Natural antisense transcript of natriuretic peptide precursor A (NPPA): structural organization and modulation of NPPA expression
© Annilo et al; licensee BioMed Central Ltd. 2009
Received: 25 March 2009
Accepted: 11 August 2009
Published: 11 August 2009
Mammalian transcriptome contains a large proportion of diverse and structurally complex noncoding RNAs. One class of such RNAs, natural antisense transcripts (NATs), are derived from the opposite strand of many protein-coding genes. Although the exact structure and functional relevance of most of the NATs is unknown, their emerging role as gene expression regulators raises the hypothesis that NATs might contribute to development of complex human disorders. The goal of our study was to investigate the involvement of NATs in regulation of candidate genes for blood pressure.
First we analysed blood pressure candidate genes for the presence of natural antisense transcripts. In silico analysis revealed that seven genes (ADD3, NPPA, ATP1A1, NPR2, CYP17A1, ACSM3, SLC14A2) have an antisense partner transcribed from the opposite strand. We characterized NPPA and its antisense transcript (NPPA-AS) in more detail. We found that NPPA-AS is expressed in a number of human tissues as a collection of alternatively spliced isoforms and that NPPA-AS and NPPA can form RNA duplexes in vivo. We also demonstrated that a specific NPPA-AS isoform is capable of down-regulating the intron-retained NPPA mRNA variant. We studied the evolutionary conservation of NPPA-AS and were able to detect the presence of Nppa-as transcript in mouse.
Our results demonstrate functional interaction of NPPA-AS with NPPA at the RNA level and suggest that antisense transcription might be an important post-transcriptional mechanism modulating NPPA expression.
A number of large-scale transcriptional mapping studies have shown that the mammalian transcriptome is extremely complex not only due to alternative splicing but also (and maybe primarily) because of the abundance of noncoding and often overlapping transcriptional units [1–4]. This has raised the hypothesis of RNA-based regulatory system that has allowed the elaboration and expansion of phenotypic complexity of multicellular organisms . It appears that the transcription from both strands in eukaryotic genomes is widespread [6–10], resulting in a large pool of complementary RNAs, or natural sense-antisense transcript pairs. The diversity and extent of antisense transcription suggests that this may be an important and common mechanism of gene expression modulation (recently reviewed in [11–13]).
Depending on the methodological approach and criteria for antisense transcript detection, the estimates of the proportion of transcripts involved in formation of sense-antisense pairs varies from 20 to 40% [2, 6–10]. Majority of the natural antisense transcripts (NATs) originate from the opposite DNA strand of the same locus as the sense transcript (cis-NATs). In some cases, NATs can be transcribed from different loci on the genome (trans-NATs) . Although high-throughput studies have investigated expression pattern and evolution of antisense transcripts on a genome-wide scale, the direct regulatory role of NATs has been demonstrated only in a few cases. The mode of NAT action includes very different mechanisms like transcriptional interference , RNA masking , and epigenetic silencing by triggering heterochromatin formation . In addition, other double-stranded RNA dependant mechanisms like RNA editing or RNA interference may be involved. It has been shown that bidirectionally transcribed loci in mouse can produce endogenous siRNAs  and therefore may regulate gene expression by means of RNAi. In the case of Zeb2 (zinc finger E-box binding homeobox 2) expression regulation, a NAT masks one of the 5' splice sites of Zeb2 pre-mRNA, thereby causing the retention of regulatory intron that is necessary for the translation of Zeb2 protein . Strong phenotypic effect of antisense transcription was shown in a specific case of thalassemia which is caused by a deletion leading to aberrant antisense transcription and silencing of a neighboring gene by CpG island methylation . The potential role of NATs in the regulation of gene expression raises the hypothesis that they might contribute to complex genetic human disorders such as cardiovascular disease, cancer, diabetes or mental disorders.
The goal of the present study was to investigate whether natural antisense transcripts are involved in regulation of candidate genes for hypertension. We proposed that the functional variation of candidate genes might be affected by the interaction with regulatory factors, including non-coding antisense RNAs. We focused on the genes with demonstrated role in familial forms of hypo- and hypertension from a salt-water homeostasis pathway [19–21].
