Download Antisense RNA Crossing Mitochondrial Membrane?

From www.bloodjournal.org by guest on August 1, 2017. For personal use only.
2994
CORRESPONDENCE
criteria proposed by the Scientific Standardization Committee Subcommittee for Standardization of Lupus Anticoagulant.3 Anticardiolipin
(aCLAb) and anti–b2-glycoprotein I (ab2GP1Ab) IgG antibodies had
been tested using commercially available assays (Quanta Lite [Inova
Diagnostics Inc, San Diego, CA] and Varelisa b2GP1 [Elias, Freiburg,
Germany]). Antiprothrombin (aFIIAb) and anti-annexinV (aAnVAb)
IgG antibodies had been assayed using enzyme-linked immunosorbent
assay research kits (gift from J. Amiral, Diagnostica Stago, Asnières,
France). All normal values were obtained using 200 control
plasmas assuming that threshold values corresponded to the 99th
percentiles. All patient plasmas were tested crude and after IgG
depletion using protein A-sepharose. We systematically performed mixing
studies using a 1:1 mixture of patient and normal plasma. Normal plasma has
been prepared pooling the 200 control plasmas and was used crude (NP) or
b2-glycoprotein I depleted after immunoaffinity chromatography (NPb2neg).
We identified 42 patients with positive antiphospholipid antibodyrelated markers: 10 patients with an isolated LA (7 with a KCT-type LA
and 3 with a dRVVT-type LA according to Galli et al1), 10 patients with
isolated aCLAb (levels ranging from 25 to 68 GPL units, normal values
lower than 14.5 GPL units), 10 patients with isolated ab2GP1Ab (levels
ranging from 42 to 123 U/mL, normal values lower than 14.5 U/mL), 2
patients with isolated aFIIAb, and 10 patients with isolated aAnVAb (Table
1). We observed that plasmas with isolated positive ab2GP1Ab were the only
ones to be associated with an acquired resistance to aPC, which disappeared
in cases of IgG depletion. These plasmas generated resistance to aPC during
mixing studies and the intensity of the inhibitory activity depended on the
b2-glycoprotein I content of the normal plasma. We thereafter supplemented
NP and NP-b2neg with IgG purified from plasmas with isolated positive
anti–b2-glycoprotein I IgG antibodies (3:1; IgG concentration, 10
mg/mL in Tris-buffered saline). An acquired plasma resistance to aPC
was induced only in the presence of plasma b2-glycoprotein I (median
values and ranges of aPC-FV ratio: NP, 1.59 [1.19-1.88]; NP-b2neg,
2.22 [1.98-2.45]; P 5 .005; control values using IgG purified from
normal plasma: NP, 2.30 [2.05-2.55], NP-b2neg, 2.28 [2.00-2.49];
nonsignificant).
Thus, the modified aPC-resistance test, together with a mixing study
performed in this test, can easily allow us to identify the acquired anticoagulant activity acting as an inhibitor of the aPC-mediated factor Va proteolysis.
Concerning women with recurrent miscarriages positive for antiphospholipid
antibody-related markers, only ab2GP1Ab is related with the impairment of
the anti-factor V activity of aPC. This finding is concordant with data
shown by Galli et al1 in their group of patients with mainly thrombotic
antecedents. If acquired aPC resistance represents a pathogenic mechanism responsible for the clinical risk in a subgroup of antiphospholipidpositive patients, the modified aPC-resistance test should allow to
identify plasmas positive for this peculiar anticoagulant activity.
Eric Mercier
Laboratoire d’Hématologie
CHU Nı̂mes
Nı̂mes, France
Isabelle Quere
Médecine Interne B et Maladies Vasculaires
CHU, Montpellier
Montpellier, France
Pierre Mares
Service de Gynécologie et Obstétrique
CHU, Nı̂mes
Nı̂mes, France
Jean-Christophe Gris
Laboratoire d’Hématologie
Faculté de Pharmacie
Montpellier, France
REFERENCES
1. Galli M, Ruggeri L, Barbui T: Differential effects of anti–b2glycoprotein I and antiprothrombin antibodies on the anticoagulant
activity of activated protein C. Blood 91:1999, 1998
2. Jorquera JI, Montoro JM, Fernandez MA, Aznar J: Modified test
for activated protein C resistance. Lancet 344:1162, 1994
3. Brandt JT, Triplett DA, Alving B, Scharrer I: Criteria for the diagnosis
of lupus anticoagulants: An update. Thromb Haemost 74:1185,1995
Antisense RNA Crossing Mitochondrial Membrane?
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.
From www.bloodjournal.org by guest on August 1, 2017. For personal use only.
CORRESPONDENCE
2995
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 Enrı́quez
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
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
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
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
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
4. Mahapatra S, Ghosh T, Adhya S: Import of small RNAs into
Leishmania mitochondria in vitro. Nucleic Acids Res 22:3381, 1994
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
7. Lye LF, Chen DH, Suyama Y: Selective import of nuclearencoded tRNAs into mitochondria of the protozoan Leishmania tarentolae. Mol Biochem Parasitol 58:233, 1993
Magnetic Resonance Imaging in Myelofibrosis
To the Editor:
The recent review about magnetic resonance imaging (MRI) of the
bone marrow in hematologic malignancies has suggested that MRI had
little or no value in the evaluation of myelofibrosis (MF).1 Although MR
findings did not also appear beneficial in the differential diagnosis of
myelofibrosis, our data showed that MRI may have some importance in
predicting the prognosis of this disorder.
Thirteen patients with MF were evaluated (11 patients with primary
MF and 2 patients with secondary MF) and diagnoses of MF were made
according to previously defined criteria.2 Before the MR examination,
bone marrow aspirates and biopsies from posterior iliac crest were
From www.bloodjournal.org by guest on August 1, 2017. For personal use only.
1998 92: 2994-2995
Antisense RNA Crossing Mitochondrial Membrane?
Götz Hofhaus, Norbert Gattermann and José Antonio Enri?quez
Updated information and services can be found at:
http://www.bloodjournal.org/content/92/8/2994.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.