Expression of a novel non-coding mitochondrial RNA in human proliferating cells.
ABSTRACT Previously, we reported the presence in mouse cells of a mitochondrial RNA which contains an inverted repeat (IR) of 121 nucleotides (nt) covalently linked to the 5' end of the mitochondrial 16S RNA (16S mtrRNA). Here, we report the structure of an equivalent transcript of 2374 nt which is over-expressed in human proliferating cells but not in resting cells. The transcript contains a hairpin structure comprising an IR of 815 nt linked to the 5' end of the 16S mtrRNA and forming a long double-stranded structure or stem and a loop of 40 nt. The stem is resistant to RNase A and can be detected and isolated after digestion with the enzyme. This novel transcript is a non-coding RNA (ncRNA) and several evidences suggest that the transcript is synthesized in mitochondria. The expression of this transcript can be induced in resting lymphocytes stimulated with phytohaemagglutinin (PHA). Moreover, aphidicolin treatment of DU145 cells reversibly blocks proliferation and expression of the transcript. If the drug is removed, the cells re-assume proliferation and over-express the ncmtRNA. These results suggest that the expression of the ncmtRNA correlates with the replicative state of the cell and it may play a role in cell proliferation.
- SourceAvailable from: Peter H. Schreier[show abstract] [hide abstract]
ABSTRACT: The complete sequence of the 16,569-base pair human mitochondrial genome is presented. The genes for the 12S and 16S rRNAs, 22 tRNAs, cytochrome c oxidase subunits I, II and III, ATPase subunit 6, cytochrome b and eight other predicted protein coding genes have been located. The sequence shows extreme economy in that the genes have none or only a few noncoding bases between them, and in many cases the termination codons are not coded in the DNA but are created post-transcriptionally by polyadenylation of the mRNAs.04/1981; 290(5806):457-465.
- [show abstract] [hide abstract]
ABSTRACT: A 3'-end proximal segment of most of the putative mRNAs encoded in the heavy strand of HeLa cell mtDNA has been partially sequences and aligned with the DNA sequence. In all cases, the 3'-end nucleotide of the individual mRNA coding sequences has been found to be immediately contiguous to a tRNA gene or another mRNA coding sequence. These and previous results indicate that the heavy (H) strand sequences coding for the rRNA, poly(A)-containing RNA and tRNA species form a continuum extending over almost the entire length of this strand. We propose that the H strand is transcribed into a single polycistronic RNA molecule, which is processed later into mature species by precise endonucleolytic cleavages which occur, in most cases, immediately before and after a tRNA sequence.Nature 05/1981; 290(5806):470-4. · 38.60 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The discovery that mutations in mitochondrial DNA (mtDNA) can be pathogenic in humans has increased interest in understanding mtDNA maintenance. The functional state of mtDNA requires a great number of factors for gene expression, DNA replication, and DNA repair. These processes are ultimately controlled by the cell nucleus, because the requisite proteins are all encoded by nuclear genes and imported into the mitochondrion. DNA replication and transcription are linked in vertebrate mitochondria because RNA transcripts initiated at the light-strand promoter are the primers for mtDNA replication at the heavy-strand origin. Study of this transcription-primed DNA replication mechanism has led to isolation of key factors involved in mtDNA replication and transcription and to elucidation of unique nucleic acid structures formed at this origin. Because features of a transcription-primed mechanism appear to be conserved in vertebrates, a general model for initiation of vertebrate heavy-strand DNA synthesis is proposed. In many organisms, mtDNA maintenance requires not only faithful mtDNA replication, but also mtDNA repair and recombination. The extent to which these latter two processes are involved in mtDNA maintenance in vertebrates is also appraised.Annual Review of Biochemistry 02/1997; 66:409-35. · 27.68 Impact Factor
Nucleic Acids Research, 2007, Vol. 35, No. 21Published online 25 October 2007
Expression of a novel non-coding mitochondrial
RNA in human proliferating cells
Jaime Villegas1,2,3, Veronica Burzio1,2,3, Claudio Villota1,2, Eduardo Landerer1,3,
Ronny Martinez1, Marcela Santander1, Rodrigo Martinez1, Rodrigo Pinto3,
Marı ´a I. Vera3, Enrique Boccardo4, Luisa L. Villa4and Luis O. Burzio1,2,3,*
1Bios Chile Ingenierı ´a Gene ´tica S.A.,2Institute for Fundamental and Applied Biology, Fundacio ´n Ciencia para la
Vida, Avenida Zan ˜artu 1482,3Laboratorio de Biologı ´a Celular y Molecular and Department of Urology, Facultad de
Ciencias de la Salud, Universidad Andre ´s Bello, Repu ´blica 252, Santiago, Chile and4Ludwig Institute for Cancer
Research, Sao Paulo, Brazil
Received August 20, 2007; Revised September 21, 2007; Accepted September 26, 2007
Previously, we reported the presence in mouse cells
of a mitochondrial RNA which contains an inverted
repeat (IR) of 121 nucleotides (nt) covalently linked
to the 5’ end of the mitochondrial 16S RNA (16S
mtrRNA). Here, we report the structure of an
equivalent transcript of 2374nt which is over-
expressed in human proliferating cells but not in
resting cells. The transcript contains a hairpin
structure comprising an IR of 815nt linked to the
5’ end of the 16S mtrRNA and forming a long double-
stranded structure or stem and a loop of 40nt.
The stem is resistant to RNase A and can be
detected and isolated after digestion with the
enzyme. This novel transcript is a non-coding RNA
(ncRNA) and several evidences suggest that the
transcript is synthesized in mitochondria. The
expression of this transcript can be induced in
resting lymphocytes stimulated with phytohaemag-
glutinin (PHA). Moreover, aphidicolin treatment of
DU145 cells reversibly blocks proliferation and
expression of the transcript. If the drug is removed,
the cells re-assume proliferation and over-express
the ncmtRNA. These results suggest that the
expression of the ncmtRNA correlates with the
replicative state of the cell and it may play a role in
The mitochondrial DNA (mtDNA) is a closed-circular,
double-stranded molecule that displays an exceptional
?16500bp of the genome encode the 12S and 16S
ribosomal RNAs, 22 transfer RNAs (tRNAs) and 13
polypeptides (2,4–7). The H-strand encodes the 12S and
16S ribosomal RNAs, 14 tRNAs and 12 polypeptides,
while the L-strand codes for 8 tRNAs and the ND6
subunit of NAD dehydrogenase (2,4–8). Between the
tRNAPheand tRNAProgenes is the D-loop that has
evolved as the major control region. Besides the H-strand
origin of replication, the D-loop contains the major
promoters that regulate transcription of the H- (HSP)
and the L-strand (LSP) (9–11). Both strands are
transcribed as polycistronic RNAs, which are then
processed to release the individual mRNAs, tRNAs and
rRNAs (3). The human mitochondrial RNA polymerase
as well as transcription factors have been extensively
Previously, we described the presence in mouse cells of a
novel mitochondrial RNA containing an IR of 121nt
linked to the 50end of the 16S mtrRNA (12,13). The IR
generates a perfect double-stranded structure of 121bp
and a loop of 120nt. In situ hybridization (ISH) revealed
that this ncRNA is over-expressed in spermatogenic cells,
especially in mouse proliferating spermatogonia (14).
