From the Renal Division and the Department of Medicine, St. Michael's Hospital and University of Toronto, Toronto, Ontario M5S 1A8, Canada
Received for publication, September 30, 2002, and in revised form, October 22, 2002
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ABSTRACT |
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Expression of the neuronal nitric-oxide synthase
(nNOS) mRNA is subject to complex cell-specific transcriptional
regulation, which is mediated by alternative promoters. Unexpectedly,
we identified a 89-nucleotide alternatively spliced exon located in the
5'-untranslated region between exon 1 variants and a common exon 2 that
contains the translational initiation codon. Alternative splicing
events that do not affect the open reading frame are distinctly
uncommon in mammals; therefore, we assessed its functional relevance.
Transient transfection of reporter RNAs performed in a variety of cell
types revealed that this alternatively spliced exon acts as a potent translational repressor. Stably transfected cell lines confirmed that
the alternatively spliced exon inhibited translation of the native nNOS
open reading frame. Reverse transcription-PCR and RNase protection
assays indicated that nNOS mRNAs containing this exon are common
and expressed in both a promoter-specific and tissue-restricted
fashion. Mutational analysis identified the functional
cis-element within this novel exon, and a secondary structure prediction revealed that it forms a putative stem-loop. RNA
electrophoretic mobility shift assay techniques revealed that a
specific cytoplasmic RNA-binding complex interacts with this motif.
Hence, a unique splicing event within a 5'-untranslated region is
demonstrated to introduce a translational control element. This
represents a newer model for the translational control of a mammalian mRNA.
Numerous tissues and distinct cell types are known to express
neuronal nitric-oxide synthase
(nNOS)1, which participates
in protean processes (1). The mRNAs for genes that play key roles
in the biology of diverse organs and developmental pathways may require
multifaceted and sophisticated regulatory mechanisms to control their
expression. One such mechanism is alternative splicing, which may
enhance the information contained within a gene and occur as a tissue-
or developmental stage-specific phenomenon or in response to cellular
stimuli (2-4). Recent studies have shown that structural and allelic
mRNA diversity is important to the regulated expression of nNOS
(5-7). Localized to 12q24.2, NOS1 is a complex locus
consisting of 29 exons and spans a region >240 kb as a single copy in
the haploid human genome (5, 8). The translational initiation and
termination sites are located in exons 2 and 29, respectively. Given
the relevance of nNOS in diverse biological systems, its complex
spatial and temporal expression patterns, and the important
consequences of altered expression, a tightly controlled regulatory
network appears to be critical.
We recently reported that multiple distinct examples of exon 1 are
selectively utilized in a tissue-specific manner (6). These exon 1 variants, arising from alternative promoters of a single gene, are
distributed over a region >105 kb and are designated exons 1a, 1b, 1c,
etc. Multiple variants of exon 1 are evident in both mouse and rat,
indicating that nNOS mRNA diversity at the 5'-end is evolutionarily
conserved across species (9). In addition, a novel promoter was
described for murine nNOS. It is located within exon 2, further
increasing the promoter diversity of this gene (10). Overall, this nNOS
mRNA diversity involves the 5'-untranslated region (5'-UTR) and
does not affect the encoded protein sequence, but has been shown to
affect the translational efficiency of the gene (6). In contrast, a
downstream promoter within intron 3 of the human nNOS gene produces a
Leydig cell-specific mRNA transcript that encodes an N-terminally
deleted nNOS protein (11, 12).
mRNA diversity may also be generated by alternative splicing, and
several variant nNOS transcripts have been previously identified (5,
7). For instance, cassette exon deletion and insertion events have been
reported to occur within the open reading frame (ORF) of human nNOS (8,
12, 13). Alternative splicing represents a mechanism to introduce
specific alterations to the nNOS protein. Distinct roles for some of
these variants have been reported, including changes in the
response to morphine (14), specialized roles in distinct regions of the
brain (9, 15), and altered nNOS biology in differentiated skeletal
muscle cells (16). Increased expression of specific nNOS splice
variants has been reported within the spinal cord of patients affected
by amyotrophic lateral sclerosis, which may contribute to the
pathophysiology of this disease (17). Dissecting the various mechanisms
of splicing regulation and the role of the nNOS splice variants will be
clearly important in understanding the contribution of this complex
gene to disease processes.
Alternative splicing events that exclusively involve the 5'-UTR of
a mammalian mRNA are distinctly uncommon. Alterations to this
region of the mRNA have the potential to introduce
post-transcriptional regulatory elements. Several mechanisms of
translational control by the 5'-UTR have been proposed. These include,
but are not limited to, the primary structure of the mRNA affecting
translational initiation (e.g. upstream AUG codons affecting
ribosome scanning), the secondary or tertiary structures modulating
translational machinery (e.g. stem-loops and pseudoknots),
and RNA-binding proteins (e.g. iron-responsive element
(IRE)-binding protein (IRP)), among others (18-21). In many of these
examples, the translational regulation mediated by the 5'-UTR plays a
key role in tissue- and developmental stage-specific expression of the
involved gene (22-26). An alternative splicing event contributing a
translational control element within a 5'-UTR would represent a newer
model for the control of mammalian gene expression. Herein, we
describe, characterize, and quantify such a splicing event in the human
nNOS gene. Importantly, we demonstrate that an alternative splicing
event affecting the 5'-UTR modifies the translational efficiency of
select nNOS mRNAs.
cDNA and Genomic Characterization
Modifications of the rapid amplification of 5'-cDNA ends
(5'-RACE) protocol were utilized to isolate the 5' termini of mRNA transcripts for human nNOS from total cellular RNA samples (12). Briefly, total cellular RNA (1 µg) derived from various normal human
tissues was reverse-transcribed with a gene-specific antisense primer
in exon 2 (5'-CCA TGG TAA CTA GCT TCC-3'), and two rounds of PCR was
performed as described (6, 12). PCR products were digested with
EcoRI/XbaI, subcloned into pBluescript, and
subjected to DNA sequence analysis. This approach allowed the
characterization of cDNA sequences upstream of exon 2 in various
human tissues. To characterize human genomic sequences corresponding to
cDNA sequences upstream of exon 2, P1 clones were isolated by
PCR-based screening from a library of human diploid genomic DNA as
described (6). Three overlapping P1 clones were isolated, partially
digested with Sau3AI, subcloned into the BamHI
site of pBluescript SK( Quantification of AS Exon-containing mRNA Transcripts
RNase Protection Assay (RPA)--
RPA was used to quantitate and
structurally characterize the tissue-specific expression of the AS
exon. A 274-nucleotide (nt) PCR fragment containing the 89-nt AS exon
and 185 nt of exon 2 was subcloned into the pCRII vector (TA cloning
kit, Invitrogen). The full-length 382-nt
[ Reverse Transcription (RT)-PCR and Southern
Analysis--
Semiquantitative RT-PCR was utilized to assess the
tissue-specific expression of the AS exon and the relationship between the presence of the AS exon and promoter utilization. First-strand cDNA was synthesized with total cellular RNA (5 µg) derived from various normal human tissues (Clontech) using
random primers and SuperScript II reverse transcriptase (Invitrogen).
