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Molecular and Cellular Biology, December 2001, p. 8236-8237, Vol. 21, No. 23
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.23.8236-8237.2001
LETTERS TO THE EDITOR
Difference between Mitochondrial RNase P and Nuclear RNase P
 |
LETTER |
We previously demonstrated that mammalian cells contain at least
two distinct RNase P activities, one nuclear and one mitochondrial (5, 7). These enzymes were shown to have different
substrate specificities and distinct molecular and enzymatic properties (5-7). Puranam and Attardi recently reported the
purification of "nuclear" RNase P from mitochondrial preparations
(2). However, in the discussion of their findings they
made incorrect and misleading reference to previous work on
mitochondrial RNase P, necessitating a reevaluation of their conclusions.
The presence of low levels of nuclear RNase P or other nuclear RNA
processing factors in purified mitochondrial preparations is not a new
observation, and digitonin treatment is well-known to drastically
reduce the levels of these contaminants (1, 3, 7).
Mitochondrial RNase P activity, however, is definitely independent of
the presence or absence of nuclear RNase P or its RNA component (H1
RNA) (7). Reduction of contaminating nuclear RNase P by
digitonin pretreatment of mitochondria or its complete removal from
mitoplast extracts by either immunoprecipitation or purification did
not result in any change of mitochondrial RNase P activity in these
preparations (7).
Yet, inevitably, Puranam and Attardi (2) failed in the
identification of this RNase P activity independent of H1 RNA: the use of Escherichia coli
pre-tRNATyrsu3+ as a substrate
throughout their purification procedure made the assay of mitochondrial
RNase P activity naturally impossible. Mitochondrial RNase P as
identified in 1995, which faithfully cleaves mitochondrial tRNA
precursors (3-7), does not process E. coli
pre-tRNATyrsu3+ (7).
On the other hand, nuclear RNase P, which is capable of cleaving
pre-tRNATyrsu3+, does not cleave
mitochondrial pre-tRNATyr (7). Given the
considerable structural differences between cytoplasmic and
mitochondrial tRNAs, this particularly useful distinction of
mitochondrial and nuclear RNase P by their substrate specificity is not
surprising but apparently is the result of the coevolution of enzymes
and substrates. Substrate recognition of both enzymes nevertheless
overlaps as long as the precursors fulfill the respective structural
requirements (7).
Thus, is it possible that mammalian mitochondria contain two forms of
RNase P, one distinct from and one identical to the nuclear enzyme?
Such a scenario of two RNase P enzymes within one cellular compartment
would be without precedent and therefore requires careful evaluation.
Of course, the relative and absolute levels of both enzymes in
mitochondria are critical in this argument. Even in small amounts of
crude mitochondrial extracts, detection of mitochondrial RNase P
activity appears to be straightforward, thereby indicating a high level
of enzyme (7). In contrast, nuclear RNase P activity could
not be detected in mitochondrial extracts (7) but required
enrichment by biochemical purification to allow assay of its activity
(2). Puranam and Attardi nevertheless provided an estimate
of the quantities of nuclear RNase P (H1) RNA as well as other snRNAs
present in purified mitochondria (2). Taking their numbers
for granted, (i) the actual levels of U snRNAs (nuclear splicing
factors) and H1 RNA are in an astonishingly similar range and (ii) the
estimated number of H1 RNA molecules in the mitochondrial matrix
(digitonin/micrococcal nuclease resistant and "corrected for losses
of mitochondrial markers") of cells in the G2 phase of
the cell cycle is only one-third of the number of mitochondria
(2). The latter implies that the mitochondria of cells
during other phases of the cell cycle do not contain any nuclear RNase
P, which would only be possible by cell cycle stage-dependent import of
nuclear RNase P followed by rapid intramitochondrial degradation or
expulsion. Moreover, assuming that this scenario is correct, the
numbers given by Puranam and Attardi would actually be overestimates of
RNase P amounts, as their preparations were "enriched in heavy
mitochondria" (2). Thus we feel that although it is
impossible to formally exclude the possibility that mitochondria contain trace amounts of nuclear RNA processing factors, contamination of mitochondrial preparations during subcellular fractionation still
appears to be the more plausible interpretation.
| |
FOOTNOTES |
*
Phone: 43 1 4277 61187
Fax: 43 1 4277 61198
E-mail: walter.rossmanith{at}univie.ac.at
| |
REFERENCES |
| 1.
|
Kiss, T., and W. Filipowicz.