We identified seven genes that are associated with cis-NATs (ADD3, NPPA, ATP1A1, NPR2, CYP17A1, ACSM3, SLC14A2). Detailed analysis was carried out for NPPA (natriuretic peptide precursor A) and its natural antisense transcript, NPPA-AS. NPPA codes for a precursor of atrial natriuretic peptide (ANP) that protects the cardiovascular system from the volume and pressure overload by decreasing vascular smooth muscle tone. Common genetic variants at the NPPA locus that are associated with the higher ANP concentration are also associated with lower blood pressure and reduced risk of hypertension . In addition, NPPA expression is tightly regulated during the embryonic heart development [23, 24], suggesting that complex regulatory mechanisms control the activity of NPPA.
Natural antisense transcripts associated with candidate genes for blood pressure regulation
Hypertension candidate genes associated with antisense transcripts.
2 exons, both 5' UTR
U92992 (total 17 ESTs)
3 exons, 99 aa
NPPA(natriuretic peptide precursor A)
BU732528 (total 12 ESTs)
1 exon, 121 aa
ATP1A1(Na+/K+ -ATPase alpha 1 subunit)
3 coding exons
AK309389 (total 27 ESTs)
NPR2(natriuretic peptide receptor B)
1 exon (coding and 3' UTR)
SPAG8 (total 37 ESTs)
8 exons, 501 aa
CYP17A1(cytochrome P450, family 17)
3 coding exons
BX100578 (total 6 ESTs)
ACSM3(acyl-coenzyme A synthetase)
1 coding exon
EXOD1 (total 49 ESTs)
11 exons, 328 aa
SLC14A2(solute carrier family 14, member 2)
1 noncoding 5' UTR exon
AK126075 (total 12 ESTs)
Structure and expression of NPPA in human tissues
We investigated the expression of NPPA using commercial Human Multiple Tissue cDNA panels MTC I and II (Figure 1B, C). Consistent with previous studies [23–25], the strongest expression of NPPA was detected in heart, but several tissues contained additional alternative products. Sequencing of these products revealed that the largest NPPA band detected in many tissues (1820 bp) represents the isoform of NPPA with retained both introns (further referred to as NPPA+Intr1+2). Visual inspection of the agarose gel (Figure 1B) indicates that the expression of this unspliced form and correctly spliced NPPA appear to be inversely correlated. Alternative product of 722 bp that was observed in liver, testis and leukocytes contains the retained intron 1 (NPPA+Intr1). In addition, a product shorter than correctly spliced NPPA mRNA (457 bp) was detected in testis. Sequencing of this product revealed that its sequence and splicing pattern are similar to the transcripts originating from the opposite strand, suggesting that this is actually an isoform of NPPA-AS, which was not represented by any of the ESTs in the database. To ensure that the amplification of NPPA+Intr1+2 is not caused by genomic DNA contamination, additional PCR experiments were performed with several primers that detect only NPPA-AS, but not NPPA [see Additional file 2 – Figure S2]. Because NPPA and NPPA-AS are both transcribed from the same genomic locus, contamination with the genomic DNA should result in amplification of unspliced NPPA-AS as well. However, these reactions yielded only the products corresponding to correctly spliced NPPA-AS, indicating no presence of the genomic contamination and demonstrating that the NPPA+Intr1+2 isoform indeed represents mRNA with retained introns.
NPPA-AS is expressed as a collection of alternatively spliced isoforms
Next, we characterized the structure and expression profile of NPPA-AS in human tissues using the panel of tissue-specific cDNAs (Figures 1C, D). Sequencing of eight identified isoforms confirmed that they all are spliced according to GT-AG consensus rule [see Additional file 2 – Figures S3 and S4]. It appears that NPPA-AS isoforms are not a result of alternative usage of different exons, but rather almost every exon displays at least two alternative splice donor/acceptor sites (Figure 1C). Majority of the alternative splicing events occur at the acceptor site of the intron. In addition, clearly identifiable polypyrimidine tract is located close to the splice acceptor site of all introns.
