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The Journal of Neuroscience, September 15, 1999, 19(18):7711-7720
Neuronal Expression of Neural Nitric Oxide Synthase (nNOS)
Protein Is Suppressed by an Antisense RNA Transcribed from an NOS
Pseudogene
Sergei A.
Korneev,
Ji-Ho
Park, and
Michael
O'Shea
Sussex Centre for Neuroscience, School of Biological Sciences,
University of Sussex, Brighton, East Sussex, BN1 9QG, United
Kingdom
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ABSTRACT |
Here, we show that a nitric oxide synthase (NOS) pseudogene is
expressed in the CNS of the snail Lymnaea stagnalis. The
pseudo-NOS transcript includes a region of significant antisense
homology to a previously reported neuronal NOS (nNOS)-encoding mRNA.
This suggested that the pseudo-NOS transcript acts as a natural
antisense regulator of nNOS protein synthesis. In support of this, we
show that both the nNOS-encoding and the pseudo-NOS transcripts are coexpressed in giant identified neurons (the cerebral giant cells) in
the cerebral ganglion. Moreover, reverse transcription-PCR experiments
on RNA isolated from the CNS establish that stable RNA-RNA duplex
molecules do form between the two transcripts in vivo.
Using an in vitro translation assay, we further
demonstrate that the antisense region of the pseudogene transcript
prevents the translation of nNOS protein from the nNOS-encoding mRNA.
By analyzing NOS RNA and nNOS protein expression in two different identified neurons, we find that when both the nNOS-encoding and the
pseudo-NOS transcripts are present in the same neuron, nNOS enzyme
activity is substantially suppressed. Importantly, these results show
that a natural antisense mechanism can mediate the translational
control of nNOS expression in the Lymnaea CNS. Our findings also suggest that transcribed pseudogenes are not entirely without purpose and are a potential source of a new class of regulatory gene in the nervous system.
Key words:
nNOS; antisense; pseudogene; translational regulation; dsRNA; RNA interface
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INTRODUCTION |
Nitric oxide (NO) is now recognized
as an intercellular signaling molecule in the nervous system
(Garthwaite et al., 1988 ). It is generated from L-arginine
and molecular oxygen by the enzyme NO synthase (NOS), the neuronal
isoform of which (nNOS) is activated by calcium/calmodulin (Bredt and
Snyder, 1990). The neuronal expression of nNOS protein was first
confirmed immunocytochemically in the rat brain (Bredt et al., 1990),
and many subsequent studies show that NO is a neurotransmitter,
although perhaps an enigmatic one (for review, see Bredt and Snyder,
1992; Dawson and Snyder, 1994; Hölscher, 1997).
To understand at cellular, molecular, and behavioral levels precisely
how NO functions as a neurotransmitter, we have exploited the
advantages of the presence of identifiable neurons in the nervous
system of the mollusc Lymnaea stagnalis. Building on the finding that NO mediates the activation of feeding behavior in Lymnaea (Elphick et al., 1995), we have now identified a
number of nNOS-expressing neurons that are involved in feeding (Korneev et al., 1998; Park et al., 1998). These include two large and readily
identifiable neurons known as the B2 motoneuron located in the buccal
ganglion and the cerebral giant cell (CGC) of the cerebral ganglion.
The existence of large identifiable nNOS-expressing neurons, together
with our molecular characterization of NOS-encoding transcripts
(Korneev et al., 1998), now allows us to investigate the expression of
different NOS transcripts at the single-cell level. Our work has
revealed a neuron-specific expression pattern for an unusual RNA
molecule that cannot be translated into nNOS protein because it is
transcribed from a NOS-related pseudogene. We show that this pseudo-NOS
transcript has a crucial role in the regulation of nNOS protein
synthesis through a natural antisense mechanism.
Pseudogenes are common in all eukaryotic genomes and are defined as
inactive versions of currently functional genes (Lewin, 1990). They
have been rendered inactive by the accumulation of deleterious
mutations that prevent any or all of the stages of gene expression, and
they are regarded as evolutionary dead ends. The pseudogene we have
identified is a member of the NOS gene family and is actively
transcribed in the Lymnaea CNS. Importantly the pseudo-NOS
RNA contains a region that is antisense to the functional nNOS mRNA,
and it was this that suggested a role for the pseudotranscript in the
regulation of nNOS protein synthesis.
Here, we show that both pseudo-NOS and nNOS-encoding transcripts are
coexpressed in identified neurons, that stable RNA-RNA duplexes form
between them, and that the antisense region of the pseudo-NOS RNA
specifically suppresses the synthesis of the nNOS protein. This
represents a novel mechanism for regulating the expression of an
important neuronal signaling pathway in the brain. Also, our findings
suggest the NOS pseudogene is a member of a new class of regulatory
genes that are derived from the pool of "nonfunctional"
pseudogenes. Pseudogenes therefore are not all relics of evolution and
entirely without purpose as the classical picture of them might suggest.
