial fraction and weak hybridization to the cytosolic and nuclear fractions. The probe for the SL RNA showed hybridization to both the nuclear and cytosolic fractions, but not to the mitochondrial fraction. The fact that the small nucleolar RNA, U6 RNA, and SL RNA probes showed no hybridization to the mitochondrial fraction indicates a high level of purity of this cell fraction, which was confirmed by the guide RNA hybridization results. Quantitation of these blots indicated that the mitochondrial fraction had undetectable levels of nuclear or cytosolic contamination, the cytosolic fraction had 0.3% nuclear contamination and <0.1% mitochondrial contamination, and the nuclear fraction had <0.1% mitochondrial contamination.

tRNATrp(CCA), tRNAIle(AUA), tRNAGln(CUG), tRNALys(UUU), and tRNAVal(CAC) Are Representative of Three Different Subcellular Distribution Patterns-- The intracellular distributions of specific tRNAs were examined using the above cell fractionation procedures. Equal amounts of tRNA from the mitochondrial and cytosolic cell fractions were separated by two-dimensional gel electrophoresis (Fig. 2A). Comparison of the patterns of the ethidium bromide-stained gels indicated that ~24 tRNAs are shared between the cytosolic and mitochondrial fractions, 20 are mainly cytosolic, and 9 are mainly mitochondrial. However, it should be noted that compartment-specific nucleotide modifications may alter the mobility of a given tRNA and may lead to the erroneous assignments of import phenotypes on ethidium bromide-stained gels. Therefore, proper assessment of import phenotype must include Northern analysis.


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  		Fig. 2.   Subcellular distribution, separation, and genomic localization of tRNATrp, tRNAIle, tRNAGln, tRNALys, and tRNAVal. A, separation of mitochondrial and cytosolic tRNAs by two-dimensional electrophoresis. ~7 µg of tRNA from either the cytosolic or mitochondrial fraction were separated by two-dimensional gel electrophoresis. The ethidium bromide-stained gels are shown (the first dimensions are shown on top) with a schematic diagram indicating the distribution of the individual tRNAs. The results of Northern hybridizations using oligonucleotides specific for each of the five tRNAs (as indicated) to specific tRNA spots are also shown. B, Southern blot of a clamped homogeneous electric field gel electrophoresis of L. tarentolae chromosomal DNA. S. cerevisiae (S.c.) chromosomes (with sizes indicated in kilobase pairs (kbp) and separated L. tarentolae (L.t.) chromosomes are shown. The results of Southern hybridizations with the different tRNA gene probes (Trp, Ile, Gln, Lys, and Val) are shown.

To establish the intracellular distribution of the five specific tRNAs, gels were blotted and hybridized with labeled oligonucleotide probes. The tRNAGln probe detected a tRNA spot that was more intense in the cytosolic RNA fraction. The tRNATrp and tRNAVal probes detected single spots of approximately equal intensity in both the cytosolic and mitochondrial RNA fractions. The tRNAIle probe detected a single spot of low relative intensity in the mitochondrial RNA fraction, and the tRNALys probe detected a single spot of high relative intensity in the mitochondrial RNA fraction. From these results, we operationally define the distribution of tRNATrp(CCA) and tRNAVal(CAC) as a "shared" pattern, the distribution of tRNAGln(CUG) as a "mainly cytosolic" pattern, and the distribution of tRNAIle(UAU) and tRNALys(UUU) as a "mainly mitochondrial" pattern. These are relative operational designations and do not include corrections for the relative amounts of tRNA in the cytosol and mitochondrion on a per cell basis.

Localization of tRNA Genes-- The genes for the tRNAs examined in this study were analyzed by clamped homogeneous electric field gel electrophoresis and Southern blotting (Fig. 2B). The tRNAIle and tRNATrp genes localized to the same chromosome of ~700 kb. This is consistent with the observations that these two tRNA genes are contained in the same genomic tRNA cluster (33) and are single-copy.2 The tRNALys probe also showed hybridization to the same chromosome of ~700 kb, which is consistent with the fact that this tRNA is in the same gene cluster as tRNAIle and tRNATrp. In addition, the tRNALys probe also showed hybridization to a chromosome of ~680 kb, which may indicate another copy of the same tRNA gene or hybridization of the probe to a different tRNA gene of similar sequence. The probe for tRNAGln hybridized to three chromosomes of ~1500, 850, and 650 kb. The hybridization pattern of this probe is consistent with the previous observation that this gene is multicopy (34). The tRNAVal probe hybridized to a single chromosome band of ~560 kb.

