Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2994-2995
CORRESPONDENCE
Antisense RNA Crossing Mitochondrial Membrane?
 |
LETTER |
To the Editor:
In a recent article in Blood, Shirafuji et al1
report the isolation of a cDNA encoding an antisense RNA for subunit I
of mitochondrial cytochrome c oxidase (MARCO). Expression of this cDNA
in hematopoietic cell lines caused morphological changes and cell
death. Although Shirafuji et al1 convincingly demonstrate that the antisense RNA induced these changes, we would take issue with
the investigators on the suggested mechanism, namely interference with
the corresponding mitochondrial gene transcript (COX I).
Animal mitochondria represent a distinct cellular compartment that has
its own mitochondrial DNA, encoding 13 proteins, including 3 subunits
of cytochrome c oxidase. The mRNAs of the mitochondrial genes are
translated on mitochondrial ribosomes. Whereas the protein components
required for mitochondrial gene expression (polymerases, ribosomal
proteins) are imported into mitochondria, all the necessary RNA
components (transfer-RNAs, ribosomal RNAs) are encoded by the
mitochondrial genome and are therefore not taken up from the cytoplasm.2 There is no indication of significant DNA or
RNA uptake into mitochondria. Even for the nucleus-encoded small RNA components of two mitochondrial riboproteins (MRP-RNAse3
and RNAse P4) the import mechanism is unclear and may
involve cotransport with the associated proteins. Nonspecific uptake of
polynucleotides is unlikely, because the inner mitochondrial membrane
is a nonpolar lipid bilayer with only a very limited permeability
towards polar molecules. To support an antisense mechanism, the
investigators would have to demonstrate that the MARCO-RNA actually
crosses the mitochondrial membrane to interfere with the corresponding mitochondrial COX I gene transcript. Second, the investigators should
have shown that other mitochondrial transcripts are not affected,
because, otherwise, the reported reduction of the COX I mRNA could be
interpreted as an unspecific feature associated with early stages of
cell death.
Finally, we would like to speculate on an alternative explanation for
the findings of Shirafuji et al.1 Because all the mitochondrial genes encode very hydrophobic membrane proteins, they
contain nucleotide sequences that are similar to a variety of other
membrane spanning proteins. It is therefore conceivable that MARCO
could affect the cytoplasmic translation of an important membrane
protein that is located in the cytoplasm rather than in the
mitochondria. Decreased expression of this protein may trigger cell
death, with an associated early change in mitochondrial gene
transcription.
Nevertheless, even the demonstration that mitochondrial (antisense)
mRNAs can induce cell death when present in the cytoplasm might open
new perspectives regarding the role of mitochondria in
apoptosis.
Götz Hofhaus
Institut für
Biochemie
Norbert Gattermann
Klinik für
Hämatologie, Onkologie, und Klinische
Immunologie
Heinrich-Heine-Universität,
Düsseldorf
Düsseldorf, Germany
José Antonio Enríquez
Dpto. Bioquimica y Biologia Molecular y
Celular
Universidad de Zaragoza, Spain
Zaragoza,
Spain
| |
REFERENCES |
1.
Shirafuji N,
Takahashi S,
Matsuda S,
Asano S:
Mitochondrial antisense RNA for cytochrome c oxidase (MARCO) can induce morphological changes and cell death in human hematopoietic cell lines.
Blood
90:4567,
1997[Abstract/Free Full Text]
2.
Attardi G,
Schatz G:
Biogenesis of mitochondria.
Annu Rev Cell Biol
4:289,
1988
3.
Chang DD,
Clayton DA:
A mammalian mitochondrial RNA processing activity contains nucleus-encoded RNA.
Science
235:1178,
1987[Abstract/Free Full Text]
4.
Doersen C-J,
Guerre-Takada C,
Altman S,
Attardi G:
Characterization of an RNase P activity from HeLa cell mitochondria. Comparison with the cytosol RNase P activity.