Similar results were obtained with human spermatogonia
using a probe complementary to the 16S mtrRNA (14).
These results suggest that human cells might contain a
transcript with similar structural features to the mouse
RNA, and that its expression correlates with cell
proliferation. In this work, we report that the human
RNA is over-expressed in several human proliferating cells
but not in resting cells. The structure of this transcript of
2374nt reveals the presence of an IR of 815nt linked to
the 50end of the 16S mtrRNA. Together with the 16S
mtrRNA, the IR forms a long double-stranded structure
The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors
*To whom correspondence should be addressed. Tel: +56 2 473 6133; Fax: +56 2 2394250; Email: email@example.com
? 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
or stem, which is resistant to RNase A digestion. On the
other hand, in silico analysis revealed that this transcript is
a new member of the expanding family of non-coding
RNAs (ncRNA) (15–17), and therefore we named this
molecule non-coding mitochondrial RNA or ncmtRNA.
suggest the mitochondrial origin of this transcript.
ISH using a probe specific for the ncmtRNA, confirmed
over-expression of this transcript in proliferating cells.
The expression of this transcript can be induced in resting
lymphocytes stimulated with phytohaemagglutinin (PHA)
(18), together with DNA synthesis and the expression of
the proliferation markers proliferating cell nuclear antigen
(PCNA), Ki-67 and phosphohistone H3 (19–21). On the
other hand, treatment of DU145 cells with aphidicolin
(22,23) reversibly blocks cell proliferation as well as the
expression of the ncmtRNA. These results suggest that the
ncmtRNA is a new marker of cell proliferation.
MATERIALS AND METHODS
HeLa, SiHa, DU145, MCF/7, H441, Caco-2 and 42/95
(melanoma) cells were cultured in Dulbecco’s Modified
Eagle’s Medium (DMEM; Gibco) containing 10% fetal
calf serum (FCS), 2mM glutamine, 50mg/ml of penicillin,
50mg/ml of streptomycin and 0.1mM non-essential amino
acids. The human promyelocitic leukemia cell line HL-60,
was cultured in Iscove’s modified Dulbecco’s medium
(IMDM; Gibco) supplemented with 20% fetal bovine
serum, 2mM glutamine, 50mg/ml of penicillin, 50mg/ml of
streptomycin and 0.1mM non-essential amino acids. All
human cell lines were maintained in a humidified cell
culture chamber at 378C and 5% CO2. To inhibit
mitochondrial transcription, HeLa cells were cultured as
described with the addition of 50ng/ml of ethidium
bromide plus 50mg/ml of uridine and 1mM sodium
pyruvate (24). For aphidicolin treatment (22,23), DU145
cells were cultured for 16h with 5mg/ml of aphidicolin
followed by 10h without the drug. Then the cells were
again treated with 5mg/ml of aphidicolin for another 16h.
To release the cells from the aphidicolin block, they were
cultured for 48h in regular medium without the drug.
Human peripheral blood lymphocytes obtained from
healthy donors were isolated by Ficoll gradient centrifu-
gation as described before (18). The cells were suspended
in RPMI 1640 medium supplemented with 10% FCS (18)
and the number of cells per ml was determined in a
Neubauer chamber. The cells were cultured for 24, 48 and
72h at 378C with 5% CO2, with or without 10mg/ml of
PHA (18). About 100000 cells in 200ml of medium were
cultured in 96-wells plates and pulsed for 16h with BrdU
starting at 0, 24 and 48h. Cells were collected at 24, 48 and
72h of culture and BrdU incorporation was measured
colorimetrically by ELISA according to manufacturer’s
instructions (Proliferation ELISA, Roche).
Formalin-fixed, paraffin-embedded human tissue samples
were obtained from diagnostic biopsies or resection
specimens from patients at the Hospital Barros Luco
Trudeau (Santiago, Chile). The tissues were used in
agreement with the ethical guidelines approved by the
ethical committee of the hospital and our institutions.
RNA and DNA isolation
Total RNA from cells was extracted with TRIzol
(Invitrogen) as described before (12,13,25). The polyA+
fraction was obtained with the Oligotex mRNA Midi kit
(Qiagen) according to the manufacturer’s instructions.
To eliminate mtDNA contamination, RNA preparations
were treated with TURBO DNA-free (Ambion) according
to the manufacturer’s instructions. Human mtDNA was
prepared from human lymphocytes, HeLa, SiHa, Hep G2
and HL-60 cells (26).
Reverse transcription was carried out with 50–100ng of
freshly prepared RNA, 50ng of random hexamers or
sequence-specific primers and 200 U of reverse transcrip-
tase (M-MLV or SuperScript II, Invitrogen) (12,13). The
cDNA was amplified by PCR and analyzed by electro-
phoresis as described before (12,13,27). The primers used
to amplify the sense 16S mtrRNA were: P1 (r) 50AAGGT
GGAGTGGGTTTGGGGC (position 11–31); P9 (f)
501–522); P10 (f) 50ACCGTGCAAAGGTAGCATAAT
CAC (position 912–935); P11 (r) 50AATAGGATTGCG
CTGTTATCCCTA (position 1260–1283); P12 (r) 50CTG
TTCTTGGGTGGGTGTG (position 1536–1554). For the
antisense 16S mtrRNA, P2 (f) 50GGGGTCTTAGCTTT
GGCTCTCC (position 1326–1347); P3 (f) 50TTGGTGG
CTGCTTTTAGGCCTA (position 1207–1227); P4 (f)
50GGTTGATTGTAGATATTGGGCT (position 833–854);
741–759); P6 (f) 50GTGATTATGCTACCTTTGCAC
GGT (position 626–649); P7 (r) 50ACCATTTACCCAA
ATAAAGTATAG (position 1483–1506); P8 (r) 50GGAC
CAATCTATCACCCTATA (position 942–962). To iden-
tify the position of each primer, the sequence from nt 1 to
1559 of the sense or antisense 16S mtRNA was used as
reference. For the region between the IR and the 16S
(807–824). For the 12S mtrRNA, P14 (f) 50AGCCTAT
ATACCGCCATCTTC (position 604–624); P15 (r) 50AA
P16 (f) 50GTGTACTGGAAAGTGCACTTG (position
927–947). For COX I, P17 (f) 50GAACAGGTTGAACA
GTCTACCCT (position 371–393) and P18 (r) 50TTCC
For the 18S rRNA, P19 (f) 50GATGCGTGCATTTAT
CAGATC (position 309–329) and P20 (r) 50AGTGGACT
CATTCCAATTACA (position 652–672). For GAPDH,
P21 (f) 50ACTCTGGTAAAGTGGATATTGT (position
131–152) and P22 (r) 50ATGATGTTCTGGAGAGCCC
50AAGAGAGGCATCCTCACCCTG (position 181–202);
P24 (r) 50GGCGACGTAGCACAGCTTCTCC (position
639–660). (f) and (r) represent forward and reverse
primers, respectively. The sequence of the primers for
Nucleic Acids Research, 2007, Vol. 35, No. 217337
the 16S, 12S and COX 1 transcripts were deduced from the
human mtDNA (GenBank Accession No. V00662).