The following sense primers were used: exon 1a (5'-CAT TCT GGA ATC CCA
TGC TC-3'), long exon 1c (5'-TTG TCT CTC CCA GGG AAG-3'), short exon 1c
(5'-CCA CCA TGC CAG GGT GAG G-3'), exon 1g (5'-GCA GCG CGA AGA GGC
AGC-3'), exon 1j (5'-GCA AAG TGC TGC GTC ACT CT-3'), and AS exon
(5'-GTT GCC AGT GCA GCC ATC T-3'), with a common antisense primer in
exon 2 (5'-GGA AGT GAT GGT TGA CCA GG-3'). The amplification of exon 22/exon 23 was performed using 5'-CAC CAT CTT CCA GGC CTT-3' and 5'-CGG
TAG GAA ACG ATG GCC-3'. PCR amplifications were performed under well
characterized semiquantitative conditions in a total volume of 100 µl
for 30 cycles as described (6). The quantity of the first-strand
template used in each reaction was adjusted to reflect
glyceraldehyde-3-phosphate dehydrogenase levels, which were determined
using real-time PCR. Products were size-fractionated by agarose gel
electrophoresis and downward-transferred to GeneScreen Plus membranes
(DuPont). Southern blots were hybridized with
[ Functional Analysis of the AS Exon upon Translation of a
Downstream Reporter ORF
Generation of Exon 1/AS Exon Reporter
Constructs--
A series of in vitro expression vectors
were constructed using a modified pSP-Luc+ plasmid
(Promega) that included a sequence of 60 adenylate residues 3' of the
luciferase ORF. Reporter constructs either included or excluded the
89-nt AS exon between exons 1 and 2 and were generated using native
restriction sites and 5'-RACE clones. AS exon-containing reporters were
produced for the major exons 1a, 1c, 1f, and 1g. Each reporter
contained sequences of previously reported representative cDNA
variants with 100, 326, 276, and 161 nt of the exon 1, respectively (GenBankTM/EBI accession numbers AF049712,
AF049714, AF049717, AF049718) (6).
Modified Exon 1c/AS Exon Reporter Constructs--
The
effects of various transcriptional start sites on the repression
mediated by the AS exon were tested using modified versions of the exon
1c/pSP-Luc reporter. A PCR-based approach was used with Vent
DNA polymerase (New England Biolabs Inc.) and three specific sense
primers. To generate reporters containing 101, 66, and 31 nt of exon 1c
sequence, the following primers were designed: 5'-CGG GGT ACC ACG CCA
GGC GGC TGA TTA-3', 5'-CGG GGT ACC AGA TGG GGA GCA CTG TCT GA-3', and
5'-CGG GGT ACC ACC ATG CCA GGG TGA GGC-3', respectively, and a common
antisense primer, 5'-GCC TTT CTT TAT GTT TTT GG-3'. PCR was performed
on existing templates either containing or lacking the AS exon. PCR
amplicons were digested with KpnI/NcoI and
subcloned into the exon 1c/pSP-Luc reporter prepared similarly.
Mutational Analysis of the Stem-Loop Structure Located within the
AS Exon--
Mutations within the AS exon stem-loop structure were
generated using the QuikChange site-directed mutagenesis kit
(Stratagene, La Jolla, CA). The following sense/antisense overlapping
primers were used: S1, 5'-GAG GCC TTG CCG GAA ATG GTT GCC AGT GCA
C-3'/5'-GTG CAC TGG CAA CCA TTT CCG GCA AGG CCT C-3'; S2, 5'-GGT GAG
GCC TTG CCG GAT GGT TGC CAG TGC AGC-3'/5'-GCT GCA CTG GCA ACC ATC CGG CAA GGC CTC ACC-3'; S3, 5'-CCA TGC CAG GGT GAG GCC TTG CCG GAG CCA GTG
CAG CCA TCT GAA TTC C-3'/5'-GGA ATT CAG ATG GCT GCA CTG GCT CCG GCA AGG
CCT CAC CCT GGC ATG G-3'; and L1, 5'-GCC TTG CCG GAG CTG GTT GCA CAA
GCA GCC ATC TGA ATT CC-3'/5'-GGA ATT CAG ATG GCT GCT TGT GCA ACC AGC
TCC GGC AAG GC-3'. Amplifications were performed using the existing
exon 1c/AS exon (31 nt)/pSP-Luc luciferase reporter as the template.
For all described reporter constructs, the subcloned regions and
subsequent mutations were confirmed using fluorescence-based DNA
sequencing as described above.
RNA Transfections--
PC12 cells (rat pheochromocytoma,
nNOS(+); American Type Culture Collection, Manassas, VA) were
maintained in RPMI 16 40 medium supplemented with 10% heat-inactivated
horse serum, 5% fetal bovine serum, and antibiotics (Invitrogen).
C2C12 cells (mouse skeletal myoblast, nNOS(+); American Type Culture
Collection) were grown in Dulbecco's modified Eagle's medium
supplemented with 15% fetal bovine serum and antibiotics. HeLa cells
(human adenocarcinoma, nNOS( Functional Analysis of the AS Exon upon Translation of the nNOS
ORF
pcDNA3 (Invitrogen)-based vectors were used to stably
integrate the full-length nNOS cDNA downstream of native 5'-UTR
sequences. PCR-generated fragments containing the exon 1 variants
containing or lacking the AS exon were subcloned into the native nNOS
XbaI site within exon 2 (upstream of the human nNOS ORF).
