High Expression Level of Tra2-β1 Is Responsible for Increased SMN2 Exon 7 Inclusion in the Testis of SMA Mice

Spinal muscular atrophy (SMA) is an inherited neuromuscular disease caused by deletion or mutation of SMN1 gene. All SMA patients carry a nearly identical SMN2 gene, which produces low level of SMN protein due to mRNA exon 7 exclusion. Previously, we found that the testis of SMA mice (smn−/− SMN2) expresses high level of SMN2 full-length mRNA, indicating a testis-specific mechanism for SMN2 exon 7 inclusion. To elucidate the underlying mechanism, we established primary cultures of testis cells from SMA mice and analyzed them for SMN2 exon 7 splicing. We found that primary testis cells after a 2-hour culture still expressed high level of SMN2 full-length mRNA, but the level decreased after longer cultures. We then compared the protein levels of relevant splicing factors, and found that the level of Tra2-β1 also decreased during testis cell culture, correlated with SMN2 full-length mRNA downregulation. In addition, the testis of SMA mice expressed the highest level of Tra2-β1 among the many tissues examined. Furthermore, overexpression of Tra2-β1, but not ASF/SF2, increased SMN2 minigene exon 7 inclusion in primary testis cells and spinal cord neurons, whereas knockdown of Tra2-β1 decreased SMN2 exon 7 inclusion in primary testis cells of SMA mice. Therefore, our results indicate that high expression level of Tra2-β1 is responsible for increased SMN2 exon 7 inclusion in the testis of SMA mice. This study also suggests that the expression level of Tra2-β1 may be a modifying factor of SMA disease and a potential target for SMA treatment.


Introduction
Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by the loss of lower motor neurons and atrophy of muscles. According to age of onset and its severity, SMA is categorized into three main types (type I-III) and two subtypes (type 0 and type IV) [1,2]. SMA occurs in approximately 1 in 6,000 to 10,000 newborns and has a carrier frequency of 1 in 35 to 50. SMA is the second most common autosomal recessive disease and the most common genetic cause of infant mortality [3].
SMA is caused by deletions or mutations of survival motor neuron 1 (SMN1) gene. All SMA patients carry a nearly identical SMN2 gene. However, SMN2 expresses only limited amount of functional SMN protein and is unable to compensate for the SMN1 gene defect [4]. The two genes differ by only five nucleotides, and the most important difference between SMN1 gene and SMN2 gene is a C-to-T transition at 6 th nucleotide of exon 7. The nucleotide transition causes exon 7 exclusion in most SMN2 mRNA [5][6][7][8].
According to previous studies, there are two putative splicing models of SMN1 and SMN2 mRNAs: the C-to-U change in SMN2 mRNA causes the loss of a serine/arginine-rich (SR) splicing factor (SF2/ASF)-dependent exonic splicing enhancer (ESE) [9] or the creation of a heterogeneous nuclear ribonucleoprotein (hnRNP) A1-dependent exonic splicing silencer (ESS) [10]. In the central part of exon 7, an AG-rich ESE is recognized by the SR-like splicing factor Htra2-β1 which can promote exon 7 inclusion [11]. The Htra2-β1 also serves as a nucleation point for several additional regulatory factors that indirectly associate with SMN exon 7, including SRp30c and hnRNP-G [12,13].
The SMN protein is complexed with several proteins, including Gemins 2-8 and Unr-interacting protein (UNRIP) [14][15][16][17][18][19][20][21][22]. The SMN complex has multiple functions. One of the most important functions of the SMN complex is as an assemblyosome that is necessary for biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) [23][24][25][26]. The snRNP complexes are known as the major components of spliceosome. The major spliceosome consists of the snRNPs U1, U2, U4/U6, and U5, which is responsible for most pre-mRNA splicing. The minor spliceosome, by contrast, comprises snRNPs U11, U12, U4atac/U6atac, and U5, which is responsible for the splicing of a rare class of introns that have non-canonical consensus sequences [27]. The SMN complex also functions as mRNA carrier that promotes β-actin mRNA transport to the axon of motor neurons. The neuron-specific function might be the reason that the ubiquitouslyexpressed SMN protein defection causes motor neuron degeneration [25,28,29].
The first SMA-like mouse models were developed independently at the same time by Li group and Burghes group [30,31]. Mice have only one smn gene, which has similar function with human SMN1. Knockout of smn gene in mice caused embryonic lethal before E6.5. The SMA-like mice were generated by crossing smn +/− mice with human SMN2 transgene, which showed motor neuron degenerative symptoms [30]. Our previous study showed that the testis expressed high levels of SMN2 full-length mRNA and SMN protein, which is different from other tissues of SMA mice. In addition, hnRNP Q, a SMN exon 7-interacting protein, was found to be expressed at higher level in the testis compared with brain and liver. Overexpression of hnRNP Q1 promoted SMN2 exon 7 inclusion, but hnRNP Q2/3 had a negative role in SMN mRNA splicing. Therefore, differential expression of hnRNP Q isoforms might intricately regulate SMN mRNA splicing [32]. To further investigate the mechanism of SMN2 mRNA splicing in the testis of SMA mice, we established the primary testis cell culture from SMA mice. We found that the SMN2 full-length mRNA decreased in 96 hour-cultured primary testis cells compared with the testis tissue and 2 hour-cultured cells. Among the splicing factors we examined in primary testis cells, we found the expression of Tra2-β1 also dramatically decreased in 96 hour-cultured cells compared with 2 hour-cultured cells. In contrast, the expression levels of hnRNP Q showed no significant difference in primary testis cells of SMA mice. In addition, overexpression of Tra2-β1 in the primary testis cells increased SMN2 full-length mRNA expression, suggesting that SMN2 mRNA splicing change in testis cells is regulated by Tra2-β1.