The sequence of the primers for the 18S rRNA, GAPDH
mRNA and b-actin mRNA were deduced from the
NM_001101, respectively. Amplified DNA fragments
were purified (Wizard SU Gel and PCR Clean-up system;
Promega), cloned in pGEM?-T Easy (Promega) or pTOPO
(Invitrogen) and the purified recombinant plasmids were
sequenced as described before (12,13).
About 1mg of total RNA from human cells treated with
TURBO DNA-free in 50ml of 2? SSC (28) was incubated
with RNase A at a final concentration of 50mg/ml for
15min at 258C. The digestion products were extracted
with phenol–chloroform, and after adding 10mg of
glycogen to the aqueous phase, the RNA was precipitated
with ethanol and recovered by centrifugation (12,13).
S1 protection assay was carried out using a single-
stranded digoxigenin-labeled DNA probe complementary
to the region between the IR and the 16S mtrRNA of the
ncmtRNA. First, an amplicon was obtained by RT-PCR
using primers 1 and 2 and this fragment was used as a
template in an asymmetric PCR reaction that contained
only primer 1 and digoxigenin-11-dUTP (Roche Non-
Radioactive ISH). S1 protection was carried out as
described (27), using 50mg RNA and 50ng digoxigenin-
labeled probe. After S1 digestion, reactions were ethanol-
precipitated and analyzed on native 2.5% agarose gels,
which were transferred onto HybondTM-XL membranes
(Amersham). After blocking with 0.02% Tween 20 plus
5% skim milk in PBS, the position of the digoxigenin-
labeled probe was revealed with anti-digoxigenin antibody
conjugated with alkaline phosphatase (Roche) and devel-
oping in BCIP/NBT alkaline phosphatase substrate
mixture (DAKO) for 10min.
HeLa cells RNA dissolved in 2?SSC was digested with
RNase A and the double-stranded structure was recovered
after phenol extraction as described before. The cDNA
was synthesized with primer P8 (Figure 2a) and tailed at
the 30end with dCTP using terminal deoxynucleotidyl-
transferase (TdT; Promega) (29). Amplification by PCR
was carried out with the specific primer 8 (Figure 2a) and
the anchor primer provided by the manufacturer. The
amplicon obtained of ?250bp was purified and both
strands were sequenced directly.
Total RNA of HeLa and MCF/7 cells (3mg each) were
electrophoresed at 75V for 90min in a native 1.0%
agarose gel prepared in TAE buffer or in an agarose gels
under denaturing conditions containing 2.2M formalde-
hyde (27). The RNAs were transferred in 20?SSC to a
nylon membrane (HybondTM-XL; Amersham) for 18h,
and exposed to UV light for 5min. The probe used was
primer 13 (Figure 2a) labeled with32P. Briefly, in a final
volume of 20ml, 100ng of the oligonucleotide were mixed
with 4ml of enzyme buffer (100mM cacodylate buffer pH
6.8, 1mM CoCl2and 0.1mM DTT), 2ml of [a-32P]-dCTP
(3000Ci/mmol) and 10U of TdT, and incubated at 378C
for 30min. The probe was purified using a Sephadex G-50
spin column (Amersham). Hybridization was carried out
for 18h at 378C with 5ml of 0.5M sodium phosphate, pH
7.1, 2mM EDTA, 7% SDS and 0.1% sodium pyropho-
sphate (30) containing32P-labeled primer 13at a ratio of
105c.p.m./cm2of membrane surface area. The blots were
washed twice for 5min at room temperature with 2?SSC
plus 0.1% SDS and once with 0.5? SSC plus 0.1% SDS.
Radioactivity on the membranes was visualized with a
phosphor imager (MolecularTMImager FX Phosphor
Imaging System, BioRad).
About 1mg of RNA digested with RNAse A as
described before, was subjected to electrophoresis on a
1.5% agarose gel containing 2.2M formaldehyde and
transferred to a nylon membrane (27). The membrane was
probed with an amplicon of ?250bp corresponding to the
double-stranded region of the ncmtRNA and obtained by
RT-PCR using primers 8 and 5 (Figure 2a) and
[a-32P]-dCTP. The probe was extracted with phenol and
precipitated with isopropanol. Hybridization was carried
out with a solution containing 8 million c.p.m./ml in
4?SSC, 10% dextran sulfate, 150mg/ml yeast tRNA,
150mg/ml herring sperm DNA, 50% formamide and
1?Denhardt’s solution (27). After hybridization at 658C
overnight, the membrane was washed for 10min at room
temperature with 2?SSC and 1?SSC, 20min at 558C
with 0.5?SSC and 20min at room temperature with
0.2?SSC. Then the membrane was exposed to X-ray films
at ?708C (12,27).
HeLa cells were grown as indicated and about 5?108cells
were harvested and recovered by centrifugation at 300g
for 5min at 48C. The cells were resuspended in about 10
volumes of a hypotonic solution containing 10mM KCl,
0.15mM MgCl2and 10mM Tris–HCl, pH 6.8, incubated
for 10min on ice and dounce homogenized with the tight
pestle (13,31). The homogenization was monitored by
phase microscopy until ?70% of the cells were broken and
the mitochondrial fraction was obtained as described
before (31). The final mitochondria fraction was resus-
pended in 2–3ml of 0.25M sucrose, 2mM MgCl2and
0.4mM sodium phosphate buffer at pH 6.8 and treated
with RNAse A at a final concentration of 50mg/ml for
15min at room temperature (32). The mitochondria
fraction was recovered by centrifugation at 10000g for
15min, suspended in 100ml of PBS containing 100 U of
RNaseOut (Invitrogen) and mitochondrial RNA was
extracted with TRIzol as described before.
Cells cultured in 8-well chamber slides (Lab-Tek?,
NUNC) for 24–48h, were washed three times with PBS
and fixed in 4% paraformaldehyde in PBS for 10min at
Nucleic Acids Research, 2007, Vol. 35, No. 21
room temperature. The slides were then washed three
times with PBS for 5min and incubated with 0.2N HCl
for 10min at room temperature. Hybridization was
carried out essentially as described before (12,14). The
cells were hybridized for 18h at 378C with 200ml of the
hybridization solution containing 3.5pmol of the anti-
sense probe (primer 6 or 13) or the corresponding sense
probes (Figure 2a), previously labeled at the 30end with
digoxigenin-11-dUTP (Roche Applied Science) (12,14).