CHO-K1 cells (American Type Culture Collection) grown in Ham's F-12
medium supplemented with 10% fetal calf serum (Invitrogen) were
transfected with the various pcDNA constructs containing either
exon 1a or 1c, each with or without the AS exon upstream of the
full-length nNOS ORF, as described (12). The mutations of the AS exon
were also inserted into the exon 1c/AS exon (31 nt)/pcDNA-based
nNOS expression vector using existing restriction sites. For all
constructs described, the subcloned regions were confirmed using
fluorescence-based DNA sequencing as described above. Three independent
pools of G418-resistant clones, each representing >300 independent
clones, were selected and maintained in 800 µg/ml G418 (Invitrogen).
nNOS protein levels were assessed as described (12). Briefly, a
monoclonal anti-human nNOS antibody, raised against an epitope
located within the C terminus (amino acids 1095-1289) of the nNOS
protein (BD Biosciences), was used with a horseradish
peroxidase-linked secondary antibody (Amersham Biosciences). Detection
was facilitated using the Supersignal West Pico chemiluminescent
substrate (Pierce) and the Fluor-S Max Multimager (Model 170-7720, Bio-Rad). Densitometric analysis was performed using QuantityOne and
ImageQuant software (Amersham Biosciences). Steady-state nNOS mRNA
levels were determined by RPA as described above using a 444-nt probe
that protected a 369-nt nNOS fragment corresponding to nNOS exons 16 and 17. To control for insertional copy number of the nNOS-containing pcDNA construct, we measured the levels of neomycin transcripts using a 151-nt probe complementary to 120 nt of the neomycin
phosphotransferase ORF.
Mapping the Exon 1c Transcriptional Start Site
Primer Extension Analysis--
An oligonucleotide (5'-GAG GCA
TCA TGA GCA GCT CCG GCA AGG-3') complementary to the junction of
exons 1c and 2 was end-labeled with [ RPA--
A uniformly [ RNA Electrophoretic Mobility Shift Assay
PCR was used to generate the riboprobe templates. The following
sense primers were designed containing a T7 promoter sequence: wild-type, 5'-TAA TAC GAC TCA CTA TAG GGA CGG AGC TGG TTG CCA G-3'; and
S3, 5'-TAA TAC GAC TCA CTA TAG GGA CGG AGC CAG TGC AGC C-3'. All
reactions were performed with the common antisense primer 5'-GAC GTG
TCC TAA GGT CAG-3', and the existing wild-type or mutant exon 1c/AS
exon/luciferase reporters were used as templates. PCR products were
ligated using the TA cloning kit into the pCRII vector.
[ Data Analysis
All experiments were performed at least three times. Unless
otherwise indicated, quantitative data are expressed as the means ± S.E. of three independent experiments, each done in triplicate. Comparisons were performed using analysis of variance, and statistical significance is defined as p < 0.05.
Cloning of the AS Exon and Its Genomic Structure--
To
characterize the structure of nNOS mRNA 5' termini, we used 5'-RACE
to clone nNOS transcripts using RNA isolated from different human
tissues. This RACE strategy was designed for cloning the 5' termini
upstream of exon 2, the translational initiation exon. We previously
reported the use of this technique to identify and characterize
distinct nNOS mRNAs that vary in their 5'-UTR sequences due to
alternative promoter usage, resulting in the transcription of different
exon 1 variants (6). Using this strategy, we detected the insertion of
a 89-bp fragment interposed between the exon 1 variants and the common
exon 2. Three overlapping P1 bacteriophage clones were isolated from a
human genomic library. These P1 clones and their plasmid subclones were
used for sequence analysis and restriction mapping of a 150-kb genomic
region of the human nNOS gene upstream of exon 2 that corresponds to
the genomic region encompassing these sequences (6). Restriction
mapping and sequence analysis indicated that genomic regions
representing the 89-bp region are located downstream of the first nine
alternative exon 1 variants and 27.5 kb upstream of the common exon 2 (GenBankTM/EMBL Data Bank accession number AY098642). The
89-bp region represents an exon flanked by genomic exon-intron
boundaries that conform to the GT/AG rule (27). Therefore, this 89-bp
sequence represents an alternatively spliced exon, which we termed the AS exon (Fig. 1). Sequence inspection of
the AS exon failed to identify the presence of AUG codons that would
contribute short upstream ORFs, which have been shown to disrupt the
scanning process of the translational machinery (19). Similarly, the
89-nt exon did not interrupt a short ORF due to the presence of
upstream AUG codons.
Tissue-specific Expression of the AS Exon--
RNase protection
analysis and a semiquantitative RT-PCR approach were used to determine
the tissue-specific expression of nNOS mRNAs and to define whether
the AS exon is expressed selectively in different tissues. As shown in
Fig. 2A, the AS exon was
present in 5-40% of nNOS transcripts within a given tissue and was
especially enriched in testis, brain, skeletal muscle, and lung. The
tissue-specific splicing pattern of the AS exon was also assessed using
RT-PCR. Amplification was performed between the AS exon and exon 2 (Fig. 2B, AS/2) and compared with overall nNOS
expression as assessed by amplification across exons 22 and 23 (22/23). As observed with the RPA, a broad tissue expression
pattern was evident.
We also characterized the association of the AS exon with particular
exon 1 variants (exons 1a, 1c, 1g, and 1j). Amplifications were
performed using exon 1-specific sense primers and a common antisense
primer located within exon 2. Two amplification sizes were evident when
PCR products were resolved by gel electrophoresis. Southern analysis
using an AS exon-specific primer (data not shown) and direct sequencing
confirmed that the larger amplicon resulted from insertion of the AS
exon between exons 1 and 2. An alternative splicing pattern was
observed for exons 1a, 1c, and 1g. Within a given tissue, the relative
abundance of AS exon-containing transcripts was seen to vary in an exon
1-specific fashion. In brain, for instance, exon 1a and 1c transcripts
rarely included the AS exon, whereas it was relatively abundant in exon
1g transcripts. An alternative splicing pattern was never observed with
exon 1j. A single amplification product was detected. Our prior and
recent characterization of the nNOS genomic locus indicated that exon 1j is located 11.1 kb downstream of the AS exon and is within the
intervening intronic sequence upstream of exon 2 (6).2 Transcripts initiating
at this genomic region could not include the AS exon. Further work will
be required to characterize this putative exon 1j promoter and the
cellular stimuli that may affect its activity.
