From the
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.
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
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) .
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 [
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
Citrate synthase activity
was determined essentially as described
(23) .
End-labeled (mt)pre-tRNA
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
[
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.
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-tRNA
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,
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
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.
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
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.
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-tRNA
su
, 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-tRNA
su
isolated in
vivo(15) .
-
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).
, 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).
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.
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.
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-tRNA
su
, 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-tRNA
su
. 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.
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-tRNA
su
, 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-tRNA
su
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-tRNA
su
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).
su
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-tRNA
su
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) .
(
)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.
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 T
C-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-tRNA
su
(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.
su
,
precursor suppressor III tRNA
; DTT, dithiothreitol; CIP,
calf intestinal alkaline phosphatase; RNP(s), ribonucleoprotein(s);
RNaseMRP, ribonuclease mitochondrial RNA processing; snRNA, small
nuclear RNA.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.