The slides were washed with 2?SSC and 1?SSC for
10min at room temperature, 0.2? SSC for 30min at 378C
and finally, with 0.2? SSC for 10min at room temperature
(14). Then the cells were incubated for 2h at room
temperature with anti-digoxigenin antibody conjugated to
alkaline phosphatase (Roche Applied Science), diluted
1:500 in the blocking buffer (1% BSA, 0.3% Triton X-100
in PBS). The color reaction was carried out with a BCIP/
NBT substrate mixture (DAKO) plus levamisol for 30min
(14). Resting or PHA-stimulated human lymphocytes
were applied on silanized slides, air dried, fixed with 4%
paraformaldehyde and subjected to the same hybridiza-
tion protocol. For FISH, after hybridization the cells were
incubated with anti-digoxigenin antibody conjugated to
FITC (Roche Applied Science). For tissue samples,
paraffin sections of ?5mm thick were collected in silanized
slides and deparaffinized by immersion in three consecu-
tive xylene baths, 10min each. The sections were
rehydrated with three washes in 100%, 90%, 70% and
50% ethanol, once in PBS and once in distilled water for
10min. Afterwards the sections were fixed with 4%
paraformaldehyde in PBS and incubated with 0.2N HCl
plus 4mg/ml of pepsin for 10min at room temperature,
and subjected to ISH as described before.
Immunocytochemistry and FISH
After ISH, melanoma cells (42/95) were incubated for
30min in blocking buffer (1% BSA, 0.3% Triton X-100 in
PBS), and then incubated for 2h at room temperature
with anti-digoxigenin antibody conjugated with fluores-
cein (Roche) and with anti-PCNA monoclonal antibody
(DAKO). The cells were washed three times with 0.05%
Tween20 in PBS, incubated for 2h at room temperature
with anti-mouse IgG conjugated with rhodamin, washed
again and mounted with DABCO. After hybridization,
HeLa cells were incubated with anti-digoxigenin antibody
conjugated with rhodamin (Roche) and with anti-cyto-
chrome c monoclonal antibody (Promega) or anti-
International). After washing with 0.05% Tween20 in
PBS, the cells were incubated with either anti-mouse IgG
conjugated with fluorescein or with anti-rabbit IgG con-
jugated with fluorescein. Fluorescence microscopy was
analyzed with a Olympus BX51 microscope. Confocal
microscopy was performed with a LSM 5 Zeiss micro-
scope equipped with a 63? objective. The analysis was
carried out with the Zeiss LSM 5 Image Browser software.
Fixed resting or PHA-stimulated lymphocytes were
incubated with the blocking solution and then with
anti-PCNA (DAKO) or anti-Ki67 antigen (DAKO) or
room temperature. After washing in PBS, the sections
were incubated for 30min with anti-mouse IgG con-
jugated to alkaline phosphatase (KPL) diluted 1:250 in
2% BSA in PBS. The color reaction was developed for
20min with BCIP/NBT substrate mixture (DAKO)
containing levamisol. Previous to immunocytochemistry,
deparaffinized tissue sections were incubated in coplin jars
for 15min at 958C in Target Retrieval Solution (DAKO)
diluted 1:10 with distilled water. The incubation with anti-
PCNA was as described before.
The human ncmt RNA
As shown in Figure 1a (panel 1), ISH with probe 6
targeted to the 16S mtrRNA (see Materials and Methods
section) revealed strong hybridization signals in HeLa,
HL-60, MCF/7, DU145, H441 and Caco-3 human cells.
As expected, hybridization with the control sense probe 10
(see Materials and Methods section) was negative
(Figure 1a, panel 2). Moreover, FISH with probe 6 in
combination with immunocytochemistry of PCNA of the
melanoma cell line 42/95 revealed that each cell expressing
the transcript was also expressing PCNA (Figure 1b). The
(Figure 1b). As a positive control, FISH was carried
with probe 20 (see Materials and Methods section) to
detect the 18S rRNA (Figure 1b).
The target of probe 6 might be a transcript homologous
to the mouse ncRNA (12). Therefore, to characterize this
transcript, a hypothetical human mitochondrial RNA was
deduced (Figure 2a). The transcript contains the complete
sequence of the 16S mtrRNA (Figure 2a, red line) linked at
its 50end to a fragment of the RNA transcribed from the
16S gene corresponding to the L-strand of the mtDNA or
antisense 16S mtrRNA (Figure 2a, blue line). Based on this
structure, primers were designed to amplify the putative
transcript by RT-PCR (Figure 2a). Primer 1 was posi-
tioned at the theoretical loop between positions 11 and 31
of the human sense 16S mtrRNA, while the forward
primer 2 was at the putative IR, corresponding to position
1326–1347 of the antisense 16S mtrRNA (Figure 2a).
RT-PCR carried out with RNA from HeLa, HL-60 and
MCF/7 cells yielded a single amplicon of ?210bp
(Figure 2c, odd lanes). No amplification was obtained
when reverse transcriptase was omitted from the reaction
(Figure 2c, even lanes). The sequence of the amplicon of
each cell line revealed that an IR of 184nt was linked to the
first 31nt at the 50end of the 16S mtrRNA (Supplementary
Figure S1a and GenBank Accession No. DQ386868).
To determine whether the IR was longer than 184nt, a
PCR-walking strategy was utilized. The cDNA from
HeLa cells was amplified between primer 1 and primers
3, 4, 5 and 6, targeted to a putative longer IR (Figure 2a).
Amplification fragments of ?500, 700 and 800bp were
obtained when primer 1 was used in combination with
primers 3, 4 and 5, respectively (Figure 2d, lanes 1, 3 and 5).
No amplification product was obtained with primers 1 and
6 (Figure 2d, lanes 7 and 8), suggesting that the 50of the
IR is positioned between primers 5 and 6 (see below).
10 wasagain negative
Nucleic Acids Research, 2007, Vol. 35, No. 217339
The sequence of the amplicon of 800bp revealed an IR of
769nt linked to the first 31nt of the 16S mtrRNA
(Supplementary Figure S1b and GenBank Access No.
DQ386868). The sequence of the region between the 16S
mtrRNA and the IR is identical to that found in the same
region of the amplicon of 215bp (compare Supplementary
Figures S1a and b), confirming that in both cases we were
amplifying the same transcript. The sequences of the 500
and 700bp fragments indicated that they were part of the
800bp amplicon. The same results and sequences were
obtained with RNA from HL-60 cells, PHA-stimulated
lymphocytes and MCF/7 cells (data not shown).
The stemof thencmtRNA is resistant toRNase digestion
The 769nt of the IR are fully complementary to the
16S mtrRNA. Therefore, the transcript should contain
18S r RNA
Figure 1. Expression of the hypothetical human ncmtRNA. (a) ISH of
the indicated cells with probe 6 (panels 1) or the control sense probe 10
(panels 2). Note cytoplasmic and perinuclear hybridization signals
(?100). (b) Melanoma cell line expressing the transcript are also
expressing PCNA. The cells were subjected to ISH with probe 6 or
merge figure confirmed the co-expression of both molecules. As a
positive control, the same cells were subjected to ISH for the 18S
rRNA and immunocytochemistry of PCNA (?63).