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MATERIALS AND METHODS |
cDNA library construction. Approximately 3 µg of
poly(A+) RNA isolated from total
Lymnaea CNS RNA by means of Dynabeads
oligo-dT25 (Dynal, Great Neck, NY) were
used to construct a cDNA library according to the manufacturer's
protocol for SuperScript Choice System (Life Technologies,
Gaithersburg, MD).
Northern hybridization.
Poly(A+) RNA (3-5 µg) isolated
from the CNS using Oligotex mRNA Mini kit (Qiagen, Hilden, Germany) were resolved in 1% denaturing formaldehyde-containing agarose gel and
transferred onto NYTRAN-N membrane (Schleicher & Schuell, Keene, NH).
Two different 32P-labeled probes were
used, one corresponding to the 3' untranslated region of
Lymnaea nNOS cDNA. The other probe was generated by
asymmetric PCR and represents a single-stranded DNA complementary to
the antisense region of the pseudo-NOS RNA and cannot therefore
recognize functional nNOS mRNA. Hybridizations were performed at 45°C
in a buffer containing 10% dextran sulfate, 5× SSPE, 5× Denhardt's
solution, 50% formamide, 0.5% SDS, and 100 µg/ml denatured and
sheared salmon sperm DNA.
Ribonuclease protection assay in vitro. Approximately 1 µg
of linearized plasmid DNA containing functional nNOS cDNA was used in
in vitro transcription reaction in the presence of
digoxigenin (DIG)-UTP and T7 RNA polymerase (Boehringer Mannheim,
Indianapolis, IN). A mixture of the DIG-labeled nNOS mRNA (200 ng) and
a synthetic pseudo-NOS RNA (2 µg) was incubated for 24 hr at 50°C
in a buffer containing 20 mM HEPES, pH 7.0, 100 mM KCl, and 1 mM EDTA and then treated with ribonuclease A (RNase A) under standard conditions (Shayiq, 1997). The products of the digestion were resolved in an
agarose gel and blotted onto a positively charged nylon membrane. This
was then subjected to an immunological detection procedure according to
the manufacturer's protocol. Crucially, a single band of ~150
nucleotides (nt) corresponding to the expected size of the duplex was
revealed, and no signal was detected in a control lane in which a
sample containing RNase A-digested nNOS mRNA without the pseudo-NOS RNA
was analyzed.
Detection of RNA duplexes in vivo. Cytoplasmic RNA was
isolated from Lymnaea CNS under nondenaturing conditions.
Briefly, the CNS was homogenized in a buffer containing 10 mM Tris HCl, pH 7.4, 3 mM
CaCl2, 2 mM
MgCl2, and 0.5% Nonidet P-40. The nuclei were
then removed by centrifugation at 500 × g and 4°C
for 10 min. The supernatant was treated with proteinase K and then
extracted with phenol-chloroform. After precipitation with isopropanol, the RNA preparation was divided into two fractions; one was treated with an excess of RNase A and RQ1 DNase (Promega, Madison, WI), and the
other was treated with RQ1 DNase (Shayiq, 1997). The two fractions of RNA were then reverse transcribed using
SuperScript II (Life Technologies) and either P1
(5'-GCATGTTGAGATGGAAGAAC-3') or P2 (5'AAAGACTGGTTTGAAAATCTC-3') primers
and amplified by means of Taq Supreme DNA polymerase (IGi)
in the presence of P3 primer (5'-CAGAGCTGTGGAGTTCTC-3') and either P1
or P2. Products of the PCR were then resolved in 3% MetaPhor agarose
gel (FMC Bioproducts, Rockland, ME), cloned, and sequenced.
Reverse transcription-PCR on isolated identified neurons.
The cell bodies of ~20 CGCs and 20 B2 neurons were identified and then individually dissected from the CNS. Total RNA was extracted from
each pool of neurons using the guanidine thiocyanate method (Chomczynski and Sacchi, 1987) and used as a template in a reverse transcription reaction in the presence of random primers
and SuperScript II (Life Technologies). The cDNA generated
was then subjected to 35 cycles of PCR amplification using the
following parameters: denaturation, 94°C, 20 sec; annealing, 55°C,
30 sec; extension, 68°C, 90 sec. For detection of functional NOS
mRNA, the PCR primers were as follows: #5, 5'-TGTGATCCTCACCGCTACAA-3';
and #8, 5'-GACTGTTGAGATGGAAGAAC-3'. For the detection of pseudo-NOS
RNA, the primers were: #1, 5'-ATCTTCCTGTCTCCGAGGC-3'; and #4,
5'-TGTGGAAATGTGTTGCCCTT-3'. Nested PCRs were then performed under the
same cycling parameters. The primers used for the nested PCR were as
follows: #6, 5'-GCTCAACACCGAAACTGCGT-3'; and #7,
5'-GAAGGTACTAGTGATTACCA-3' for detection of functional NOS mRNA. For
detection of pseudo-NOS RNA, we used the following: #3,
5'-GCTAGTAGCCCAAGTCTCTT-3'; and #2, 5'-CACTATGGCATCTAAATGTTAAG-3'. For
location of the primers, see Figure
1B.