We performed a more detailed analysis of the expression of exon 1c
transcripts because mapping of the exon 1c promoter transcriptional start site revealed that initiation occurs over a large span of genomic
DNA (see Fig. 5). Two sense primers were used to amplify exon
1c-containing nNOS transcripts. These primers detected either shorter
(Fig. 2B, 1c (Short)) or longer (1c
(Long)) versions of exon 1c. The RT-PCR results indicated that
expression of the longer forms of exon 1c was restricted to skeletal
muscle. This contrasted with the broad expression pattern of the
shorter forms. The AS exon was detected with both the longer and
shorter exon 1c transcripts.
Functional Analysis of the AS Exon upon Translation of Reporter
RNAs--
We postulated that the functional contribution of this novel
5'-UTR exon might be to selectively introduce an RNA-based control element that acts to regulate nNOS expression at the
post-transcriptional level. The functional consequences of the AS exon
on the translational efficiency of luciferase-based reporter constructs
were examined. The complete nNOS 5'-UTR sequences of various nNOS
mRNAs were inserted upstream of the luciferase ORF. Constructs were
prepared in which the AS exon was either included or omitted following exons 1a, 1c, 1f, and 1g (Fig.
3A). We performed transient
RNA transfections that yield efficient expression of in
vitro synthesized, capped RNA in eukaryotic cells within 4-6 h.
The advantages of this RNA-based transfection approach over
conventional DNA transfection methods include the elimination of
varying transcriptional efficiencies, and early harvesting minimizes
the interference from RNA degradation. Experiments were performed in
two representative cell lines with respect to nNOS expression patterns:
the C2C12 myoblast, a murine skeletal muscle cell type, nNOS(+); and
the rat PC12 pheochromocytoma, a neuronal lineage cell type, nNOS(+).
In both the C2C12 and PC12 cells, the AS sequence had marked effects on
translation of the reporter RNA (Fig. 3B). The presence of
the AS exon downstream of exons 1a and 1c caused a marked repression
( Functional Analysis of the AS Exon upon Translation of the Native
nNOS ORF in Stably Transfected Cell Lines--
Given our finding that
the AS exon repressed the translation of a reporter gene, we were
motivated to assess the effect of the AS exon on nNOS expression.
Therefore, we generated stable CHO-K1 cell lines expressing the
full-length nNOS ORF downstream of various nNOS 5'-UTRs. The 5'-UTR
sequences of the nNOS transcripts of exons 1a and 1c either lacking or
containing the AS exon were inserted upstream of the native exon 2 and
the complete human nNOS ORF. As shown in Fig.
4, translated nNOS protein levels were assessed by Western blot analysis, and the steady-state levels of both
nNOS mRNA and transcripts from the neomycin resistance cassette
were determined by RPA. A comparison of translational efficiencies
between each of the various nNOS mRNAs was performed by
quantitating the nNOS protein levels relative to nNOS mRNA levels
(neomycin mRNA was unchanged). Consistent with our findings using
RNA transient transfection of reporter constructs, the AS exon
repressed translation of the nNOS ORF when combined with either exon 1a
or 1c by 40 ± 5 and 45 ± 11% (mean ± S.E.),
respectively. This conclusion was valid whether or not nNOS
immunoreactive protein levels were normalized for nNOS mRNA or for
neomycin mRNA. The amount of translated nNOS protein was reduced
despite equivalent mRNA levels, indicating that the AS exon
represses translation of the nNOS ORF.
Translational Repression of the AS Exon Is Dependent upon Cap
Proximity--
It has been previously demonstrated that the functional
effects of a 5'-UTR cis-element may be modified by its
distance from the 5'-cap site (29). We further explored the
contribution of the upstream exon 1 sequence to the AS exon effect
using exon 1c as a model because we previously found that start
site location varies across cell types (6). Our 5'-RACE data indicated
multiple transcriptional start sites in skeletal muscle, kidney,
testis, and brain (data not shown). Primer extension and RPA were
utilized to refine the transcriptional start site of the human exon 1c promoter (Fig. 5, B and
C). The exon 1c promoter is TATA-less; and not surprisingly,
our data derived from these three independent techniques indicated that
transcription initiates at various sites over a 300-nt span within the
exon 1c genomic region (Fig. 5A). These experiments and the
work of others (30, 31) also indicated that the majority of exon 1c
transcripts initiate significantly downstream of the site tested
in our initial translational assays ( Mutational Analysis of the RNA Stem-Loop
Structure--
Sequence analysis and modeling using multiple
secondary structure prediction software programs of the AS exon
structure revealed the presence of a stem-loop structure. The mFold
algorithm (Version 3.1) revealed a Gibbs free energy value of
We postulated that the AS exon may represent a mechanism of
translational feedback involving the bioactive product of nNOS, NO. We
therefore tested each of the mutant and wild-type AS exon-containing constructs in the presence of the NO donors
S-nitroso-N-acetylpenicillamine (100 µM) and S-nitrosoglutathione (100 µM). Luciferase transient transfections were performed
following pretreatment with either S-nitroso-N-acetylpenicillamine or
S-nitrosoglutathione for 8, 24, or 48 h. The stable
CHO-K1 cell lines were also exposed to each NO donor for 24 h,
followed by extraction of total cellular protein and RNA. In both the
transient and stable transfection experimental models, the relative
levels of translation for each of the various 5'-UTR constructs was not
significantly modified by the addition of NO donors (data not
shown). We therefore concluded that NO does not modify the
translational effects of the AS exon. Future studies will be necessary
to define whether other models of cellular activation modify the
translational effect of the AS exon.