RT + −
RT + −
Primers1-3 1-4 1-5 1-6
RNase A −
1 2 3
RNase A − + − + − +
Figure 2. The human ncmtRNA. (a) Theoretical structure of the
human ncmtRNA. The 50end of the sense 16S mtrRNA (red line) is
linked to a fragment of the antisense 16S mtrRNA or IR (blue line)
forming a double-stranded structure and a loop of unknown length.
The position of the reverse primers (under the lines) and the forward
primers (over the lines) are indicated (see Materials and Methods
section). (b) Digestion of the loop and the single-stranded region of the
ncmtRNA by RNase A is also illustrated. (c) Amplification of the
cDNA obtained from three tumor cell lines using primers 1 and 2. An
amplicon of ?210bp was obtained only when the reaction was carried
out with reverse transcriptase (c, odd lanes). (d) Amplification of the
cDNA of HeLa cells by PCR using primers 1 in combination with
primers 3 (lanes 1 and 2), 4 (lanes 3 and 4), 5 (lanes 5 and 6) and 6
(lanes 7 and 8), respectively. Amplicons of ?500, 700 and 800bp were
obtained. No amplification products were generated with primer 1 and
6 (lanes 7 and 8) or without reverse transcriptase (d, even lanes). M,
100bp ladder. (e) RNA from HeLa cells in 2? SSC was incubated
without (odd lanes) or with 50mg/ml of RNase A for 15min at 258C
(even lanes). The RNA was recovered and amplified by RT-PCR using
primers 1 and 5 (lanes 1 and 2), primers 10 and 11 (lanes 3 and 4) or
with primers 7 and 5 (lanes 5 and 6). Amplicons of 800 and 350bp were
obtained with primer 1 and 5, and 10 and 11, respectively, only with
untreated RNA (lanes 1 and 3, respectively). Amplification of the
fragment of 750bp obtained with primers 7 and 5 was not affected by
the nuclease treatment (lanes 5 and 6). (f) Amplification of the 12S
mtrRNA (lanes 1 and 2), 18S rRNA (lanes 3 and 4) and GAPDH
mRNA (lanes 5 and 6) after digestion with RNase A (mock
experiment, odd lanes). (g) About 1mg of RNA from the indicated
cell lines was digested with RNase A and the digestion products were
resolved by electrophoresis on a 1.5% agarose gel. After blotting, the
membrane was probed with a32P-labeled PCR fragment targeted to the
double-stranded region of the ncmtRNA (see Materials Methods
section). A single hybridization band corresponding to a transcript of
?800nt was detected.
Nucleic Acids Research, 2007, Vol. 35, No. 21
a double-stranded structure or stem of at least 769bp
resistant to RNase A digestion as predicted in Figure 2b.
On the other hand, the loop and the single-stranded region
that extends beyond the hairpin structure should be
digested by the enzyme (Figure 2b, dotted lines). To test
this possibility, RNA from HeLa and HL-60 cells was
digested with RNase A as described in Materials and
Methods section. The cDNA from the non-digested or
digested RNA was then amplified by PCR using the
primers described in Figure 2a. The amplicon of 800bp
obtained with primers 1 and 5, was not amplified after
RNase digestion (Figure 2e, lanes 1 and 2), and the same
was true for the fragment of 360bp obtained with primers
10 and 11 (Figure 2e, lanes 3 and 4), indicating that the
single-stranded regions of the transcript were digested.
Similarly, the 12S mitochondrial rRNA, 18S rRNA and
GAPDH mRNA, were not amplified after digestion
(Figure 2f lanes 2, 4 and 6). On the other hand,
amplification of a fragment of 750bp, corresponding to
the double-stranded structure of the ncmtRNA and
obtained using primers 7 and 5 (Figure 2a), was not
affected by RNase A digestion (Figure 2e, lanes 5 and 6).
The sequence of this fragment was identical to that of the
amplicon of 800bp obtained with primers 1 and 5, except
for the absence of the first 31nt of the 16S mtrRNA.
Northern blot also confirmed the presence of the stem
after RNase A digestion. A single hybridization band of
?800nt was detected in digested RNA of HeLa, HL-60
and MCF/7 cells (Figure 2g).
To determine the 50end of the transcript using 50
RACE, HeLa cell RNA was digested with RNase A and
the nuclease-resistant stem was isolated as described
before, and used as template for 50RACE. The sequence
of the amplicon of 250bp obtained by PCR with primer 8
and the anchor primer (see Materials and Methods
section) revealed that the 769nt of the IR were extended
by 46 additional nt (Supplementary Figure S1b, under-
lined sequence), indicating that the total length of the IR
The 16SmtrRNA is contiguous withthe IR
Similarly to the 12S and 16S mtrRNAs (33), the
ncmtRNA is also polyadenylated. Total RNA from
HeLa, HL-60 and MCF/7 cells was separated into
polyA+ and polyA? fractions (see Materials and
Methods section). The cDNA obtained from both
fractions was then amplified with primers 1 and 2
(Figure 2a). As shown in Figure 3a, amplification of the
215bp fragment revealed that the transcript was enriched
in the polyA+ fraction (lanes 2, 6 and 10), compared to
the polyA? fraction (Figure 3a, lanes 4, 8 and 12). The
cDNA was synthesized from total RNA from HeLa and
MCF/7 cells using either oligo dT or primer 12, which is
complementary to the 30end of the 16S mtrRNA
(Figure 2a). Then, each cDNA was amplified by PCR
between primer 1 positioned at the loop and primer 3
positioned on the IR. In both cases, the expected amplicon
of 500bp was obtained (Figure 3b, lanes 2, 4, 6 and 8),
indicating that the cDNA primed with oligo dT or with
primer 12 comprised the complete 16S mtrRNA plus
the IR. No amplification product was obtained without
reverse transcriptase (Figure 3b, odd lanes).