Next, we mapped the 3' end of NPPA-AS by RACE using RNA from HeLa cell line. We designed the gene-specific primers that would identify the 3' variants that are important in respect of complementarity with NPPA. Sequencing of the 3' RACE products identified two alternative 3' ends of NPPA-AS. One of the RACE products confirmed the presence of 3'-terminal exon that was predicted based on EST sequences CD368210 and BU732528 (3'RACE.1, Figure 1C). In addition, we identified a novel 3'-terminal exon that overlaps with the second intron and third exon of NPPA (3'RACE.2, Figure 1C). Expression analysis of the 3'RACE.2 isoform showed that among the sixteen tissues analysed, it is expressed only in testis (Figure 1D). Isoform 3'RACE.1 contains a suboptimal AGTAAA poly(A) signal 16 nucleotides upstream of the cleavage site, in the position where the majority of poly(A) signals are located . 3'RACE.2 isoform does not contain a detectable polyadenylation signal, but an A-rich element (AAAGAGAACACAGACATA), similar to the element found in PAPOLG gene , that is also lacking any poly(A) signal variant, is located 19 nucleotides upstream of polyadenylation site. This suggests that in addition to alternative splicing, the processing of the NPPA-AS transcript might be regulated also at the level of polyadenylation.
Primary sequence of NPPA-AS is not evolutionarily conserved
The last exon of NPPA-AS 3'RACE.1 isoform (Figure 1C, represented also by ESTs GenBank:CD368210 and BU732528) contains an open reading frame (ORF) that is predicted to code for a protein of 121 amino acids [see Additional file 2 – Figure S5]. The multiple alignment and in silico translation demonstrate that in all organisms except human and chimpanzee, the predicted ORF is interrupted by at least one frameshift and one stop codon [see Additional file 2 – Figures S5A and S6]. Since the translated amino acid sequence does not contain any conserved domains and has no significant identity above 30% to any known protein, the function of the predicted protein in human and chimpanzee cannot be assessed based on the primary sequence.
Positive correlation between the expression levels of intron-retained NPPA and specific NPPA-AS isoforms
NPPA-AS as a modulator of expression of NPPA splicing isoforms
We cotransfected NPPA expression construct into the mouse embryonic fibroblast cell line NIH3T3 in pairs with individual NPPA-AS constructs and quantified correctly spliced and intron-retained variants of NPPA by real-time RT-PCR. Mouse cell line was selected for transfection in order to eliminate the possible effect of endogenous expression of NPPA and NPPA-AS. The expression of specific NPPA-AS isoforms in transfected cells was confirmed by quantitative RT-PCR (data not shown). As shown in Figure 4B, expression level of intron-retained NPPA variant was significantly downregulated after transfection with pNPPA-AS-1 (P = 0.002, Mann-Whitney test). Although all constructs caused slight changes in expression levels of both spliced and intron-retained NPPA, the effects did no reach statistical significance in other experiments.
The important regulatory role of endogenous noncoding RNAs, including antisense transcripts, has been proposed based on a number of large-scale transcription profiling studies. Because of the variety of functional mechanisms and lack of direct experimental support, the biological meaning of most of this noncoding transcription is still unclear. In the present study we have investigated NPPA/NPPA-AS sense-antisense transcript pair.
Several studies have addressed the question whether some fraction of antisense transcripts may in fact be artifacts of reverse transcription reaction [28, 29]. To exclude such artifacts from our study, we considered only NATs with two or more exons and consensus splice sequences. In addition, 3' RACE reactions further confirmed the strand-specificity of NPPA-AS by identification of two alternative polyadenylated 3' terminal exons (Figure 1C).