1992.
Evidence against a mitochondrial location of the 7-2/MRP RNA in mammalian cells.
Cell
70:11-16[CrossRef][Medline].
|
| 2.
|
Puranam, R. S., and G. Attardi.
2001.
The RNase P associated with HeLa cell mitochondria contains an essential RNA component identical in sequence to that of the nuclear RNase P.
Mol. Cell. Biol.
21:548-561[Abstract/Free Full Text].
|
| 3.
|
Rossmanith, W.
1996.
Ph.D. thesis.
University of Vienna, Vienna, Austria.
|
| 4.
|
Rossmanith, W.
1997.
Processing of human mitochondrial tRNASer(AGY): a novel pathway in tRNA biosynthesis.
J. Mol. Biol.
265:365-371[CrossRef][Medline].
|
| 5.
|
Rossmanith, W., and R. M. Karwan.
1998.
Characterization of human mitochondrial RNase P: novel aspects in tRNA processing.
Biochem. Biophys. Res. Commun.
247:234-241[CrossRef][Medline].
|
| 6.
|
Rossmanith, W., and R. M. Karwan.
1998.
Impairment of tRNA processing by point mutations in mitochondrial tRNALeu(UUR) associated with mitochondrial diseases.
FEBS Lett.
433:269-274[CrossRef][Medline].
|
| 7.
|
Rossmanith, W.,
A. Tullo,
T. Potuschak,
R. Karwan, and E. Sbisà.
1995.
Human mitochondrial tRNA processing.
J. Biol. Chem.
270:12885-12891[Abstract/Free Full Text].
|
| | | | |
Walter Rossmanith*
Institute of Anatomy
University of Vienna
Währinger Str. 13
1090 Vienna, Austria
|
| | | | |
Thomas Potuschak
ICMB
University of Edinburgh
Edinburgh EH9 3JR, Scotland, United Kingdom
|
| |
AUTHORS' REPLY |
In earlier work (1), we showed that HeLa cell mitochondria
carry a micrococcal nuclease-sensitive RNase P activity representing a
fraction of a percent of the total activity in the postmitochondrial cytosol fraction (consisting mostly of the leaked-out nuclear activity). In agreement with this result, we recently demonstrated that
highly purified HeLa cell mtRNase P contains an intact RNA identical to
H1 RNA of nuclear RNase P, representing ~0.3% of that associated
with the nucleocytosolic compartment; crucially, this RNA can be
removed from mitochondria only by treatments destroying the integrity
of the organelles (6). That mitochondria carry such a
small amount of RNase P is not surprising, knowing that they contain
only <1% of the cell tRNAs (2). Most significantly, an
analysis of the expected functional requirements for tRNA processing in
HeLa cell mitochondria showed that the detected level of mtRNase P
fully satisfies those requirements (6).
Our conclusions are in apparent contrast with the results of Rossmanith
and colleagues, who described an abundant reportedly RNA-free mtRNase P
(7, 8). Our failure to detect such enzyme was not due to
using E. coli ptRNATyr as a substrate. In fact,
contrary to a statement in their letter, this substrate was clearly
cleaved by their mtRNase P, although incorrectly, i.e., four
nucleotides upstream of the mature 5' end (7). No such
processing activity was observed at any stage during purification of
our mtRNase P, and we would have definitely been able to see it
(6).
In answer to another statement in the letter, the proportions of total
cell- and washed mitochondrion-associated U snRNAs (which is a more
appropriate parameter than "levels") that we found in purified
mitochondria were, respectively, 1 and 2 orders of magnitude lower than
the corresponding proportions of H1 RNA. Also, our most conservative
estimate of mitochondrial H1 RNA amount per cell in unsynchronized
cells, which quantifies only intact H1 RNA (~175 molecules), when
referred to transcriptionally active cells (5), would be
about twice the number of mitochondria per cell.