If the IR of 815nt is linked to the 1559nt of the 16S
mtrRNA, one would expect a transcript of 2374nt. Total
RNA from HeLa and MCF/7 cells was subjected to
polyA + + − − + + − − + + − −
M 1 2 3 45 611 1210 987
OligodT + + −
Primer 1535 −
+ + −
− + + −
+ − +
Figure 3. The IR is contiguous with the 16S mtrRNA. (a) Total RNA
from HeLa (lanes 1 to 4), HL-60 (lanes 5 to 8) and MCF/7 (lanes 9 to
12) cells were separated into polyA+ and polyA? fractions. A total of
100ng of each fraction was used to synthesize cDNA, which was then
amplified by PCR using primers 1 and 2, to generate the 215bp
amplicon as indicated. Odd lanes correspond to reactions carried out in
the absence of RT. (b) cDNA was synthesized from the polyA+
fraction of HeLa or MCF/7 cells, using oligo dT (lanes 1, 2, 5 and 6) or
primer 12 (lanes 3, 4, 7 and 8). A single amplicon of 500bp was
obtained after PCR amplification of the cDNAs with primers 1 and 3
only when reverse transcriptase was included in the reaction mixture
(even lanes). (c) About 3mg of HeLa and MCF/7 cells RNA was
resolved by electrophoresis on a 1% native agarose gel and subjected to
northern blot. The membrane was probed with32P-labeled primer 13
(Figure 2a). The probe hybridized with a single transcript, which
migrated below the 1353bp DNA marker (M=?DNA/HindIII and
fDNA/HaeIII). The size of this transcript, deduced from the dsDNA
ladder, corresponds to 2280nt. (d) Northern blot was carried out with
RNA from the indicated cells, under denaturing electrophoretic
conditions. In this case, probe 13 hybridized with a single band that
migrated on top of the 18S rRNA and corresponding to a transcript of
2200nt. (e) For S1 protection assay, an asymmetric PCR fragment 215
nts containing the 31nt of the sense 16S mtrRNA plus 184nt of the IR
was synthesized and labeled with digoxigenin (see Materials and
Methods section). After denaturation at 1008C for 5min, the probe was
incubated overnight at 508C either alone (lane 2), with 20mg of HeLa
RNA (lane 3) or with 20mg of yeast RNA (lane 4). After hybridization,
the products were digested with S1 nuclease and the products resolved
by 2.5% native agarose gel electrophoresis and blotted to a nylon
membrane. The products of digestions were reveled with anti-
Materials and Methods section). Lane 1 represents the probe alone
Nucleic Acids Research, 2007, Vol. 35, No. 217341
northern blot under native electrophoretic conditions
using as a probe oligonucleotide 13 labeled with
Probe 13 contains 9nt complementary to the 50end of the
16S mtrRNA followed by 9nt complementary to the 30
end of the IR (Figure 2a), and therefore is specific for the
ncmtRNA. A single hybridization band that migrates just
below the 1353bp DNA marker was observed (Figure 3c).
It is pertinent to mention that dsDNA and dsRNA have
the same behavior on native electrophoretic conditions
(34). The size of this component corresponds to a
transcript of ?2280nt, close to the expected size of the
ncmtRNA. Northern blot with probe 13 under denaturing
electrophoretic conditions revealed a single band on top of
the 18S rRNA and corresponding to a transcript of
2200nt (Figure 3d).
S1 protection assay also confirmed that the IR was
contiguous with the 16S mtrRNA. An asymmetric PCR
product labeled with digoxigenin (see Materials and
Methods section) and complementary to a region of the
transcript comprising the first 31nt of the 16S mtrRNA
linked to 184nt of the IR (Supplementary Figure S1a),
was protected from S1 digestion after hybridization with
HeLa cell RNA (Figure 3e, lane 3) but not when
hybridization was carried out without RNA or with
yeast RNA (Figure 3e, lanes 2 and 4, respectively).
Synthesis of thencmtRNA requires mitochondrial
To determine whether the ncmtRNA was present in
mitochondria, the organelles were isolated from HeLa
cells and treated with RNase A to eliminate cytoplasmic
RNA contamination (32). RNA was extracted from the
isolated mitochondria and then amplified by RT-PCR
using primer 1 in combination with primers 4 or 5. The
expected amplicons of 700 and 800bp, respectively, were
obtained (Figure 4a, lanes 1 and 3), and their sequences
were identical to those described before (Supplementary
Figure S1b). No amplification was obtained with primers
1 and 6 (Figure 4a, lane 5), similarly to the results
described before (Figure 2d). To ascertain that RNase
treatment eliminated cytoplasmic RNA contamination,
total RNA was extracted from two different preparations
of HeLa cell mitochondria treated with the nuclease or
untreated. As shown in Figure 4b, treatment with
RNase did not affect the amplification of the amplicon
corresponding to the ncmtRNA (215bp) or to the mRNA
of COX I (390bp). In contrast, amplification of the
18S rRNA and b-actin mRNA was abolished after
nuclease-treatment (Figure 4b, lanes 2 and 4). To confirm
the presence of thencmtRNA
co-localization studies were carried out. HeLa cells were
subjected to FISH with digoxigenin-labeled probe 6 and
anti-digoxigenin antibody conjugated to rhodamin to
detect the ncmtRNA, and to immunocytochemistry with
fluorescein-labeled anti-mouse antibody to detect the
mitochondrial marker cytochrome c or with fluorescein-
labeled anti-rabbit antibody to detect endonuclease
G (35). As shown in Figure 4c, confocal microscopy
revealed co-localization of the transcript with both
To determine whether mitochondrial transcription is
required forthe synthesis ofthis transcript, HeLa cellswere
cultured with EtBr (24,36,37). Total RNA from HeLa cells
cultured with or without EtBr for 28 days was RT-PCR-
amplified using primers 1 and 2 or 9 and 6. The expected
amplicons of 215 and 430bp, respectively, were obtained
cells (Figure 5a, lanes 3 and 4, and 5 to 6, respectively).
RT + − + − + −
215 bp ncmtRNA
390 bp COX I
360bp 18S rRNA
480 bp b-actin
Rnase A −
Figure 4. Mitochondrial localization of the ncmtRNA. (a) The
mitochondrial fraction from HeLa cells was treated with RNase A,
previously to RNA extraction. Amplification of the mitochondrial
RNA with primer 1 in combination with primer 4 (lanes 1 and 2) or
primer 5 (lanes 3 and 4) yielded the expected amplicons of 700 and
800bp, respectively. No amplification was obtained with primers 1 and
6 (lanes 5 and 6) or when the reaction was carried out without reverse
transcriptase (RT?, lanes 2, 4 and 6). (b) Total RNA extracted from
two different preparations of HeLa mitochondria (lanes 1and 2, and 3
and 4) treated without (lanes 1 and 3) or with RNase A (lanes 2 and 4)
was used to amplify the 215bp fragment of the ncmtRNA, and the
indicated amplicons of COX I mRNA, 18S rRNA and b-actin mRNA.
Note that RNase A treatment abolished contamination with cytoplas-
mic transcripts. (c) Co-localization of the ncmtRNA with the
mitochondrial markers cytochrome c and endonuclease G. HeLa cells
were subjected to FISH to detect the ncmtRNA and immunocyto-
chemistry to detect cytochrome c or endonuclease G (see Methods
section) and analyzed by confocal microscopy.
Nucleic Acids Research, 2007, Vol. 35, No. 21
This treatment also inhibited the expression of other
mitochondrial transcripts (data not shown). In contrast,
EtBr did not affect the nuclear expression of the 18S
rRNA (Figure 5a, lanes 1 and 2).