NPPA is a functional candidate gene for elevated blood pressure, coding for an atrial natriuretic peptide (ANP), a member of a small family of endogenous peptide hormones. It is produced primarily by atrial cardiocytes in response to increasing cardiac wall tension. Association of specific NPPA variants with increased ANP levels as well as with lower blood pressure and reduced risk of hypertension  strongly support the central role of NPPA in the maintenance of blood pressure homeostasis. In addition, a region harbouring NPPA was among the eight loci identified in the meta-analysis of blood pressure genome-wide association studies . Transcriptional regulation of NPPA and maturation of ANP have been studied quite extensively (for recent reviews see [31, 32]) and our results add further evidence to the elaborate control of NPPA expression.
During the expression analysis of NPPA mRNA we found the strongest expression of correctly spliced NPPA in heart and moderate expression in a number of human tissues, including prostate, pancreas and small intestine, for example (Figure 1B). In addition, we observed the expression of NPPA isoforms with retained introns (NPPA+Intr1 and NPPA+Intr1+2) in several tissues (Figure 1B). Normally, ANP is synthesized as a 153-amino acid preprohormone. Removal of the signal peptide creates a 126-amino acid prohormone that is further cleaved to form a mature C-terminal 27-amino acid ANP. In the case of NPPA+Intr1 and NPPA+Intr1+2, the ORF that starts with the first methionine (Met-1) of prepro-ANP encodes only for a signal peptide region and is terminated after the frameshift caused by the intron retention. An alternative ORF of intron-retained NPPA isoforms, starting with the methionine Met-51 includes the mature ANP sequence, but since the peptide encoded by this putative ORF does not contain a signal sequence, its proper processing and biological activity is doubtful.
The natural antisense transcript of NPPA, that we named NPPA-AS, is widely present in human tissues and displays a complex pattern of alternative splicing (Figure 1D). All different NPPA-AS splicing isoforms overlap with both exonic and intronic regions of NPPA, including intron-exon boundaries (Figure 1C). Such overlap pattern raised the hypothesis that NPPA-AS may be involved in the regulation of NPPA expression.
Overlapping antisense gene pairs are preferentially co-expressed or inversely expressed in human tissues [6, 33, 34], supporting a model of negative control by antisense RNA that proposes state of balance in case of co-expression or up- or downregulation in case of inverse expression. Interestingly, the expression of NPPA-AS was strongly correlated with the intron-retained, rather than correctly spliced form of NPPA (Figure 3), indicating that it may play a functional role in posttranscriptional regulation of NPPA expression. Such correlation, however, does not necessarily indicate causal relationship, because it can be affected by many factors including regulation of transcription of both NPPA and NPPA-AS, or modulation of NPPA splicing by other factors.
Conservation often reflects functional significance of a nucleotide sequence. Among sense-antisense pairs, less than 7% are found to be conserved between human and mouse [35, 36]. This may indicate that antisense transcripts are mostly species-specific, or alternatively, that the process of transcription, secondary structure or organization of the transcript rather than the primary sequence is functionally important. Among conserved sense-antisense pairs, about one third have identical expression pattern in mouse and human . We found that expression profile in mouse and human is different for both NPPA and NPPA-AS (Figure 2). In mouse, Nppa is strongly expressed in some tissues (brain, lung, liver) where human NPPA is expressed weakly. We did not detect any alternatively spliced or intron-retained Nppa forms in mouse. In case of NPPA-AS, we found that neither the primary structure nor the expression pattern is conserved between mouse and human: in human it is expressed in all tissues examined, while in mouse the expression was observed only in brain (Figure 2A). However, both mouse and human antisense transcripts overlap with the exon-intron boundaries of NPPA (Figure 2B), implying that such genomic arrangement might be functionally significant.