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Figure 1.
Molecular cloning of the pseudo-NOS transcript.
A, Sequence of a full-length cDNA clone isolated from a
Lymnaea CNS cDNA library. The antisense region from 93 to 238 bp is indicated in bold type. The core region of
high homology (>80%) to the nNOS-encoding transcript is
shaded. A polyadenylation signal is
underlined. Stop codons within the core region are
marked by circles: white, frame 1;
shaded, frame 2; and black, frame 3. B, Schematic representation of the pseudo-NOS and
nNOS-encoding transcripts. The antisense region in the pseudo-NOS
transcript and its complementary counterpart are shown by
black and hatched boxes, respectively.
Regions of high homology are shaded, and the unfilled
areas have no significant homology to one another. The positions of the
numbered primers used in RT-PCR experiments on isolated identified
neurons are shown by arrows.
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Hybrid arrest of translation. Lymnaea NOS cDNA cloned into
pcDNA 3.1 was used as a template for an in vitro
transcription reaction to produce NOS cRNA, which was then capped
according to the manufacturer's protocol (Boehringer Mannheim).
Similarly, cRNAs corresponding to the antisense region of the
pseudo-NOS transcript were synthesized in either the sense or the
antisense orientation with respect to the functional NOS cRNA, but they were not capped. Two hybridization reactions were performed: the first
contained 0.5 µg of NOS cRNA and 0.2 µg of either sense or
antisense pseudo-NOS cRNAs, whereas in the second, these values were
0.2 and 2 µg, respectively. Both mixtures were then incubated for 1 hr at 50°C in a buffer containing 20 mM HEPES,
pH 7, 100 mM KCl, and 1 mM
EDTA. The products of the hybridization were then translated in
vitro using a wheat germ cell-free translational system (Promega)
in the presence of 35S-methionine
following the procedures of Schulz-Aellen et al. (1989). Labeled
proteins were resolved using SDS-PAGE.
NADPH-diaphorase histochemistry. A previously described
modified diaphorase method (Park et al., 1998) was used to reveal nNOS
activity in fixed whole-mount preparations of the snail buccal ganglion
(n = ~100). Thirty cerebral ganglia were processed in exactly the same way to compare diaphorase staining in the CGCs with
that in the B2 neurons.
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RESULTS |
Paradoxical expression of nNOS mRNA
Here, we report that a functional nNOS mRNA is expressed by two
uniquely identified neurons in the Lymnaea CNS, the B2
motoneuron, and the CGC (see below). The expression of NOS protein by
the B2 motoneuron was first reported by Moroz et al., (1994a,b), who showed that they are stained by the NOS-selective NADPH-diaphorase histochemical method (Matsumoto et al., 1993) and immunolabeled by an
antibody to the neuronal isoform of NOS. The NADPH-diaphorase method
responds to NOS enzyme activity in aldehyde-fixed cells and is an
established indicator of the NOS enzyme in the mammalian nervous system
(Dawson et al., 1991; Hope et al., 1991). We have verified that this
technique also works reliably in invertebrate preparations (Elphick et
al., 1995), and to confirm the findings of Moroz et al.
(1994a,b), we have used the NADPH-diaphorase technique on whole-mount
preparations on the entire Lymnaea CNS (Park et al., 1998).
Below, we show that, in 100% of the preparations examined, the cell
bodies of the paired B2 motoneurons in the buccal ganglion are strongly
NADPH-diaphorase-positive, as expected. Paradoxically, the CGC is
almost always NADPH-diaphorase-negative. This result is puzzling
because it shows that the nNOS transcript can be present without the
nNOS enzyme, suggesting translational control operates in the CGC to
regulate nNOS protein expression. Results described below provide an
unprecedented natural antisense mechanism involving a pseudogene that
accounts for this paradoxical neuronal expression pattern.
An antisense-containing NOS pseudogene is transcribed in
the CNS
We recently reported the cloning and expression of a full-length
(5070 nt) mRNA encoding the first molluscan NOS (Korneev et al., 1998),
the enzyme responsible for the calcium-regulated synthesis of the
gaseous neurotransmitter NO (Bredt and Snyder 1992; Garthwaite and
Boulton 1995). While screening a snail CNS cDNA library for other
NOS-related transcripts, we isolated a smaller 2345 nt transcript, the
sequence of which is shown in Figure 1 (GenBank accession number AF
165914). This small transcript shows >80% sequence identity over at
least half its length with the larger nNOS transcript and would appear
to be derived from another member of the same gene family. Although the
transcript possesses features of a functional mRNA, such as the
polyadenylation signal and a poly(A) tail, it cannot be translated into
protein because of the presence of multiple stop codons in all
three reading frames. This suggested to us that we had cloned a
transcript from a NOS pseudogene, a conclusion confirmed by performing
long-distance PCR on genomic DNA. The genomic sequence (data not shown)
contains the exact sequence of the small transcript and includes
introns, indicating that an unprocessed NOS pseudogene is actively
transcribed in the snail CNS.