The presence of the long IR in the ncmtRNA might be
explained if some anomalous molecules of mtDNA
contains the IR of 815bp inserted between the genes of
Transcription of this mtDNA followed by processing of
the H-strand polycistronic RNA would then generate the
ncmtRNA. However, PCR amplification of the human
mtDNA from five different cell types between primer 16
(positioned at the 30end of the 12S mtrRNA, see
(Figure 2a) yielded only the expected amplicon of 128bp
(Figure 5c), corresponding to the normal sequence of the
The ncmtRNA is expressedin proliferating cells
As shown in Figure 1, seven different human cell
lines over-expressed a transcript that was complementary
to probe 6. However, this probe does not distinguish
between the 16S mtrRNA and the ncmtRNA. On the
other hand, we demonstrated that probe 13 (Figure 2a),
targeted to the region between the IR and the 16S
mtrRNA, was specific for the ncmtRNA. As described
before, probe 13 hybridized only with the ncmtRNA in
northern blots carried out under native or denaturing
conditions. Moreover, probe 13 supported the amplifica-
tion of the transcript between the target region and the IR.
This amplification was abolished after digestion with
RNase A (Supplementary Figure S2). Therefore, ISH with
probe 13 was carried out with HeLa, MCF/7, Du145 and
SiHa cells. The hybridization signals were the same as that
observed with probe 6, revealing strong cytoplasmic and
perinuclear staining (Figure 6a), and confirming that the
ncmtRNA is expressed in proliferating cells. Then we
asked whether this transcript is expressed in resting cells.
As shown in Figure 6b, no staining of human brain,
smooth muscle and liver cells was observed after ISH with
probe 13 or with probe 6 (data not shown). The absence of
proliferating cells in these tissues was confirmed by the
null expression of PCNA (Figure 6b).
Since human circulating lymphocytes, as resting cells,
do not express the ncmtRNA (Figure 7b, ?PHA), we
asked whether the expression of this transcript can be
induced if lymphocytes are stimulated to enter the S phase.
Peripheral human lymphocytes stimulated with PHA for
72h were actively engaged in DNA synthesis as measured
by BrdU incorporation (Figure 7a). As expected, the
stimulated cells were also expressing the proliferating
(Figure 7b, +PHA). At the same time, ISH with probe
Et B r
12 S RNA tRNAVal
Figure 5. Synthesis of the ncmtRNA requires mitochondrial transcrip-
tion. (a) Total RNA was extracted from HeLa cells incubated without
(odd lanes) or with (even lanes) 1mg/ml of ethidium bromide for 28
days. The RNA was amplified by RT-PCR using specific primers for
18S rRNA (lanes 1 and 2) (see Methods section), primers 9 and 11
(lanes 3 and 4) and primers 1 and 2 for the ncmtRNA (lanes 5 and 6).
(b) Theoretical structure of an anomalous mtDNA containing an insert
of 815bp between the tRNAval
(c) mtDNA from the indicated cell lines was amplified between
primer 16 positioned close to the 30end of the 12S gene and primer
1 positioned on the 16S mtrRNA gene as shown in (b). All samples
yielded a single amplicon of 128nt. (a) M, 100bp ladder. (c) 50bp
and the 16S mtrRNA genes.
Figure 6. Expression of the ncmtRNA in proliferating cells. (a) The
indicated cells were subjected to ISH with probe 13 (panels 1) or with
corresponding control sense probe (panels 2). Strong cytoplasmic and
perinuclear hybridization signals were found (?100). No hybridization
was found with the control sense probe. (b) ISH and immunocyto-
chemistry to determine the expression of the ncmtRNA and PCNA,
respectively, in brain, smooth muscle and liver cells. Each panel of
tissues was also stained with hematoxylin-eosin (?40).
Nucleic Acids Research, 2007, Vol. 35, No. 217343
13 revealed that the PHA-stimulated lymphocytes were
also over-expressing the ncmtRNA (Figure 7b, +PHA).
At low magnification, hybridization of PHA-stimulated
lymphocytes with probe 13 show a strong signal in most of
the cells (Figure 7c, ncmtRNA), while no staining was
observed with the corresponding control sense probe
(Figure 7c, Control sense). Similar results were obtained
with lymphocytes obtained from 11 different healthy
donors (data not shown).
Next we asked whether the arrest of the cell cycle at G1
with aphidicolin would affect the expression of the
ncmtRNA. As shown in Figure 7d, treatment of DU145
cells with the drug induces both, a block on cell
proliferation and a marked down regulation of this
transcript when compared with the untreated control
cells. When the DU145 cells are removed from the drug-
containing medium to normal culture medium for 2 days,
cell proliferation was re-initiated and the level of expres-
sion of the transcript is recovered and become similar to
the expression rate of the control cells (Figure 7d).
This is the first report on a novel non-coding mitochon-
drial RNA over-expressed in human proliferating cells but
not in resting cells. Based on the hypothetical structure
shown in Figure 2a, we established that the human
ncmtRNA contains an IR of 815nt covalently linked to
the first 865nt of the 16S mtrRNA. To confirm the
existence of the ncmtRNA additional experiments were
carried out. First, northern blot with probe 13 revealed
a transcript of 2280nt (native conditions) or 2200nt
(denaturing conditions), which are close to the expected
size of 2374nt of the ncmtRNA. Second, amplification of
the region comprising the loop and the IR of the
ncmtRNA was possible after using a cDNA primed with
oligo dT or with a primer targeted to the 30end of the
transcript. This experiment was similar to the strategy
used to establish that different regions of the long ncRNA
Tsix were contiguous (38). Third, the long double-
stranded structure of 815bp was isolated after RNase A
digestion, and its presence in the digestion products was
demonstrated by amplification between the 50and 30ends
of the stem or by northern blot analysis. Fourth, S1
protection assay demonstrated the presence in the
ncmtRNA of the sequence region between the IR and
the 16S mtrRNA. Altogether, these results support the
conclusion that the ncmtRNA contains an IR of 815nt
contiguous with the 16S mtrRNA.
Translation in silico of the 2374nt of the ncmtRNA
(GenBank Accession No. DQ386868) using either the
standard or the vertebrate mitochondrial codes, yielded
several open reading frames corresponding to peptides
ranging from 15 to 60 amino acids. These results agree
with the definition of ncRNAs, in which open reading
frames greater than 100 amino acids are rarely present
A search of the ncmtRNA was conducted in human
Blast.cgi). Although several mitochondrial cDNA entries
were present, including the 16S mtrRNA, the sequence of
the ncmtRNA or at least part of the sequence containing
the region between the IR and the 16S mtrRNA was not
found. We believe that the absence of this sequence is due
to both, the double-stranded structure of the transcript and
replication slippage of the polymerases commonly used to
construct EST libraries. Escherichia coli DNA polymerases
I, II and III as well as polymerases of phages T4 and T7,
havepoor strandseparation activityresultinginreplication
slippage of DNA templates carrying hairpin structures
(39,40). As a consequence, deletion mutations of hairpin
sequences have been observed in vitro and in vivo (41,42).