Although many antisense transcripts overlap with the intron-exon boundaries of the sense mRNA, the effect of endogenous antisense transcripts as splicing regulators is studied in detail only in a few cases [16, 38]. Modulation of mRNA splicing by exogenous antisense oligonucleotides has gained more attention and its therapeutic potential has been established in clinical trials involving patients with Duchenne's muscular dystrophy . Much less is known about the role of endogenous antisense RNAs in regulation of splicing or stability of different mRNA isoforms. The complementarity of NPPA and its antisense exons suggests that if NPPA-AS has a function in regulation of NPPA, it depends on the mechanisms that involve the interactions at the RNA level. Our results show (Figure 4B) that at least one NPPA-AS isoform can modulate the ratio of unspliced and spliced NPPA variants, by decreasing the levels of intron-retained NPPA form. Since we were using minigene expression system, we excluded the effects of such possible regulatory mechanisms like transcriptional interference and heterochromatin formation . Currently we do not know what is the exact mechanism of intron-retained NPPA downregulation, but it is possible that the formation of the duplex RNA due to the complementary regions can lead to post-transcriptional regulation via different mechanisms like RNA masking (in which case the binding of factors required for splicing or export is blocked), RNA editing or RNA interference . Although the role of RNA interference in NAT-mediated regulation in mammals has been controversial, Watanabe et al.  identified seventeen loci in mouse where siRNAs arose from interaction of sense-antisense transcripts of the same locus. Considering the large number of NATs in mammals, the real extent of siRNA biogenesis via endogenous sense-antisense RNA interaction in mammalian cells remains still unknown. It is also possible that since antisense transcription extends through entire NPPA locus and into the promoter region, the mechanisms like transcriptional interference and modulation of NPPA promoter elements can occur and affect the expression of NPPA independently.
Although the biological role of NPPA antisense transcription needs further investigation, the regulatory role of NPPA in both adult cardiovascular system and in heart development during embryogenesis [23, 24] suggest that NPPA-AS may be involved in fine-tuning of NPPA expression during embryonic development or in response to specific stimulus.
We have identified the natural antisense transcripts of human blood pressure candidate genes and provide a detailed characterization of an antisense transcript associated with the NPPA gene. Our data support the biological significance of NPPA-AS by demonstrating that it (i) is widely expressed as a collection of canonically spliced isoforms, (ii) can directly interact with NPPA at the RNA level and (iii) is able to influence the levels of intron-retained NPPA variants.
Identification and in silico analysis of natural antisense transcripts
Candidate genes (n = 38) were selected according to the prior evidence of involvement in blood pressure regulation [see Additional file 1 – Table S1]. Most of the genes were selected based on the published data on the biology and genetics of blood pressure regulation. The selection included also genes responsible for the Mendelian forms of hypertension or hypotension, location near linkage peaks or quantitative trait loci (QTLs), reports on animal models and human association studies. Additional information was obtained from different resources (OMIM, http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim; NCBI GeneBank and NCBI Locuslink http://www.ncbi.nlm.nih.gov/; Ensembl http://www.ensembl.org/index.html). Candidate gene list was also supplemented with loci involved in other cardiovascular diseases like myocardial infarction, coronary artery disease and stroke. The candidate genes were screened for antisense transcripts using recently published sense-antisense transcript data . The structure and direction of the transcripts was verified using UCSC Genome Browser http://genome.ucsc.edu/. To avoid random „transcriptional noise” and genomic DNA contamination, we considered only transcripts with at least two exons, canonical splice sites (GT/AG) and overlap with at least one exon of the sense gene. In addition, several transcripts had a polyadenylation signal and poly(A) tail, supporting together with canonical splice sites their strand-specificity.
Multiple alignment of sequences corresponding to predicted ORF of NPPA-AS from different species was performed using ClustalW2 at http://www.ebi.ac.uk/Tools/clustalw2/index.html. Sequence database searches were performed using BLAST programs at http://www.ncbi.nlm.nih.gov/.
PCR and sequencing
Expression analysis of NPPA and NPPA-AS was carried out using Human Multiple Tissue cDNA panels MTC I and II (BD Biosciences) and primers [see Additional file 1 – Table S2] that were designed using Primer3 program http://frodo.wi.mit.edu/. G3PDH primers were included with MTC panels. PCR on mouse tissue-specific cDNAs was performed using oligonucleotides designed according to mouse Nppa gene and EST BQ771223 (for Nppa-as). PCR conditions were: 75 mM Tris-HCl (pH 8.8), 20 mM (NH4)2SO4, 0.01% Tween 20, 2.5 mM MgCl2, 250 μM dNTPs and 2.5 u per 100 μl Taq DNA Polymerase (Fermentas). Cycling conditions followed the touch-down procedure, namely initial denaturation at 94°C for 2 m, followed by 11 cycles at 94°C for 30 s, annealing for 30 s at temperatures decreasing from 62 to 57°C (with 0.5°C decremental in each cycle), 72°C for 60 s, and 30 cycles at 94°C for 30 s, 57°C for 30 s, 72°C for 60 s, and ending with an extension step at 72°C for 5 m.