We sought to determine whether a cytoplasmic ribonucleoprotein complex
forms on the identified stem-loop element located within the AS exon. A
32P-labeled riboprobe representing the wild-type exon 1c/AS
exon stem-loop was incubated with the cytoplasmic protein extracts and
resolved by nondenaturing PAGE. Preliminary electrophoretic mobility
shift assay results demonstrated the formation of specific protein-RNA
complexes using cytoplasmic extracts of skeletal muscle (C2C12) and
neuronal (PC12) cells (data not shown). Specificity of this interaction
was assessed by generating a riboprobe corresponding to the S3 mutant,
which lacked the ability to repress translation. We failed to detect
significant binding of cytoplasmic proteins to the S3 probe compared
with the wild-type probe. We concluded that an RNA-binding
protein(s) exists that interacts with the putative stem-loop present
within the AS exon, although further studies will be required to
characterize this complex.
We have reported the existence of an alternatively spliced exon
within the 5'-UTR of the human nNOS mRNA. The finding of an exon
insertion/deletion event within a 5'-UTR that does not affect the ORF
is a rare event, especially in mammals. Insertion of the AS exon occurs
upstream of the authentic translational initiation codon for nNOS; and
as predicted, we have demonstrated that it leaves the nNOS ORF
unaltered. Reports of other mammalian mRNAs containing an
alternatively spliced, independent exon solely within the 5'-UTR are
sparse, but include the connexin-32, nuclear respiratory factor-1, and
ganglioside (M3) synthase genes (35-37). The functional relevance of these splicing events has not yet been addressed. In this
work, we found that the AS exon markedly repressed the translation of
the human nNOS ORF and a heterologous ORF. Our findings emphasize the
many facets of control that are operative in gene expression by
providing a newer model of 5'-UTR-mediated regulation of translational efficiency.
In considering nNOS expression, a mode of translational regulation is
fitting given that the human gene spans 240 kb and is therefore
predicted to be kinetically inefficient with respect to transcriptional
initiation and elongation, requiring 1.6-2.5 h at 100-150 kb/h (38).
Therefore, the ability to modify the translational competency of
existing nNOS mRNAs represents an efficient mechanism to control
the expression of very large transcription units. Evidence for the
translational control of nNOS expression has been observed both
in vitro and in vivo. In cellular models, the
translational efficiency of nNOS mRNAs varied in an exon 1-specific fashion. The translation of exon 1a (but not exon 1c) is increased 2-fold in differentiated C2C12 murine skeletal myocytes compared with
undifferentiated C2C12 murine skeletal myoblasts (6). In animal
models, high levels of dietary salt increase the expression of nNOS
protein in the inner medullary collecting duct of the kidney, yet the
mRNA levels remain unchanged (39). This also suggested a model of
translational control, although alterations in nNOS protein stability
could not be excluded.
There is a short, but growing, list of genes containing RNA
cis-elements that modulate translation: 15-lipoxygenase,
fibronectin, fibroblast growth factor-2, lipoprotein lipase, folate
receptor- Sequence and secondary structure analyses predicted a stem-loop
structure at the 5'-end of the AS exon. We also noted a structural similarity between the nNOS stem-loop and the classical IRE stem-loop, especially in the loop sequence (CAGUGN). This led us to consider the
possibility that the identified element could function via interaction
with an IRP. We initially favored an IRE/IRP hypothesis for the
regulation of nNOS given the known effects of NO in augmenting IRP
binding to IRE sequences (62-64). This would incorporate a translational feedback inhibition loop into the regulation of select
human nNOS mRNAs. Additionally, the observation that IRP-1 (aconitase) co-localizes with nNOS in a subset of neurons made for an
appealing model (65). Studies of the IRE stem-loop have defined
structural features that are important for IRP recognition, such as the
loop sequence and the position and sequence of the bulge located within
the stem (33, 34). We utilized a mutational strategy to study the
importance of the putative stem-loop identified within the AS exon and
to assess its structural features. Three mutations (S1, S2, and L1)
were generated, and each failed to disrupt the translational blockade
imposed by the AS exon. These results, especially the failure of the
loop sequence mutation (L1) to affect translational repression, suggest
that the AS exon does not contribute a classical IRE. The failure of
the loop mutation to disrupt the function of the AS exon also indicates
that the stem-loop structure is not involved in the formation of a
pseudoknot, as the loop sequence is typically critical to this tertiary
structure. This is an important observation because a pseudoknot
structure can form a 5'-UTR translational control element (28, 66, 67). Mutational analysis revealed that deletion of a major portion of the
stem (S3) completely abrogated the translational blockade, indicating
that the stem of this element is required for translational inhibition.
Interestingly, based on the predicted structures, exons 1c and 1a
donate to the stem component of the overall structure, whereas exons 1f
and 1g fail to contribute to the stability of the stem. This provides a
simple model to explain why the translational repression of the AS exon
is observed in an exon 1-dependent fashion.
The identification of a stem-loop structure present within the 5'-UTR
and acting as a translational repressor strongly hinted at the
involvement of an interacting trans-factor. Preliminary electrophoretic mobility shift assay studies using riboprobes corresponding to the putative stem-loop present within the AS exon
detected the presence of a bound cytoplasmic protein(s). We failed to
detect protein interaction with the S3 mutant probe, a finding
consistent with the functional analysis of this mutated 5'-UTR
sequence. We posit that the repression exerted by the AS exon is not
merely due to a structural feature of the mRNA. Rather, an
RNA-binding protein(s) appears to recognize this element and generates
the observed translational impediment. Future studies will be needed to
address the cellular stimuli that may modulate the translational
properties of AS exon-containing transcripts by affecting the formation
of protein-RNA complexes. The identification of the associated
trans-factor(s) will also be critical to understanding the
regulation of intracellular nNOS levels.
In summary, we explored the functional effects associated with the
inclusion of an 89-nt exon in human nNOS transcripts. We conclude that
it acts as a translational repressor in an exon 1-specific context.
This represents a newer model for the translational control of
mammalian mRNAs; a highly regulated splicing event within the
5'-UTR introduces a translational control element.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), and subjected to DNA sequence analysis.
Exonic sequences and exon-intron boundaries corresponding to the
alternatively spliced (AS) exon were determined on both strands of
genomic DNA with an ABI PRISM 377 automated DNA sequencer (PerkinElmer
Life Sciences).