tRNA Precursors Localize to the Nucleus-- To address the question of whether 5'-precursors are in vivo substrates for import into the mitochondrion (17), RT-PCR of the mitochondrial and shared tRNAs (tRNATrp, tRNAIle, tRNALys, and tRNAVal) was performed with RNAs isolated from the nuclear, cytosolic, and mitochondrial cell fractions using primers specific for the flanking genomic sequences (Fig. 3, A-E). For comparison, cytosolic tRNAGln was also assayed. Control amplifications using internal primers yielded products corresponding to the mature tRNA sequences in each case. We found that 5'-precursors of each tRNA could be amplified only from the nuclear fraction, a result consistent with 5'-end processing occurring prior to export from the nucleus, as is the case in other eukaryotic cells (35). Three tRNAs (tRNATrp, tRNAIle, tRNAGln) were assayed in addition by RT-PCR for the subcellular localization of 3'-precursors, and these also localized solely to the nuclear fraction. These data suggest that both 5'- and 3'-end processing of the mitochondrial tRNAs occurs prior to export from the nucleus.


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  		Fig. 3.   RT-PCR detection of pre-tRNAs. Shown are the results from RT-PCR analysis of tRNATrp (A), tRNAGln (B), tRNAIle (C), tRNALys (D), and tRNAVal (E) from total, cytosolic (cyto), mitochondrial (mito), and nuclear (nuc) RNAs (as indicated). Each panel contains a schematic (upper left panel) showing the placement of the primers used for RT-PCR. In each gel, the marker lane (M) is a 10-base pair ladder (Life Technologies, Inc.). The most intense band is 100 base pairs. pos and neg are positive and negative PCR controls, respectively. The oligonucleotides used for each PCR are indicated below each gel. A control RT-PCR of the mature tRNA sequence is shown (upper right panel), in which reactions were performed with (+) and without (-) reverse transcriptase. The bottom portion of each gel shows the RT-PCR of pre-tRNAs using RNA from each cell fraction (only reactions with reverse transcriptase are shown).

3'-CCA Addition Precedes Nuclear Export-- To analyze the subcellular localization of 3'-CCA addition to a tRNA targeted to the mitochondrion, 3'-RACE of tRNATrp was performed using RNAs isolated from the nuclear, cytosolic, and mitochondrial cell fractions. Of the clones obtained from the nuclear RNA fraction, 58 had mature 3'-CCA ends, and two had unprocessed 3'-ends (Fig. 4B). One of the latter sequences terminated at an oligo(T) stretch just downstream of the 3'-end of the mature tRNA (Fig. 4A, Nuc-1), and the other terminated at another oligo(T) stretch 14 nucleotides farther downstream (Nuc-2). The positions of these unprocessed 3'-ends are indicated in Fig. 5 (black-diamond ). This lack of precise transcription termination may provide an explanation for the observed PCR amplification of 3'-extended precursor molecules in the tRNATrp RT-PCR experiment (Fig. 3A). Similar analysis of RNAs isolated from the cytosolic and mitochondrial fractions revealed that tRNAs in these fractions contained mature 3'-CCA ends (Fig. 4). This evidence suggests that 3'-CCA addition occurs in the nucleus, even in the case of tRNAs that eventually localize in the mitochondrion.


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  		Fig. 4.   3'-RACE analysis of tRNATrp shows two unprocessed 3'-ends in the nuclear fraction. A, sequences of representative clones from the 3'-RACE of tRNATrp from different cell fractions. Sequencing lanes are labeled (A, C, G, T). The following are indicated on each sequencing ladder: the sequence of the 5'-PCR primer (S-2820); the sequence of the 3'-PCR primer (S-3123); the nucleotide at position 34 (arrows); and the location of the 3'-end of the RNA sequence, either unprocessed (unprocessed 3'-end) or processed (CCA). The sequences shown are as follows: the clones of unprocessed tRNATrp from the nuclear fraction (Nuc-1 and Nuc-2), unedited tRNATrp from the cytosolic fraction (Cyto), and edited tRNATrp from the mitochondrial fraction (Mito). B, summarization of the data from the 3'-RACE analysis of tRNATrp from the different cell fractions.


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  		Fig. 5.   The portion of the L. tarentolae tRNA gene cluster (nucleotides 2461-2653; GenBankTM/EBI accession number L20948) that contains the tRNATrp gene (33). The mature tRNATrp sequence is underlined and in boldface. The CCA anticodon is indicated (down-arrow ++), with C34 that is edited indicated by the arrow (down-arrow ). The locations of the unprocessed 3'-ends determined by 3'-RACE (Nuc-1 and Nuc-2; as shown in Fig. 4A) are indicated (black-diamond ). The locations of the oligonucleotide primers used in this study are indicated.