For sequencing, PCR products were extracted from agarose gel using NucleoSpin Extract II (Macherey-Nagel) and either cloned into the pTZ57R vector using InsT/Aclone Kit (Fermentas) or sequenced directly after ExoI/SAP (both Fermentas) treatment. Sequencing reactions were performed using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to manufacturer's instructions and analyzed on ABI Prism™ 3730xl DNA Analyzer. Sequencing results were manually analyzed using Bioedit software http://www.mbio.ncsu.edu/BioEdit/bioedit.html and mapped to the genome using BLAT alignment tool  at the UCSC Genome Browser http://genome.ucsc.edu/. Novel sequences obtained in this study have been submitted to GenBank database (FJ706070–FJ706079).
3' RACE (rapid amplification of cDNA ends)
The 3' RACE reactions were performed using GeneRacer™ Kit (Invitrogen) and 1 μg of total RNA isolated from semi-confluent HeLa cells grown in 5% CO2 at 37°C. Reverse transcription was performed using SuperScript III Reverse Transcriptase and GeneRacer™ Oligo dT Primer (both included in the kit). Amplification was carried out using GeneRacer™ or 3' primer and the following gene-specific primers: NPPA-AS-F1, -F2 or -F5. Nested PCR was performed using nested GeneRacer™ primer and suitable gene-specific primers.
Construction of plasmids
The vector pQM-Ntag/A (Quattromed, Estonia) was used to create expression constructs under the control of CMV promoter. NPPA gene was amplified from human genomic DNA using primers NPPA-GFXba and NPPA-GRBam and inserted into the Xba I and Bam HI sites of pQM-Ntag/A. These primers amplify a 2069 bp fragment (chr1:11828358-11830426 according to human genome assembly hg18) of genomic DNA, including all three NPPA coding exons and both UTRs. Four constructs representing different NPPA-AS splicing isoforms were generated. pNPPA-AS-1 and -2 represent splicing isoforms NPPA-AS.1 and NPPA-AS.2 obtained with oligonucleotides NPPA-AS-F4 and NPPA-AS-R1. For generation of pNPPA-AS-3 and -4, 3' RACE products 3'RACE.1 and 3'RACE.2 were utilized. All constructs were sequenced using internal primers as well as universal primers flanking the insert cloning site.
Transfection of expression constructs
Cells were grown in 5% CO2 at 37°C and on the day before transfection were plated into 24-well plates using medium without antibiotics. Next day, cells were transfected using Lipofectamine™ 2000 reagent (Invitrogen) and 1.0 μg of total plasmid DNA (0.2 μg of pNPPA-mg2 and 0.8 μg of any of the NPPA-AS constructs or empty vector). Cells were incubated at 37°C and RNA was isolated 24 hours later. For relative quantitation experiments, transfections were carried out in triplicate three times (total nine replicates).
Total RNA from cell lines and from mouse (male C57bl/6) tissues was isolated using TRIzol reagent (Invitrogen) and the quantity and quality of RNA was assessed using Nanodrop ND-1000 (Thermo Scientific). DNase treatment was performed using TURBO DNA-free Kit™ (Ambion) with 1 μg of RNA in a volume of 30 μl. cDNA was synthesized using 2 μg of total RNA and First Strand cDNA Synthesis Kit (Fermentas) according to manufacturer's instructions.
