Translational Regulation of Human Neuronal Nitric-oxide Synthase by an Alternatively Spliced 5'-Untranslated Region Leader Exon*

Derek C. NewtonDagger, Siân C. Bevan, Stephen Choi, G. Brett Robb, Adam Millar, Yang Wang, and Philip A. Marsden§

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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(-), 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).

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 [alpha -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.).

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 [gamma -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.

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(-); 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 beta -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 beta -galactosidase activity and protein content as described (6).

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 [gamma -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).

RPA-- A uniformly [alpha -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).

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. [alpha -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).

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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.

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.


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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.

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 (approx 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 beta -galactosidase activity derived from cotransfected, capped, polyadenylated beta -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.

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.


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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.

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 (-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.

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 -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.

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-alpha , interferon-gamma , transforming growth factor-beta 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.

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.

    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.

Dagger 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.

    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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ABSTRACT
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RESULTS
DISCUSSION
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