Mature tRNAIle Is Imported into Isolated Mitochondria More Efficiently than 5'-Extended Precursor tRNAIle-- The data presented so far are consistent with a model in which the mature tRNAGln, tRNAIle, tRNATrp, tRNAVal, and tRNALys molecules are the in vivo substrates for importation into the mitochondrion. Rubio et al. (16) showed that in vitro importation of synthetic tRNAs into isolated mitochondria shows a high level of selectivity that, at least in the case of tRNAIle and tRNAGln, is correlated with the in vivo subcellular localization. We therefore assayed the in vitro importation efficiency of T7-transcribed tRNAIle with and without a 50-nucleotide 5'-leader. The results shown in Fig. 6 indicate that mature tRNAIle is the preferred substrate for mitochondrial importation. This selective in vitro importation is probably not due to the pre-tRNA folding into an unusual structure since digestion of 5'-extended tRNAIle with E. coli RNase P RNA (44) was shown to cleave the 5'-leader at the expected position (Fig. 6).


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  		Fig. 6.   Pre-tRNAIle is not efficiently imported into L. tarentolae mitochondria in vitro. Uniformly labeled pre-tRNAIle and mature tRNAIle (as indicated) were incubated with isolated mitochondria and then digested with micrococcal nuclease to remove RNA that was not imported (Import). The following were included as controls: RNA digested with micrococcal nuclease (MN), 10% of the RNA used as input in the importation reaction (1/10 IN), and pre-tRNAIle incubated with E. coli RNase P RNA (Eco P). The migration of each full-length input RNA is indicated by the corresponding arrows.

Nucleus-localized Precursors of tRNATrp Are Unedited-- The C34 to U34 editing event of mitochondrial tRNATrp can be detected by resistance to digestion of the edited cDNA with HinfI since editing destroys a HinfI site (36). As shown in Fig. 7, the amplified cDNAs from each of the tRNATrp precursors were completely digested by HinfI. The HinfI-digested precursor tRNATrp RT-PCR products were further analyzed for possible HinfI-resistant molecules by extracting the region of the gel that would contain undigested amplification products and using this as a template for additional cycles of PCR. No amplification products were obtained in this second PCR (data not shown), indicating that the initial RT-PCR of tRNATrp precursors did not amplify any molecules with edited anticodons. This indicates that editing occurs after 5'- and 3'-end maturation and is consistent with an intramitochondrial localization of this reaction.


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  		Fig. 7.   Pre-tRNATrp RT-PCR products are digested with HinfI, indicating that they are not edited at C34. A, schematic diagrams of the precursor RT-PCR products. The precursor sequence is indicated by the thinner line, and the position of the mature tRNATrp sequence is indicated by the thicker horizontal arrow. The position of the HinfI site is indicated by the vertical arrow. B, ethidium bromide-stained gel of the HinfI-digested tRNATrp RT-PCR products. The marker lanes are 100- and 10-base pair ladders (as indicated), and the positions of the 100- and 20-base pair bands are indicated. Digestion reactions were carried out with (+) and without (-) HinfI. bp, base pairs.

T7-transcribed Unedited tRNATrp(C34CA) Is More Efficiently Imported in Vitro into Isolated Mitochondria Than Edited tRNATrp(U34CA)-- Since mature unedited tRNATrp appears to represent the in vivo substrate for importation into the mitochondrion, we decided to analyze the specificity of the in vitro import system for unedited versus edited tRNA. A comparison of the in vitro importation of T7-transcribed unedited and edited tRNATrp is shown in Fig. 8. Unedited tRNATrp reached a higher saturation level than edited tRNATrp, indicating that the import mechanism can distinguish between these two isoforms.


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  		Fig. 8.   In vitro importation of unedited tRNATrp saturates at a higher concentration than edited tRNATrp. Increasing amounts of unedited and edited tRNATrp were incubated with isolated mitochondria, and importation was assayed and quantitated. The graph shows the amount of RNA protected when increasing input unedited tRNATrp (black-square) or edited tRNATrp () was used.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

There appear to be at least three overlapping classes of tRNAs in L. tarentolae in terms of subcellular localization: 1) mainly cytosolic, 2) mainly mitochondrial, and 3) shared between the two compartments. This is based on two-dimensional gel analysis of cytosolic and mitochondrial tRNAs and on Northern blot analysis of five specific tRNAs: tRNATrp, tRNAIle, tRNAGln, tRNALys, and tRNAVal. We showed in this study that tRNALys represents a second mitochondrion-localized tRNA in addition to the previously identified tRNAIle. This differs from the previous result of Suyama and co-workers (33, 37), who classified tRNALys as having a shared phenotype.