Real-time quantitative PCR
For investigation of expression levels of NPPA and NPPA-AS in human tissues, the Human Multiple Tissue cDNA panels MTC I and II (BD Biosciences) were used. Amplification was performed with the following primer pairs: NPPA-Ex1Ex2-F and NPPA-Ex2-R for quantification of spliced NPPA; NPPA-In1Ex2-F and NPPA-Ex2-R for detection of unspliced NPPA. For quantification of NPPA-AS, primers NPPA-ASRT-F4 and NPPA-ASRT-R4 were used. GAPDH was used as an endogenous control and amplified with primers GAPDH-S and GAPDH-AS. For quantification of spliced and unspliced NPPA from transfection experiments, RNA from mouse NIH3T3 cells was isolated and DNase-treated as described above. Primers were designed avoiding binding to mouse sequences to prevent nonspecific amplification. Correctly spliced NPPA mRNA was detected using primers NPPA-Ex1Ex2-F and NPPA-Ex2R, unspliced NPPA was detected using primers NPPA-In1Ex2-F and NPPA-Ex2-R. Two endogenous reference genes were used: GAPDH (amplified with primers GAPDH-S and GAPDH-AS) and HPRT1 (amplified with primers HPRT1-S and HPRT1-AS). The reactions were performed in the 96-well microtiter plate using ABI PRISM® 7900 Real-Time PCR cycler. The 25 μl reaction mixture consisted of 3 μl of 1:10 cDNA dilution, 12.5 μl of ABsolute™ QPCR SYBR® Green ROX Mix (Thermo Scientific), 70 nM forward and reverse primer. The cycling parameters were: enzyme activation at 95°C for 15 m followed by 40 cycles 95°C for 15 s, 60°C for 30 s, 72°C for 30 s. Reactions were performed in triplicates for each biological replicate. As negative controls for DNA contamination, reactions without the reverse transcriptase were carried out. We performed control and optimization experiments for all primer pairs and selected for actual quantitation experiments primer pairs with amplification efficiency of 100 ± 10%. During optimization, serial dilutions of template were used and the specificity of the PCR products was confirmed by the presence of a single peak during the dissociation curve analysis. Amplification efficiency of the reactions was 100 ± 10% and intra- and inter-assay variation coefficients were below 3% and 8%, respectively. PCR efficiencies were calculated from ten-fold dilution series and relative expression of correctly spliced and intron-retained NPPA was calculated according to Pfaffl  by taking PCR efficiency into account. The geometric mean of GAPDH and HPRT1 was used as an endogenous control. Statistical significance of the results was analyzed using two-tailed Mann-Whitney test.
RNA from NIH3T3 cells cotransfected with constructs pNPPAmg2 and pNPPA-AS-1 or pNPPA-AS-3 was isolated as described previously and treated with DNase (TURBO DNA-free Kit™, Ambion) according to manufacturer's instructions and 0.5 μl of RNase A (10 mg/ml, Fermentas). RNase was inactivated with 0.5 mg/ml proteinase K treatment at the presence of 1% SDS for 30 min at 37°C. RNA was extracted with phenol/chloroform treatment and cDNA was synthesized using primers In1R (for NPPA-AS-1 duplex detection) or duplR (for NPPA-AS-3) and First Strand cDNA Synthesis Kit (Fermentas). In parallel, control experiments using RNAs not treated with RNase A and reverse transcriptions without reverse transcriptase were performed. PCR was performed with primers In1R and Ex1In1F for detection of NPPA::NPPA-AS-1 duplex and with primers duplF and duplR for detection of NPPA::NPPA-AS-3 duplex.
We thank Kristiina Rull for providing placental RNA, prof. Toivo Maimets for providing cell culture facilities, Urmo Võsa and Viljo Soo for technical assistance and Martti Laan, Kai Kisand and Sulev Kuuse for mouse tissue samples. We also thank Mart Speek for stimulating discussions and Jaana Männik and Ana Rebane for critical review of the manuscript. This work was supported by Wellcome Trust grant no. 070191/z/03/z (to M.L.), Howard Hughes Medical Institute grant #55005617 (to M.L.), Estonian Ministry of Education and Science core grant no. 0182721s06 (to M.L.) and Estonian Science Foundation grant ETF6597 (to T.A.).
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