An unexpected feature of the pseudo-NOS transcript is the presence of a
region ~150 nt in length that is antisense to a region close to the
middle of the functional nNOS mRNA (Fig.
1A,B). This means that, in addition
to being a pseudo-NOS RNA, the small transcript is also an example of a
trans-encoded natural antisense RNA (Vanhe-Brossolleet and
Vaquero, 1998). In Figure 2, we show an
alignment of the antisense region in the pseudo-NOS transcript with the
complimentary region of the functional nNOS mRNA. Note that the
complementarity between the two RNA strands reaches 80% over a
distance of ~150 nt, and this should enable a stable duplex to form
between the two transcripts in vivo.
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Figure 2.
Alignment of the antisense region (93-238 nt) of
the pseudo-NOS transcript with its complementary counterpart in the
nNOS mRNA. In this alignment, there is ~80% complementarity. The
non-Watson-Crick G-U base pairs that are common in RNA secondary
structure are shown by dots. The positions of three
primers used in the identification of RNA-RNA duplexes are
underlined and named. Primers in positions expected to
be protected from ribonuclease A (within the proposed duplex) are P2
and P3. The primer located outside of the protected area is called P1.
Further explanation of the ribonuclease A protection experiment is
provided in Figure 5. Details of procedures are in Materials and
Methods.
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Natural antisense RNAs have been proposed to mediate the regulation of
gene expression in eukaryotes (Wightman et al., 1993; Shayiq, 1997),
and so it was of considerable interest to determine whether our
pseudo-NOS RNA regulates nNOS expression. To address this question, we
first showed that a transcript corresponding to the cloned pseudo-NOS
cDNA is actually expressed in the CNS. This was achieved by Northern
blot hybridization using a probe specific to the antisense region of
the pseudo-NOS transcript. Results of this experiment are shown in
Figure 3 in which the expression of
transcripts containing the antisense region is compared with the
expression of the larger 5070 nt functional nNOS mRNA. Importantly, the
RNA revealed by the probe complementary to the antisense region of
pseudo-NOS is of the expected size (2345 nt), indicating that the
pseudo-NOS transcript we cloned is full-length and is indeed expressed
in the CNS.
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Figure 3.
The pseudo-NOS and nNOS-encoding transcripts are
expressed in the Lymnaea CNS. Northern blot analysis of
Lymnaea CNS poly(A+) RNA using a
probe specifically recognizing the antisense region of the pseudo-NOS
transcript identifies a prominent band of the expected size (~2500
nt) in lane A (arrow). A less prominent
transcript of ~3200 nt is also revealed in the experiment. This
suggests that there are other RNA molecules in the CNS that are
antisense to the nNOS-encoding transcript. In lane B,
the result of the hybridization with a probe recognizing the 3'
untranslated end of nNOS mRNA is shown. As expected, a single
transcript of ~5000 nt is revealed (arrow).
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Neuron-specific coexpression of nNOS mRNA and pseudo-NOS RNA
The extensive complementarity between the antisense region of the
pseudo-NOS RNA and a corresponding region of the nNOS-encoding mRNA
(Fig. 2) suggests strongly that a stable RNA-RNA duplex will form in
neurons that contain both transcripts. But do neurons that coexpress
pseudo-NOS and functional nNOS transcripts actually exist in the CNS of
the snail? To answer this question, we have exploited the advantage of
the existence in the snail CNS of two large identifiable
nNOS-expressing neurons. These are the paired B2 motoneuron of the
buccal ganglion (Park et al., 1998) and the paired CGCs of the cerebral
ganglion (Korneev et al., 1998). In the present study, RNA was isolated
from up to 20 individually dissected cell bodies of the CGC and B2
neurons, and reverse transcription (RT)-PCR experiments designed to
identify pseudo-NOS and functional nNOS transcripts were performed (see
Materials and Methods). Using this approach, both transcripts were
detected in the CGC but not in the B2 neuron in which only the
functional nNOS transcript is present (Fig.
4). The identity of all PCR products was
confirmed by cloning and sequencing.
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Figure 4.
A uniquely identified neuron (the CGC)
coexpresses functional NOS mRNA and pseudo-NOS RNA. In
A, the results of RT-PCR experiments on RNA purified
from isolated identified CGCs are illustrated. Lane 4
shows that a PCR product of the expected size (598 bp) is generated by
primers specific to nNOS mRNA. Similarly, in lane 2, a
PCR product of the expected size (431 bp) generated from the same RNA
sample by primers specific to the pseudo-NOS RNA is detected.
Lanes 1 and 3 show the results of PCR
experiments designed to control for possible DNA contamination of the
samples analyzed in lanes 2 and 4,
respectively. In these experiments, reverse transcriptase
was omitted, and as a consequence, no products were generated. In
B, the results from isolated B2 motoneurons are
presented. Lane 4 shows a PCR product of the expected
size (598 bp) generated by the same nNOS-specific primers as used in
A. Note that there is no PCR product in lane
2 in which the pseudo-NOS RNA-specific primers were used.