-32P]CTP-labeled antisense riboprobe was generated
using the T7 MAXIscript kit (Ambion Inc., Austin, TX). 105
cpm of gel-purified probe was hybridized for 16 h at 42 °C with 25 µg of total cellular RNA. Multiple independent samples of human skeletal muscle RNA were extracted using guanidinium-based protocols from tissue procured from the National Disease Research Interchange (Philadelphia), and all other RNAs were obtained commercially (Clontech, Palo Alto, CA). RPA was performed using
the RPA IIITM kit (Ambion Inc.) and sized using
CENTURYTM RNA markers (Ambion Inc.).
-32P]ATP-labeled oligonucleotides positioned
internally to flanking PCR primers. Human glyceraldehyde-3-phosphate
dehydrogenase mRNA levels were assessed as a quantitative control
(sense, 5'-GAA GGT GAA GGT CGG AGT C-3'; and antisense, 5'-GAA GAT GGT
GAT GGG ATT TC-3') by real-time PCR using the ABI PRISM 7900HT sequence detection system (Applied Biosystems). The TaqMan probe (5'-CAA GCT TCC
CGT TCT CAG CC-3') was 5'-labeled with VIC and 3'-labeled with tetramethylrhodamine (TAMRA). Reactions were performed in triplicate.
); American Type Culture Collection) were
grown in Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum and antibiotics. In vitro transcription
of capped RNA was carried out using the Sp6 mMessageMachine kit (Ambion
Inc.). The quality of in vitro synthesized,
7mGpppG-capped RNA was assessed on 0.66 M
formaldehyde-containing 1% agarose gels. Cells were maintained in
60-mm dishes and transfected at 50-70% confluency with in
vitro synthesized, quantified, capped RNA using Lipofectin
(Invitrogen). 3 µg of RNA and 12 µl of Lipofectin were used for
each 60-mm dish along with 5 µg of
-galactosidase RNA to control
for transfection efficiency. Cellular extract was harvested 4-6 h
after RNA transfection, at which time reporter activity was increasing
linearly as a function of time (6). Multiple independent RNA
preparations were used. Northern blot analysis using total cellular RNA
harvested 4-6 h post-transfection and a random primer-labeled
luciferase DNA probe was carried out to assess steady-state levels of
transfected reporter RNA molecules. Luciferase activity was measured
with a luminometer (Monolight 2010C, Analytical Luminescence
Laboratory, San Diego, CA) and normalized for
-galactosidase
activity and protein content as described (6).
-32P]ATP
(specific activity of 6000 Ci/mmol; PerkinElmer Life Sciences). 8.0 × 104 cpm of labeled primer was hybridized to 25 µg of total cellular RNA for 16 h at 25 °C. Human total
cellular RNA from cerebellum and skeletal muscle was extracted using
guanidinium-based protocols from tissues procured from the National
Disease Research Interchange. Reverse transcription was carried out
using Superscript II. Primer extension products were sized using a
35S-labeled dideoxynucleotide chain termination sequencing
ladder as described (8).
-32P]CTP-labeled
antisense riboprobe was synthesized using an existing 5'-RACE genomic
clone. Linearization at an internal StyI site resulted in a
318-nt probe complementary to 200 nt of exon 1c and 91 nt of exon 2. 105 cpm of PAGE-purified probe was hybridized for 16 h
at 42 °C with yeast tRNA (50 µg) (data not shown) or human total
cellular RNA (50 µg) (Clontech). RNase protection
was as described (8).
-32P]CTP (specific activity of 800 Ci/mmol;
PerkinElmer Life Sciences) probes were synthesized using the T7
MAXIscript kit and PAGE-purified. Cytoplasmic protein extracts were
prepared from C2C12, PC12, and CHO-K1 cell monolayers in lysis buffer
(10 mM HEPES, 10 mM KCl, 5% glycerol, and
0.3% Nonidet P-40, pH 7.4) containing CompleteTM EDTA-free
protease inhibitor (Roche Molecular Biochemicals). The supernatant was
collected following centrifugation at 16,000 × g for 1 min. 4.0 × 104 cpm of riboprobe was incubated with
cytoplasmic protein extract (30 µg) at 4 °C for 30 min. Binding
reactions were resolved using nondenaturing (1× Tris borate/EDTA) 6%
polyacrylamide gel and analyzed using a PhosphorImager with
ImageQuant software (Version 1.2).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Genomic organization of the AS exon within
the human nNOS gene locus. A, the 89-nt AS exon is
located immediately downstream of the neuronal cluster (exons 1f, 1g,
and 1h) of exon 1 variants (6, 28). The ATG codon, located within exon
2 (Ex 2), is indicated as the start of the ORF
(shaded). The defined exonic (uppercase) and
flanking intronic (lowercase) sequences of the AS exon are
shown with the splice sites. B, potential splicing pattern
of mature human nNOS mRNAs containing the AS exon. One of the
upstream exon 1 variants (1x) splices with the AS
exon, which subsequently splices with the common exon 2. The figure is
not to scale.
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Fig. 2.
Quantitation of human nNOS mRNAs
containing the AS exon. A, RPA of total cellular RNA
extracted from various human tissues. The probe was complementary to
the entire AS exon (89 nt) and the contiguous 185 nt of exon
(Ex) 2. The proportion of AS exon-containing transcripts
were calculated as a percentage of the exon 2-containing transcripts.
B, tissue-specific expression of the AS exon assessed by
RT-PCR. The locations of the PCR primers are indicated for each blot on
the left and amplicon size on the right (bp). The probe
oligonucleotide used for Southern blotting of the exon 1x/exon 2 amplicons was located within exon 2. The probe oligonucleotide used for
Southern blotting of the exon 22/exon 23 amplicon was located within
exon 23. Representative results of three independent experiments are
shown in A and B. B, brain;
S and K skeletal muscle; K, kidney;
A, adrenal gland; H, heart; T, testis;
Lu, lung, L, liver.
50% decrease) of translational efficiency. The repressive effect
of the AS exon on translational efficiency was independent of cell type
and not simply a function of the length of the exon 1 variants. The
observation that repression occurred within the context of specific
exon 1 variants (exons 1a and 1c), but not with others (exons 1f and 1g), may suggest that sequence elements from both the first exon 1 and
the AS exon combine to form a functional RNA cis-element. Transient RNA transfection of CHO-K1 and HeLa cells (data not shown)
recapitulated the results obtained with C2C12 and PC12 cells.