Thermophilic DNA polymerases, including Taq polymer-
ing hairpin structures (43), with fateful consequences for
amplification and DNA sequencing (44–46).
Synthesis ofthe ncmtRNA requires mtDNA transcription
Several results strongly suggest that the ncmtRNA is
synthesizedin mitochondria.Thesequenceof the
Figure 7. Reversibility of the ncmtRNA expression. (a) Human
lymphocytes were incubated without (white bars) or with PHA
(hatched bars) for 24, 48 and 72h. At each time period, the cells
were incubated with BrdU for 16h and the incorporation of the
nucleoside was determined by ELISA (see Materials and Methods
section). The incorporation of BrdU is expressed as the absorbance
at 450nm. (b) About 100000 lymphocytes incubated without (?PHA)
or with PHA (+PHA) for 72h were subjected to immunocytochemistry
to determine the expression of Ki-67, PCNA and phospho-histone
H3 (PO4-H3), and to ISH with probe 13 to determine the expression
of the ncmtRNA (?40). (c) PHA-stimulated lymphocytes for 72h were
hybridized with probe 13 (ncmtRNA) or with the corresponding
sense control probe (Control sense) (?20). (d) DU145 cells were
subjected to ISH together with a control culture. The blocked cells
were changed to normal medium for 48h and then subjected to ISH
(see Methodssection) and
Nucleic Acids Research, 2007, Vol. 35, No. 21
transcript exhibits a minimum of 99.9% identity with
several haplotypes of the human mtDNA (see http://
www.ncbi.nlm.nih.gov/gquery/). Moreover, we showed
that the transcript was present in isolated mitochondria
ncmtRNA co-localized with cytochrome c and endonu-
clease G, two specific markers of the organelle (35). On the
other hand, treatment of HeLa cells with EtBr abolished
the presence of the ncmtRNA as well as other mitochon-
drial transcripts, indicating that the synthesis of the
(24,36,37). These results also indicate that the ncmtRNA
is not a transcription product of a mitochondrial
pseudogene (47). Searching for the sequence of the
nih.gov/BLAST/Blast), yielded only fragments of the
16S mtrRNA (sense and antisense orientations) having
an identity of 82–94% with contigs corresponding to
several human chromosomes. However, the sequence of
the ncmtRNA was not found (data not shown). This result
is consistent with the results of amplification of the
transcript by RT-PCR. As shown here, we have never
observed amplification between the loop and the IR when
Since there is not a contiguous sequence in the mtDNA
encoding the ncmtRNA, it is reasonable to hypothesize
that the synthesis of this transcript involves post-
transcriptional reactions. Although other possibilities
cannot be discarded, the synthesis of the ncmtRNA
might involve either a trans splicing reaction or a RNA
ligase reaction as reported in other organisms (48–50).
Although the synthesis of the ncmtRNA warrants future
work, it is interesting to mention the similarity between
the structures of this transcript with the presence of
double-stranded RNAs in HeLa cell mitochondria. This is
a heterogeneous fraction sedimenting from 4S to 17S and
resistant to RNase A digestion (28,51). Unfortunately, no
reported, and perhaps one of these double-stranded
structures corresponds to the ncmtRNA.
Besides the ribosomal RNAs 16S and 12S, and the
tRNAs, the ncmtRNA is not the only ncRNA present in
mitochondria. Recently, a new family of small ncRNAs
was reported to be present in isolated mouse mitochon-
dria, consisting of six transcripts ranging in size between
23 and 68nt (52). The authors also reported the presence
of similar molecules in isolated chloroplast of Nicotiana
tabacum. Unfortunately, no information on the biological
function was provided (52).
of thesetranscripts was
Expression ofthe ncmtRNA andcell proliferation
The most striking results of the present work are the close
correlations between expression of the ncmtRNA and cell
proliferation. Besides the cell lines reported here, over-
expression of the ncmtRNA was observed in 16 additional
tumor and normal cell lines (data not shown). In contrast,
this transcript was not expressed in non-proliferating cells
the expression of this transcript can be induced in
PHA-stimulated lymphocytes together with DNA synth-
esis and the expression of the proliferation markers Ki-67,
PCNA and phosphohistone H3 (19–21). Moreover,
treatment of DU145 cells with aphidicolin confirmed the
previous results. After treatment with the drug, the cells
stop proliferating as described before (22,23). At the
same time, the expression of the ncmtRNA was down-
regulated. However, after removal of the drug the
expression of this transcript was re-established together
with cell proliferation. Altogether, these results strongly
suggest that the ncmtRNA is a new marker of cell
proliferation. However, the precise function that this
transcript may play in the cell cycle remains unclear and
warrants future studies.
Similarly to the present work, other ncRNAs have been
reported to be involved in cell proliferation. ncRNAs are
involved in different cellular and molecular events,
including differentiation and development, imprinting,
regulation of X chromosome silencing and human diseases
(17). Moreover, microRNA, an important group within
the expanding family of ncRNAs (53), are also involved in
cell proliferation and the regulation of the cell cycle.
In Drosophila melanogaster, germline stem cell division is
regulated by microRNAs. This conclusion was reached
after observing a reduction of the germline stem cell
proliferation in the dicer-1 mutant, the RNase III essential
microRNAs involved were not identified, the authors
proposed that the function of these molecules is to inhibit
Dacapo, which in turn negatively regulates the G1/S
transition (54). In human lymphoma, c-Myc up-regulates
the expression of six microRNAs encoded by the c13orf24
cluster of chromosome 13 (55). Two of these molecules,
microR-17-5p and microR-20a, negatively regulate the
translation of the transcription factor E2F1, which in turn
is also up-regulated by c-Myc. The authors propose that
the microRNAs play a fine-tuning role to regulate
proliferative signals (55). MicroRNAs also regulate cell
proliferation or differentiation. Thus, microRNA-133
enhances proliferation of the mouse myoblast C2C12
cells, while microRNA-1 promotes differentiation and
Supplementary Data are available at NAR Online.
We thank Dr Giuseppe Attardi, California Institute of
Technology, Pasadena, CA, for suggesting the treatment
of HeLa cells with ethidium bromide. We also thank
Dr Marc Shuman from UCSF for providing several
human cell lines and for his enthusiastic support. We
thank Dr Bernardita Mendez, Dr Pablo Valenzuela,
Dr Arturo Yudelevich and Dr Mario Rosemblatt for
their continuous support and enriched discussions. This
work was supported by Millennium Scientific Initiative N8
P04-071-F, Grant D04I1338, FONDEF, CONICYT,
Santiago, Chile, and Grants DID-32-03, DID-26-04 and
Nucleic Acids Research, 2007, Vol. 35, No. 217345
DID-57-04, Universidad Nacional Andre ´ s Bello. Funding
to pay the Open Access publication charges for this article
was provided by MIFAB P04-071-F.
Conflict of interest statement. None declared.
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