Lanes 1 and 3 represent control
experiments in which reverse transcriptase was omitted. The absence of
any PCR products in these lanes proves that the RNA sample used in the
RT-PCR experiments was free from DNA contamination. All RT-PCR products
shown have been cloned and sequenced to confirm their identity.
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From the data obtained from identified neurons, two major conclusions
can be drawn: first, that nNOS mRNA and pseudo-NOS RNA are coexpressed
in the CGC, and secondly, that the transcription of the corresponding
genes can be independently regulated in a neuron-specific manner.
Stable RNA-RNA duplexes form in vivo
The neuron-specific coexpression of the nNOS-encoding and
antisense NOS transcripts is strongly supportive of a role for the pseudo-NOS gene in the translational regulation of nNOS expression. However, this could only occur if the nNOS and pseudo-NOS transcripts form stable RNA-RNA duplex molecules. A method commonly used to detect
such duplexes involves the treatment of purified cellular RNA with
RNase A, an enzyme that specifically cleaves single-stranded RNA. Such
treatment ought to leave our hypothesized RNA-RNA duplex intact. It is
known, however, that RNase A can attack mismatched areas in
double-stranded RNA, and because complementarity between the nNOS and
pseudo-NOS transcripts does not reach 100%, RNase A might have some
activity within the proposed duplex. If this activity were significant,
all or most of the duplexed RNA molecules might be destroyed, making
this method for detecting the duplex inappropriate. Therefore, before
analyzing the results of experiments on RNase A-treated cytoplasmic
RNA, we first performed a ribonuclease protection assay using synthetic
nNOS mRNA and synthetic pseudo-NOS RNA (see Materials and Methods for
details). Importantly, this in vitro control experiment
showed that the RNase A activity was substantially suppressed within
the RNA-RNA duplex (results not shown) under standard conditions for
RNase A treatment (Shayiq, 1997). Therefore, we were confident that, by
using the same conditions, it would be possible to detect the natural
RNA-RNA hybrid in cytoplasmic RNA isolated from the snail CNS.
The logic of the experiment using cytoplasmic RNA is explained in
Figure 5. Briefly, cytoplasmic RNA was
purified under nondenaturing conditions to preserve possible RNA-RNA
hybrids and then treated with RNase A. To identify the duplex, we first
performed two reverse transcription reactions using two primers
specific to nNOS mRNA. One primer was located within the duplex area
(primer P2), and the other was located on single-stranded RNA outside
the duplex area (primer P1). The cDNA generated by the P2 primer was
then amplified by PCR in the presence of primer P2 and a second primer located within the duplex (primer P3). The cDNA generated by P1 was
similarly amplified using P1 and P3 primers. If the duplex exists, a
single product would be generated by the RNase A-treated sample (from
P2 and P3), whereas nothing would be generated in the presence of the
P1 and P3 primers (Fig. 5).
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Figure 5.
A schematic diagram showing the major steps of the
ribonuclease protection procedure used to detect RNA-RNA duplexes
in vivo. To preserve possible RNA-RNA hybrids,
cytoplasmic RNA was purified from the CNS under nondenaturing
conditions. To identify our hypothesized RNA-RNA duplex, the RNA has
to be treated with RNase A, an enzyme that cleaves single-stranded but
not double-stranded RNA molecules. After RNase A treatment, reverse
transcription reactions in the presence of either P2 primer (located
within the protected area) or P1 primer (located outside the protected
area) are performed. After adding the P3 primer, a cDNA generated in
the first reaction could be amplified using PCR and then will be
revealed as a single band of the expected size by electrophoresis. In
contrast, no cDNA could be produced in the second reverse transcription
reaction, and subsequently, no PCR product is expected. In the
left column, the predicted results of the control
experiments (no RNase A treatment) are summarized. Two RT-PCR products
should be detected in the reverse transcription reaction: one generated
by P2 and P3 and the other by P1 and P3.
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The results of the experiment are shown in Figure
6. A single product of the expected size
(139 bp) is detected in the experiment in which the P2 and P3 primers
were used (Fig. 6, lane 2). In contrast, no product was
detected with the P1 and P3 primers (lane 4). Similar
experiments were performed in parallel on purified RNA that was not
treated with RNase A. As expected, a PCR product of 139 bp was
generated by the P2 and P3 primers, and a 340 bp product was generated
by the P1 and P3 primers (lanes 1, 3). The correct identity of the 139 and 340 bp products have been confirmed by
cloning and sequencing. An exactly complementary experiment to that
illustrated in Figure 5 was performed using primers designed to amplify
the pseudo-NOS strand of the duplex. In this experiment, the antisense
region of the pseudo-NOS transcript was protected from RNase A
action.
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Figure 6.
The predicted RNA-RNA duplex exists in the CNS.