Measurements of
-galactosidase activity derived from cotransfected, capped, polyadenylated
-galactosidase reporter RNAs were used to
control for transfection efficiency. Results were the same whether the
data were normalized or not. Total RNA isolated from transfected cells
was subjected to Northern blot analysis using a luciferase probe (Fig.
3B). It is clear that differences in the amounts of
transfected RNA across constructs do not explain the differences in
luciferase enzymatic activity. Therefore, we concluded that differences
in the luciferase activities reflect alterations in the translational
efficiency of the reporter mRNAs and not alterations in mRNA
stability or transfection efficiency.
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Fig. 3.
The AS exon represses the translation of a
luciferase reporter in an exon 1-specific manner. A,
design of the luciferase (Luc) reporter constructs. Human
nNOS 5'-UTR sequences including or excluding the AS exon were placed
upstream of the luciferase ORF. The lengths of the sequences for exons
(Ex) 1a, 1c, 1f, and 1g were 100, 326, 276, and 161 nt,
respectively. B, effect of the various nNOS leader sequences
on the translational efficiency of in vitro transcribed,
capped nNOS 5'-UTR/luciferase reporter RNAs transiently translated in
cultured cells. The activity profiles of nNOS 5'-UTR sequences in C2C12
or PC12 cells were normalized for protein content and expressed
relative to the activity of the luciferase ORF lacking any nNOS 5'-UTR
sequences (vector). Data are expressed as the means ± S.E. of
three independent experiments, each performed in triplicate. Northern
blot analysis was performed on total RNA extracted from transiently
transfected cells using a 32P-labeled luciferase
probe.
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Fig. 4.
The AS exon represses translation of the nNOS
ORF in stably transfected CHO-K1 cells. A,
design of the constructs expressing the native nNOS ORF downstream of
the various 5'-UTRs. B, Western blot (15 µg of total
cellular protein) of nNOS protein and RPA (15 µg of total cellular
RNA) of nNOS and neomycin cassette transcripts. Representative blots
are shown; three independent transfections were performed and assayed.
C, densitometric analysis of nNOS protein levels normalized
to mRNA levels. Data were derived from the three independent pools
of transfected cells and reflect the means ± S.E. Vct,
vector; CMV, cytomegalovirus; BGH pA, bovine
growth hormone polyadenylylation signal; Ex, exon.
326 nt with respect to the exon
1c/exon 2 boundary) (Fig. 3). Therefore, mRNAs of exon 1c exist in
both short (more abundant) and long (less abundant) versions. Our
RT-PCR results indicated that the AS exon is associated with both short
and long exon 1c transcripts. We therefore inquired whether the
translational repression of the AS exon is modified by the length of
preceding exon 1c sequence. To address this issue, we designed
luciferase reporter constructs to represent the common and shorter
5'-UTRs. In addition to the initially tested 326-nt exon 1c/AS exon
reporter, we selected cap sites corresponding to biologically relevant
mRNA transcripts of
101,
66, and
31 nt with respect to the
exon 1/exon 2 junction (Fig. 5A). Reporter constructs with
these lengths of exon 1c sequence were generated with and without the
AS exon. Transient RNA transfections in C2C12 (Fig.
6) and PC12 (data not shown) cells
revealed an increased potency in the relative repression due to the AS
exon as the distance from the cap was reduced. The translational
efficiency of the RNAs lacking the AS exon was enhanced by >450% as
the 5'-UTR was shortened (326 versus 31 nt), possibly due to
an increase in the rate of ribosome scanning. The mRNAs possessing
the AS exon failed to follow this trend and remained relatively
translationally repressed. Therefore, mapping of the exon 1c start
sites resulted in a detailed assessment of the translational effect of
the AS exon and confirmed that this novel exon imposes a significant translational blockade. The magnitude of the translational repression imposed by the AS exon was especially profound when the AS exon was
located close the cap.
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Fig. 5.
Mapping of the human nNOS exon 1c promoter
transcriptional start sites. A, genomic sequence of the
promoter and exonic sequences of human nNOS exon 1c. The first residue
of exon 1c upstream of exon 2 is numbered 1. Arrows denote
the start sites determined using primer extension analysis.
B, primer extension analysis of the human nNOS exon 1c
promoter transcriptional start sites. The extension products of a 27-nt
32P-labeled antisense oligonucleotide primer complementary
to
14 to +13 were separated by denaturing PAGE. Products were sized
with a 35S-labeled T7-primed pBluescript SK(
) I ladder
(representative result, n = 3). The locations of the
transcriptional start sites are numbered on the left (relative to
A). C, RPA of human nNOS exon 1c promoter
transcripts. An exon 1c antisense riboprobe was hybridized to total
cellular RNA isolated from human skeletal muscle or to yeast tRNA (not
shown) and digested with RNase. Protected fragments were
size-fractionated alongside RNA markers on denaturing polyacrylamide
gels; nucleotide lengths are indicated on the left. The
bracket indicates that the majority of transcripts initiated
between
80 and
30 nt (representative RPA, n = 3).
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Fig. 6.
Effect of cap site position on the
translational repression mediated by the AS exon. Reporter
constructs were generated either including or excluding the AS exon
inserted between exons 1c and 2. Numbering indicates the nucleotide
length of exon 1c 5'-UTR sequence separating the 5'-methyl-G cap site
and the start of the AS exon. Effects of the various leader sequences
on the translational efficiency of in vitro transcribed,
capped nNOS 5'-UTR/luciferase reporter RNAs were determined in
transiently transfected cultured C2C12 cells. Luciferase activity was
normalized for protein content and expressed as a percentage of the
vector. The data from these transient transfections represent the
means ± S.E. of four independent experiments, each performed in
triplicate.
12.8
kcal/mol for this putative structure (Fig.
7A) (32). We postulated that
this structure, or components of it, may harbor the functional RNA cis-element responsible for the associated translational
repression. To test this hypothesis, we mutated various parts of the
putative stem-loop structure and assayed the functional effects using
transient RNA transfections and stably transfected cell lines. Our
mutations of the exon 1c/AS exon sequences reflected, in part, existing data regarding the IRE stem-loop and the importance of its structural features (33, 34). In the predicted structure of the exon 1c/AS exon
stem-loop, the initial 8 nt of the 5'-stem are derived from exon 1c
sequences (Fig. 7A). Therefore, exon 1 determines, in part,
the primary RNA sequences that are predicted to be incorporated into
secondary RNA structures. This model is consistent with the observation
that the repressive effect of the AS exon is critically dependent upon
which exon 1 variant is upstream of the AS exon. We generated one
mutation of the loop (L1), altering its sequence from CAGU to ACAA.