J Biol Chem
260:5942,
1985[Abstract/Free Full Text]
| |
RESPONSE |
I thank Dr Hofhaus for the interest in my study of expression cloning
and characterization of MARCO (mitochondrial antisense RNA for cyto-c
oxidase).1 He points out that it is doubtful that antisense
RNA can cross mitochondrial membrane. It has been reported that tRNAs
in the cytoplasm can cross mitochondrial membrane using their specific
receptors, which can recognize specific nucleotide sequences and
integrate tRNAs into mitochondria in accordance with ATP and TAS factor
(sequence-specific RNA binding factor) or other cytoplasmic
proteins.2 As I referred to in my report, small RNAs,
including antisense RNAs, can also cross mitochondrial membrane with
this mechanism.3,4 These receptors have been reported to
recognize nucleotide sequence GAAA A/G G in Leishmania mitochondria
system,4 GAAGGG, CGAGAGG, and CGAAGGG in trypanosomes and
crithidia in 180 nt srRNA system,5 CGAGAG in 140 nt srRNA system,5 CGAATG in trypanosome 5S RNA system,6
and GGCAGAG and GGUAGAG in L tarentolae tRNA
system.7 These observations have not been reported in the
human mitochondria system; however, it is possible that also in the
human system similar mechanisms work to integrate tRNAs and small RNAs
into mitochondrial organella across mitochondrial membrane because of
very high homology of mitochondrial nucleotide sequences beyond species
specificity. The nucleotide sequence of MARCO contains three GAAAGG and
one GGTAGAG.1 These sequences may be recognized by
mitochondrial membrane receptor, and MARCO may be able to cross
membrane. Also, it is not clear how large nucleotides can cross the
mitochondrial membrane. It may be possible that, after MARCO is
transcribed in the cytoplasm, partial digestion of RNA occurred, and
small fragments of MARCO with receptor-recognized sequence can cross the mitochondrial membrane, as reported in the Kinetoplastid srRNA system in which cytoplasmic 28S rRNA precursor is processed
posttranscriptionally.5 Otherwise, it may be possible that
the entire MARCO RNA can cross mitochondrial membrane after binding to
the receptor.
Furthermore, as Dr Hofhaus points out, other mechanisms should be
possible to induce cell death when MARCO is expressed in the cytoplasm.
Thus, I agree with Dr Hofhaus that precise experiments are required to
make clear the mechanism of the action of MARCO.
Naoki Shirafuji
Department of Hematology/Oncology
The Institute of
Medical Science
The University of Tokyo
Tokyo, Japan
| |
REFERENCES |
1.
Shirafuji N,
Takahashi S,
Matsuda S,
Asano S:
Mitochondrial antisense RNA for cytochrome c oxidase (MARCO) can induce morphologic changes and cell death in human hematopoietic cell lines.
Blood
90:4567,
1997
2.
Tarassov IA,
Entelis NS:
Mitochondrially-imported cytoplasmic tRNALys (CUU) of Saccharomyces cerevisiae: In vivo and in vitro targetting system.
Nucleic Acids Res
20:1277,
1992[Abstract/Free Full Text]
3.
Ghosh A,
Ghosh T,
Ghosh S,
Das S,
Adhya A:
Interaction of small ribosomal and transfer RNAs with a protein from Leishmania donovani.
Nucleic Acids Res
22:1663,
1994[Abstract/Free Full Text]
4.
Mahapatra S,
Ghosh T,
Adhya S:
Import of small RNAs into Leishmania mitochondria in vitro.
Nucleic Acids Res
22:3381,
1994[Abstract/Free Full Text]
5.
White TC,
Rudenko G,
Borst P:
Three small RNAs within the 10kb trypanosome rRNA transcription unit are analogous to domain VII of other eukaryotic 28S rRNAs.
Nucleic Acids Res
14:9421,
1986
6.
Lenardo MJ,
Dorfman DM,
Reddy LV,
Donelson JE:
Characterization of the Trypanosoma brucei 5S ribosomal RNA gene and transcript: The 5S rRNA is a spliced-leader-independent species.
Gene
35:131,
1985[Medline]
[Order article via Infotrieve]
7.
Lye LF,
Chen DH,
Suyama Y:
Selective import of nuclear-encoded tRNAs into mitochondria of the protozoan Leishmania tarentolae.
Mol Biochem Parasitol
58:233,
1993[Medline]
[Order article via Infotrieve]