The experiment was performed according to the procedure described in
Figure 5. Lanes 1 and 3 show the products
of RT-PCR generated by RNA treated with DNase only. Lanes
2 and 4 show the products of RT-PCR generated on
RNA treated with both DNase and RNase A. In lanes 1 and
2, RNA was reverse transcribed with the P2 primer
located within the predicted duplex and then amplified in the presence
of the P2 and P3 primers (see Figs. 3 and 5 for location of the
primers). Lanes 3 and 4 show the results
generated when RNA was reverse transcribed with the P1 primer located
outside the predicted duplex and then amplified in the presence of P1
and P3 primers. A product of the same predicted size (139 bp) is
generated by the RNA sample that was treated with DNase only and with
DNase plus RNase A (lanes 1 and 2). The
band shown in lane 3 is the size predicted (340 bp) of a
product generated by the P1 and P3 primers. Note that there is no
product in lane 4. Importantly, these results correspond
precisely to those predicted in Figure 5. All of the RT-PCR products
shown have been cloned and sequenced to confirm their identity.
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These experiments leave little doubt that stable RNA-RNA duplex
molecules do form in vivo between the antisense pseudo-NOS transcript and the nNOS mRNA in Lymnaea CNS.
Antisense regulation of nNOS protein synthesis
in vitro
To show that this RNA-RNA duplex is able to prevent the synthesis
of nNOS protein, we have performed in vitro translation experiments. Using T7 RNA polymerase, we generated large quantities of
the major part of the nNOS-encoding mRNA and the complete antisense region of the pseudo-NOS RNA. When translated alone, the synthetic nNOS
cRNA provides a template for the synthesis of an nNOS protein of the
correct size, and this can be seen in Figure
7 (lane 1). When the nNOS cRNA
is incubated with the antisense pseudo-NOS transcript, the synthesis of
nNOS protein is completely suppressed (lane 2). Furthermore,
when the antisense pseudo-NOS cRNA was replaced with sense pseudo-NOS
cRNA, which cannot form a duplex with nNOS cRNA, there is no inhibition
of translation (lane 3).
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Figure 7.
Synthesis of nNOS protein in vitro
is suppressed by the antisense pseudo-NOS RNA. Lane 1
represents the result of the translation of NOS cRNA and shows the main
labeled product is a protein of the expected (93 kDa) size
(arrow). Lane 2 illustrates the effect of
incubating the 0.2 µg of NOS cRNA with 2 µg of antisense pseudo-NOS
cRNA before translation. Note strong suppression of translation of the
nNOS protein. As a control for any effects on translation that are not
related to the formation of a duplex, we also preincubated the 0.2 µg
of NOS cRNA with a 2 µg of a sense version of the pseudo-NOS cRNA
(lane 3). No inhibition of NOS protein synthesis is
observed. Similar results, although weaker, have been obtained, even
when the ratio between nNOS cRNA and pseudo-NOS transcripts was 2.5:1.
A second major protein of ~50 kDa can be seen in each
lane. This protein is present in the cell-free wheat
germ system and performs a useful function as an internal control. Note
that it is not diminished in intensity in lane 2.
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Importantly, the results of in vitro translation experiments
show that the duplex formed between the pseudo-NOS transcript and nNOS
mRNA is sufficient to block the synthesis of nNOS protein. They do not,
however, show that an antisense mechanism suppresses the synthesis of
nNOS protein in particular snail neurons in vivo.
In vivo antisense regulation of nNOS
protein expression
It is the existence of two large, identified nNOS mRNA-containing
neurons in the CNS (the paired B2 and the CGC) that has provided an
opportunity to test our idea that a natural antisense RNA can prevent
the expression of nNOS protein in vivo. Crucially, as
described above, although both identified neurons express nNOS mRNA,
only the CGC coexpresses the NOS pseudogene transcript. If our
conclusions are valid, there should be significant suppression of nNOS
enzyme activity correlated with the neuronal coexistence of the
functional and pseudo-NOS transcripts in the CGC.
Enzyme activity associated with nNOS can be rapidly and reliably
localized to cells using the NOS-specific NADPH-diaphorase histochemical method. In both nervous and peripheral tissue and in both
vertebrates and invertebrates, NADPH-diaphorase and NOS activity are
regarded as identical (Dawson et al., 1991; Matsumoto et
al., 1993; Elphick et al., 1995). Our previous work on the buccal ganglion (Park et al., 1998) shows that the B2 neuron is strongly and consistently NADPH-diaphorase-positive (100% of B2 neurons observed in >100 preparations), indicating that all B2 neurons
always contain an active nNOS protein (Fig.
8A). Using exactly the
same NADPH-diaphorase protocol, experiments were performed on cerebral
ganglia preparations from 30 animals, each of which contains a pair of
CGCs. Remarkably, in only one preparation did we find bilateral and
strongly positive staining, comparable with staining in the B2 neuron.