This change was not predicted to significantly affect the structure or
stability of the element, which maintained a comparable Gibbs free
energy value of
12.8 kcal/mol. We also generated three mutations of
the stem component. The first (S1) modified the GC sequence of the
2-nucleotide bulge located on the lower half of the stem to AA; and the
second (S2) deleted the 2-nucleotide bulge entirely. Both of these stem
mutations had minor effects on the overall structure of the stem-loop
and maintained Gibbs values of less than or equal to
12.7 kcal/mol. The third stem mutation (S3) removed 7 nucleotides from the 5'-side of
the stem (UGGUUGC), completely disrupted the predicted structure, and
reduced the stability to
2.5 kcal/mol. Each mutation was introduced
into the previously described exon 1c/AS exon (31 nt)/luciferase reporter constructs and tested in transient RNA transfections of C2C12,
PC12, and CHO-K1 cells. The results were similar across cell types;
only the results obtained with C2C12 are shown (Fig. 7B).
The mutations of the AS exon were also incorporated into the construct
used to express the nNOS ORF downstream of the exon 1c/AS exon (31 nt)-containing nNOS 5'-UTR stably within the CHO-K1 cell line. As in
Fig. 4, nNOS protein was quantified and normalized to nNOS and neomycin
mRNA levels. The relative expression levels, reflective of the
translational efficiencies of the various mRNAs, are presented in
Fig. 7C. The effects of the mutations were comparable in
both the transient RNA and stable DNA transfections. We observed that
mutations of the loop (L1) or bulge (S1 and S2) failed to reverse the
translational blockade imposed by the AS exon. These sequences are
therefore not critical to the function of the AS exon. Disruption of
the stem (S3) completely abrogated the translational blockade imposed
by the AS exon. In both the transient and stable transfections, the
translational readout was comparable to the RNA lacking the AS exon.
Therefore, using independent methodologies, we have demonstrated that
the translational repression of the AS exon is dependent upon the
putative stem-loop structure. We have also determined that the
nucleotide sequences of the loop and bulge do not appear to be
critical, whereas the overall stem component is required for the
function of this element.
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Fig. 7.
A putative stem-loop motif mediates the
translational repression of the AS exon. A, predicted
structures present within the 5'-UTR of exon 1c/AS exon mRNAs and
the tested mutations. Altered nucleotides are boxed, and
deleted nucleotides are indicated (arrows). The structure is
composed of nucleotides from exon 1c (lowercase) and the AS
exon (uppercase). B, mean luciferase activities
of transiently transfected exon 1c (31 nt) reporter RNAs. Luciferase
RNAs with 5'-UTRs either excluding (black bars) or including
(gray bars) the AS exon or a mutated stem-loop
(hatched bars) were transfected into C2C12 cells. Activities
are relative to the normalized wild-type (wt) exon 1c (31 nt) construct lacking the AS exon. Data are the means ± S.E. of a
representative experiment, performed in triplicate (n = 3). C, translational profile of nNOS mRNAs within stably
transfected CHO-K1 cell lines. Transcripts with native or mutant exon
1c/AS exon 5'-UTRs were assessed. Results from densitometric analysis
of steady-state nNOS protein levels normalized to mRNA are
expressed relative the exon 1c (31 nt) construct. A representative
Western blot is shown; sample order reflects that of the
graph.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, interferon-
, transforming growth factor-
1, and
m-numb, among others (40-48). Perhaps the best
characterized model of translational control mediated by a 5'-UTR
cis-element is the IRE (49-55). This stem-loop structure is
the binding site for IRP-1 and IRP-2 (56-58). The binding of IRP-1 to
the IRE is stimulated by reduced intracellular iron levels and inhibits
the translation of the mRNA. This couples the expression of genes
involved in iron metabolism to available cellular iron levels (59, 60).
We noted that the AS exon is associated with both short and long exon
1c transcripts. Interestingly, the relative inhibitory effect of the AS
exon is more potent when it is closer to the 5'-methyl cap. This
finding is supported by prior work addressing the positional effect of
RNA cis-regulatory sequences with respect to the cap
mRNA structure. The binding of IRP-1 to an IRE located close to the
cap structure of mRNAs represses translation by precluding the
recruitment and formation of the 43 S preinitiation complex (29). This
mechanism is also position-dependent; reporter mRNAs
bearing IREs located farther downstream exhibit diminished
translational control and lose the ability to respond to cellular iron
levels via IRP binding (61). Cap-distal IREs still retain a repressive
effect on translation, although the mechanism is distinct and
represents interference with productive ribosome scanning. Our findings
using increasingly shorter 5'-UTRs fit this model of translational
blockade and suggest that the AS exon, in conjunction with exon 1c,
would exist as a major translational deterrent.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY098642.
Recipient of a Canadian Institutes of Health Research Doctoral
fellowship award.
§ Recipient of a Heart and Stroke Foundation Career investigator award and supported by Grant T-3688 from the Heart and Stroke Foundation of Canada. To whom correspondence should be addressed: Dept. of Medicine, University of Toronto, Medical Sciences Bldg., Rm. 7358, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-2441; Fax: 416-978-8765; E-mail: p.marsden@utoronto.ca.
Published, JBC Papers in Press, October 25, 2002, DOI 10.1074/jbc.M209988200
2 Y. Wang, S. C. Bevan, D. C. Newton, S. X. Zhang, S. VanDamme, M. Lorenzo, T. L. Miller, and P. A. Marsden, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are: nNOS, neuronal nitric-oxide synthase; 5'-UTR, 5'-untranslated region; ORF, open reading frame; IRE, iron-responsive element; IRP, iron-responsive element-binding protein; 5'-RACE, rapid amplification of 5'-cDNA ends; AS, alternatively spliced; RPA, RNase protection assay; nt, nucleotide(s); NO, nitric oxide; RT, reverse transcription.
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