There are two obvious differences in the experiments by Rossmanith et
al., as compared to ours. (i) The first difference is the quality of
their mitochondrial preparations, which exhibited a massive degradation
of their abundant nuRNase P contaminant, as well as of their
mitochondrial RNAs (as exemplified by tRNAGlu). This
degradation, which resulted presumably from those authors' use of
frozen cells for cell fractionation and from the expected consequent
molecular damage, contrasts dramatically with our patterns of mostly
intact RNA. This damage could account for the differences in enzymatic
and physical properties and in RNA content of their RNase P. (ii) The
second difference is the lack of any clearly interpretable
quantification of RNase P activity and RNA content. In particular, in
their most recent paper (8), Rossmanith et al.
acknowledged the presence of degraded RNA in their partially purified
mtRNase P, but no quantification was presented. It is well established
that yeast mtRNase P retains partial activity after extensive
degradation of its mtDNA-encoded RNA (3).
As to the physiological significance for HeLa cells of the presence of
a limiting, though adequate, amount of RNase P in mitochondria, which
we determined by two independent approaches, it is important to point
out that the RNase P tRNA processing sites are the sites of
polyadenylation, and resulting stabilization, of the upstream encoded
RNAs (4, 6). Thus, limiting the amount of RNase P in
mitochondria to the minimum required for tRNA synthesis would not allow
stabilization of the 10- to 15-fold excess of fast turning-over nascent
L-strand transcripts and prevent both a disproportionately high rate of
synthesis of the eight L-strand-encoded tRNAs relative to the
H-strand-encoded tRNAs, which could negatively affect translation (2), and an accumulation of abundant antisense RNA.
| |
REFERENCES |
| 1.
|
Doersen, C.-J.,
C. Guerrier-Takada,
S. Altman, and G. Attardi.
1985.
Characterization of an RNase P activity from HeLa cell mitochondria. Comparison with the cytosol RNase P activity.
J. Biol. Chem.
260:5942-5949[Abstract/Free Full Text].
|
| 2.
|
King, M. P., and G. Attardi.
1993.
Post-translational regulation of the steady-state levels of mitochondrial tRNAs in HeLa cells.
J. Biol. Chem.
268:10228-10237[Abstract/Free Full Text].
|
| 3.
|
Morales, M. J.,
C. A. Wise,
M. J. Hollingsworth, and N. C. Martin.
1989.
Characterization of yeast mitochondrial RNase P: an intact RNA subunit is not essential for activity in vitro.
Nucleic Acids Res.
17:6865-6881[Abstract/Free Full Text].
|
| 4.
|
Ojala, D.,
J. Montoya, and G. Attardi.
1981.
tRNA punctuation model of tRNA processing in human mitochondria.
Nature
290:470-474[CrossRef][Medline].
|
| 5.
|
Pica-Mattoccia, L., and G. Attardi.
1972.
Expression of the mitochondrial genome in HeLa cells. IX. Transcription of mitochondrial DNA in relationship to the cell cycle.
J. Mol. Biol.
57:615-621.
|
| 6.
|
Puranam, R. S., and G. Attardi.
2001.
The RNase P associated with HeLa cell mitochondria contains an essential RNA component identical in sequence to that of the nuclear RNase P.
Mol. Cell. Biol.
21:548-561.
|
| 7.
|
Rossmanith, W.,
A. Tullo,
T. Potuschak,
R. Karwan, and E. Sbisà.
1995.
Human mitochondrial tRNA processing.
J. Biol. Chem.
270:12885-12891.
|
| 8.
|
Rossmanith, W., and R. M. Karwan.
1998.
Characterization of human mitochondrial RNase P: novel aspects in tRNA processing.
Biochem. Biophys. Res. Commun.
247:234-241.
|
| | | | |
Giuseppe Attardi
Division of Biology
California Institute of Technology
Pasadena, CA 91125
|
| | | | |
Ram S. Puranam
Department of Medicine (Neurology)
Duke University Medical Center
Durham, NC 27710
*Phone: (626) 395-4930
Fax: (626) 449-0756
E-mail: attardi{at}caltech.edu
|
Molecular and Cellular Biology, December 2001, p. 8236-8237, Vol. 21, No. 23
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.23.8236-8237.2001
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