In four preparations, there was trace staining in one member of the
pair, and for the remaining 54 neurons, no staining above background
was seen. At best therefore, in the animals examined, only <10%
(n = 60) of the CGCs show nNOS enzyme activity,
although we know from the results of published in situ hybridization experiments (Korneev et al., 1998) and from the single
neuron RT-PCR experiments reported here that this neuron does contain
the nNOS transcript (Figs. 4, 8B,C). An illustration of nonexpression and the exceptional sporadic expression of nNOS enzyme
activity in the CGC is provided in Figure
9, A and B.
Importantly, these results confirm the existence of translational
mechanism-suppressing nNOS expression in the CGC and demonstrate that,
under some circumstances, it is possible for the neuron to synthesize
nNOS protein.
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[in a new window]
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Figure 8.
Antisense RNA-mediated regulation of nNOS protein
expression in vivo. A, NADPH-diaphorase staining of the
buccal ganglia. A pair of symmetrical NOS-containing
diaphorase-positive B2 motoneurons is indicated by
arrows. B, A diagram of the
Lymnaea CNS showing the positions of identified neurons
referred to in this investigation. Dark cell bodies of B2 motoneurons
reflect the fact that the neurons are strongly and consistently
NADPH-diaphorase-positive, i.e., they always contain an active nNOS
protein (A, C). In contrast, <10% of
all CGCs examined showed nNOS enzyme activity. To emphasize this
result, the cell bodies of CGCs are shown in light
color. C, A summary of our experiments on the
expression of nNOS mRNA, antisense RNA, and nNOS protein in the
identified neurons B2 and CGC. The asterisk indicates
that nNOS protein in the CGC is not entirely absent but is detected
only in ~10% of the cells observed (n = 60).
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View larger version (51K):
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Figure 9.
NADPH-diaphorase staining of the cerebral ganglia.
A, The majority of CGCs shows no NADPH-diaphorase
activity. B, One of the very few clearly
NADPH-diaphorase-positive CGCs. Cell bodies of the CGCs are indicated
by arrows.
|
|
To summarize, an identified neuron that contains the nNOS mRNA but not
the pseudo-NOS RNA consistently expresses a functional NOS protein. In
contrast, in a neuron in which both transcripts are colocalized, NOS
enzyme activity is practically undetectable. These in vivo
observations support the view that the antisense pseudo-NOS transcript
suppresses the translation of functional nNOS mRNA in neurons in which
the two transcripts are colocalized. We conclude that the suppression
of nNOS enzyme activity in the CGC is caused by the hybrid arrest of
translation mediated by an endogenous antisense-containing RNA
transcribed from a NOS pseudogene (see Discussion).
| |
DISCUSSION |
Our results have implications on two fronts. First, they show
that, in a eukaryotic system, a natural antisense mechanism can
regulate the synthesis of an important neuronal signaling molecule.
Second, they suggest that novel regulatory functions for some
transcribed pseudogenes can arise during the course of evolution.
Although our findings are conclusive with respect to the ability of a
natural antisense-containing transcript to suppress nNOS protein
synthesis, they do not speak to the mechanism of antisense
interference. Two quite different mechanisms have been proposed to
explain natural antisense-mediated regulation of translation: hybrid
arrest of translation and digestion of double-stranded (ds) RNA with
specific ribonucleases (Nellen and Lichteinstein, 1993). Although the
second possibility cannot be fully excluded, there is a number of
observations that cannot be explained if our RNA duplex is a target for
dsRNA-activated RNases. For example, and most significantly, we
have demonstrated the existence of the RNA duplex in vivo
and that both transcripts involved in duplex formation are present in
the CGCs. We therefore favor a mechanism involving antisense-mediated
hybrid arrest of translation. The precise steps in the chain of events
leading to the inhibition of nNOS protein synthesis, however, are not
clear. They might include, for example: (1) steric alterations in the
sense RNA structure that prevent translation; (2) trapping of specific
RNA-binding proteins by the antisense transcript; and (3) inhibition of
initiation of protein synthesis by activated dsRNA-dependent protein
kinases, etc. (Vanhe-Brossolleet and Vaquero, 1998). Whatever its
precise mechanism, this compelling example of natural
antisense-mediated suppression suggests that the phenomenon of RNA
duplex formation is an important mechanism of translational regulation
in eukaryotes. Recently, this conclusion has found support in
unexpected observations that the introduction of dsRNA molecules can
effectively and specifically suppress gene expression in
Caenorhabditis elegans and Drosophila (Fire et
al., 1998; Kennerdell and Carthew, 1998).
Our results show that nNOS protein synthesis is usually suppressed in
the CGC by an antisense-mediated mechanism, but also that there is
sporadic use of nNOS protein by this neuron (Fig. 9). According to our
model (Fig. 10), these changes in nNOS
expression are mediated by the differential transcription of the NOS
pseudogene. Specifically, the active transcription of the pseudogene
will lead to the suppression of nNOS protein synthesis, and on the other hand, the inhibition of pseudogene transcription will permit nNOS
production. Importantly, a switch from the "off" to the "on" mode of nNOS expression would be achieved rapidly because the functional nNOS gene is already active in both modes and nNOS mRNA
could be available immediately for translation once the suppressive effect of the NOS pseudogene is removed. We therefore propose that in
the CGC antisense-mediated translational control, supplemented by
transcriptional regulation of the NOS pseudogene, provides an effective
molecular mechanism for achieving rapid changes in nNOS protein
production in response to some internal or external signals.