The signals for mitochondrial targeting are not known, although we have shown previously that swapping the D-arm between mitochondrion-localized tRNAIle and cytosolic tRNAGln reversed the importation phenotypes of these molecules both in vivo and in vitro (5, 16), suggesting that the tertiary structure of the molecule is involved in the specificity of targeting. In this study, we have tested the hypothesis of Hancock et al. (17) that the signal for mitochondrial importation resides within the 5'-flanking region and that 5'-extended precursor tRNAs are the native substrates for mitochondrial importation. We have shown that 5'- and 3'-end processing of tRNAs of all three classes occurs in the nucleus prior to export to the cytosol. These results imply that the substrates for importation into the mitochondrion are the mature tRNAs, at least in terms of end processing. The proposal that mature tRNAs are the import substrates in vivo is further supported by the finding that a T7-transcribed mature tRNAIle is imported into isolated mitochondria more efficiently than the 5'-extended precursor molecule.

The lack of editing at nucleotide 34 of the tRNATrp precursor molecules is consistent with a mitochondrial localization of this editing reaction and also with the importation of mature unedited tRNATrp. This observation is in contrast with the editing of tRNAAsp in marsupials (38, 39) and mitochondrial tRNA editing in plants (40), where editing precedes tRNA 3'-processing. This difference might reflect the fact that in L. tarentolae, the edited tRNA is encoded in the nucleus and thus required for translation in both the cytosol and the mitochondria, whereas in plants and marsupials, the edited tRNAs are encoded in the mitochondria and only function in intra-organellar translation. In addition, tRNA editing in plants leads to stabilization of the acceptor stem and may be responsible for providing the proper recognition substrate for the 3'-processing activity.

The finding that edited L. tarentolae tRNATrp is poorly imported in vitro compared with unedited tRNATrp suggests a subtle molecular discrimination of isoacceptors by the transport apparatus. This discrimination of a single nucleotide change in the anticodon is reminiscent of the results of Rusconi and Cech (2, 3) in Tetrahymena, in which a single nucleotide change in a tRNAGln anticodon conferred a mitochondrial importation phenotype in vivo.

The presence of an intramitochondrial RNase P-like activity and a ATP/CTP:tRNA nucleotidyltransferase activity (16, 17) is not inconsistent with importation of mature tRNAs since the former activity could be involved in other aspects of RNA metabolism (41-43), and the latter activity could play a role in the repair of the 3'-ends of tRNAs. It should be noted, however, that our results with the mitochondrion-localized tRNAs we have examined do not eliminate the possibility that there are alternate pathways for importation of other tRNAs into the mitochondrion in these cells (19, 20). A complete cataloging of all mitochondrial tRNAs in the cell is required to address this question.

The data presented in this report suggest a model for a tRNA import pathway in L. tarentolae, which is diagrammed in Fig. 9. The tRNAs are transcribed in the nucleus as precursors with extended 5'- and 3'-ends, and the precursors are end-processed prior to export from the nucleus. The mature tRNAs are then exported to the cytosol, where a fraction is retained for cytosolic translation, and another fraction is imported into the mitochondrion for organellar translation.


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  		Fig. 9.   Proposed model of L. tarentolae tRNATrp transcription, processing, and editing. The tRNA is transcribed from the nuclear genome as a precursor with extended 5'- and 3'-ends. The ends are processed, and the 3'-terminal CCA is added to the tRNA in the nucleus. The processed tRNA is then exported to the cytosol. A portion of the exported tRNA is maintained in the cytosol, and a potion is imported into the mitochondrion. Within the mitochondrion, a fraction of the tRNATrp is edited at the first position of the anticodon (C34 to U34).


    ACKNOWLEDGEMENTS

We thank all members of the Simpson laboratory for discussion and assistance. We thank Dr. David Campbell and Gusti Zeiner for supplying the U6 snRNA sequence and the U6-1 oligonucleotide. We also thank Dr. Norman Pace for providing an E. coli RNase P RNA plasmid.

    FOOTNOTES

* This work was supported in part by a Human Frontier Science Program grant (to L. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported in part by Institutional National Research Service Award T32 GM08375 from the United States Public Health Service (UCLA Biotechnology Training Program).

|| Supported in part by a National Science Foundation Graduate Research Fellowship.

Dagger Dagger To whom correspondence should be addressed: Howard Hughes Medical Inst., University of California, 675 Charles E. Young Dr. South, Los Angeles, CA 90095. Tel.: 310-825-4215; Fax: 310-206-8967; E-mail: simpson@hhmi.ucla.edu.

Published, JBC Papers in Press, September 18, 2000, DOI 10.1074/jbc.M007838200

2 J. D. Alfonzo, S. T. Kapushoc, and L. Simpson, unpublished data.

    ABBREVIATIONS

The abbreviations used are: RT-PCR, reverse transcription-polymerase chain reaction; PIPES, 1,4-piperazinediethanesulfonic acid; snRNA, small nuclear RNA; RACE, rapid amplification of cDNA ends; kb, kilobases.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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