Functionally, the important issue is to determine when and why the CGC
in Lymnaea needs to express nNOS. At present, we can only
speculate on the basis of what is known about the role of this neuron
and its homologs in other molluscs. Crucially, we need to gain an
understanding of the natural processes that cause sporadic expression
of nNOS protein in the CGC of Lymnaea to occur. Comparison
with other molluscan species might be helpful in this respect, and
specifically, it is of some interest that the CGC homolog in the
gastropod Pleurobranchaea consistently expresses nNOS enzyme
(Moroz and Gillette, 1996). In Lymnaea, the CGCs are very
well studied serotonergic neurons that have "gating" and modulatory
functions in the neural circuit controlling feeding behavior (Kemenes
et al., 1994). Their homologs in Aplysia [the metacerebral
giant cells (MCCs)] and in Pleurobranchaea mediate the effects of arousal on feeding behavior (Gillette and Davis, 1977;
Kupfermann and Weiss, 1982). Thus, it would appear that neurons
of this type play important roles in aspects of behavioral plasticity
related to feeding. Perhaps in Lymnaea, these or related functions might be reinforced by NO when the CGCs express the nNOS
enzyme. In Aplysia, the homologous MCC, which does not stain using NADPH-diaphorase (Jacklet and Gruhn, 1994), generates a very slow
EPSP in response to the release of NO after the stimulation of an
identified presynaptic nitrergic neuron (Jacklet, 1995). This
NO-induced slow depolarization of the MCC would appear to be mediated
by cGMP (Koh and Jacklet, 1999). Thus, in Aplysia, the
homolog of the CGC is sensitive to NO, and this might contribute to
MCC-mediated arousal in the feeding system. If the CGC of
Lymnaea is also activated by NO, NO might function as an
autostimulatory neurotransmitter when the nNOS protein is expressed in
this neuron.
Natural antisense-mediated regulation of gene expression is well known
to exist in prokaryotic systems (Wagner and Simons, 1994) but is far
less well recognized in eukaryotes (Vanhe-Brossolleet and Vaquero,
1998). Our experiments provide a clear example of a natural antisense
mechanism operating in an eukaryotic system. The singular advantage of
our system that has allowed us to demonstrate this is the ability to
perform molecular experiments on single identified neurons. This has
enabled us to show that a trans-encoded endogenous antisense
RNA is involved in the translational regulation of the nNOS in a
particular neuron. Of considerable additional interest is the finding
that the endogenous antisense RNA molecule is derived from a
pseudogene. Importantly, among the published examples of natural
antisense RNAs in eukaryotes, none represent transcripts derived from
pseudogenes. This new finding has fundamental implications for future
investigations of antisense regulation. In particular, it suggests that
a greater than expected number of antisense-containing transcripts may
be encoded in the genomes of eukaryotes.
With respect to the evolution of regulatory functions of pseudogenes,
we must now conclude that transcribed pseudogenes are not necessarily
without function. Indeed, they would appear to be especially suited to
roles involving the antisense regulation of the active genes to which
they are related (Fig. 10). If this is true, there must be other
examples of pseudogenes that are not translated but that may regulate
the expression of proteins encoded by related functional genes.
Although a comprehensive search of the available databases could
provide an answer to this question, this is beyond the scope of the
current paper. This is because an enormous amount of information would
have to be analyzed without the benefit of a simple ready-to-use
algorithm for detecting such sequences. Our future plans do include
such an investigation, and as a prelude to this, we have recently
analyzed the sequence ofjust 10 pseudogenes and their corresponding
functional genes picked at random in the GenBank database. In this
limited search, another example of a transcribed pseudogene (a
cytochrome P-450-like pseudogene, accession numbers M12280 and M12287) (Zaphiropoulos et al., 1986) that has a region of significant antisense
homology to the functional gene was detected. This suggests strongly
that the regulatory role we attribute to the NOS pseudogene is unlikely
to be an isolated example, and we may have uncovered the first member
of an entirely new class of regulatory gene. We therefore believe that
antisense-mediated regulation of gene expression is far more widespread
in eukaryotic systems than currently recognized. In the brain, an organ
in which the pattern of gene expression is highly complex and labile,
such an expansion of the diversity of ways in which gene activity can
be regulated is of particular significance.
| |
FOOTNOTES |
Received April 21, 1999; revised June 14, 1999; accepted June 28, 1999.
This work is supported by the Biotechnology and Biological Sciences
Research Council. We thank E. I. Korneeva, R. Philips, and
M. Piper for their technical assistance and S. Saunders for her helpful
comments on this manuscript.
Correspondence should be addressed to Michael O'Shea at the above address.
| |
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TOP
ABSTRACT
INTRODUCTION