Materials and Methods Animals
18-20-week-old type 3 SMA mice were used for the experiments. The mice were sacrificed by high concentration of carbon dioxide and the tissues were harvested after sacrifice. All breeding and subsequent use of animals, including mice sacrifice, in this study were approved by the Institutional Animal Care and Use Committee of Kaohsiung Medical University. The IACUC approval number is 98033.

Primary culture of testis cells from SMA mice
The testes were harvested from adult type 3 SMA mice (smn −/− SMN2 + ) [30] and decapsulated in PBS. Then the tissues were incubated with type IV collagenase (2.5 mg/ml) for 30 minutes at 37°C in serum-free culture medium (DMEM/F12) supplemented with 15 mM HEPES. The tissues and cells were centrifuged (5 minutes, 400g) and resuspended in 2 ml of Hank's balanced salt solution containing 0.1% Trypsin-EDTA and 40 unit DNaseⅠ and incubated for 10 minutes at 37°C. After centrifugation at 400g, the cells were washed 5 times by PBS and then resuspended in culture medium containing 5% horse serum and 2.5% fetal bovine serum. The cells were seeding on 0.1% gelatin-coated dishes and incubated at 37°C with 5% CO 2 .

Primary culture of spinal neuron cells from SMA mouse embryos
The spinal cord were harvested from E17.5 embryos of SMA mice (smn −/− SMN2 + ). Six to eight embryos from the same litter were collected together. The spinal cord were digested with 0.025% Trypsin-EDTA in GHEBS buffer (137 mM NaCl, 2.7 mM KCl, 22.2 mM glucose, 25 mM HEPES, pH 7.4, and 20 U/ml penicillin plus 20 mg/ml streptomycin) for 10 minutes at 37°C. Then the tissues were homogenized by mechanical dissociation and moved to 4% BSA in GHEBS buffer. The cells were centrifuged (10 minutes, 520g) and resuspended in 1 ml GHEBS buffer. The cells were isolated by centrifugation (10 minutes, 520g) on an Iodixanol (OptiPrep, Axis-Shield) gradient solution. Iodixanol solution (11.5%) was freshly prepared in GHEBS buffer. A sharp band on the top of the Iodixanol solution was the neuron fraction. The fraction was collected and resuspended in GHEBS buffer. The cells were centrifuged (10 minutes, 520g) in 4% BSA again and resuspended in culture medium (Neurobasal supplemented with B-27) containing 2% horse serum, 0.5 mM L-glutamine, and 25 μM 2-mercaptoethanol). The cells were seeded in 6-well plates and incubated at 37°C with 5% CO 2 for 3 days. The plates were coated with poly-ornithine (30 μg/ml) for 30 minutes and laminin (2 μg/ml) for 60 minutes.

Cell culture and transfection
Mouse testis cell lines, GC-1 spermatogonia (from Bioresource Collection and Research Center; BCRC Number: 60312) were grown in Dulbecco's Modified Eagle Medium with 4 mM Lglutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 1.0 mM sodium pyruvate, and 10% fetal bovine serum at 37°C in a humidified atmosphere containing 5% CO 2 . TM3 Leydig cells (from Bioresource Collection and Research Center; BCRC Number: 60475) and TM4 Sertoli cells (from Bioresource Collection and Research Center; BCRC Number: 60254) were grown in Dulbecco's Modified Eagle Medium/F12 with 4.5 g/L glucose, 2.5 mM Lglutamine, 0.5 mM sodium pyruvate, 1.2 g/L sodium bicarbonate, 15 mM HEPES supplemented with 5% horse serum and 2.5% fetal bovine serum at 37°C containing 5% CO 2 . The mouse testis cell lines, primary testis cells and primary neuron cells were transfected with pIRES-SMN2 minigene-luc plasmid (provided by Dr. Minlei Zhang [33]) by Lipofectamine 2000 Transfection Reagent (Invitrogen) according to manufacturer's instructions. The primary testis cells were transfected with Tra2-β1 siRNA (Ambion, siRNA ID: s73772) by Lipofectamine 2000 Transfection Reagent. After 48 hours of transfection, total RNA was extracted from transfected cells.

snRNP complex assembly
The cell lysate was extracted from primary testis cells using RSB-100 buffer (100 mM NaCl, 10 mM Tris-HCl, pH7.4, 2.5 mM MgCl 2 , and 0.1% NP-40) containing protease inhibitor cocktail (Roche) and phosphatase inhibitors (50 mM NaF, 0.2 mM Na 3 VO 4 ). After pre-cleaning with protein-G agarose for 1 h at 4°C, immunoprecipitations with anti-Sm (Smith Antigen) antibodies (Y12, Thermo Scientific) from cell extracts (30 μg) were carried out in RSB-500 buffer (500 mM NaCl, 10 mM Tris-HCl, pH 7.4, 2.5 mM MgCl 2 and 0.1% NP-40) containing protease inhibitor cocktail and phosphatase inhibitors for 2 h at 4°C. After five washes with the same buffer, bound RNAs were isolated from immunoprecipitates by TRIzol Reagent extraction and ethanol precipitation. snRNA-specific reverse primers were used to generate cDNA using the SuperScript III Reverse Transcriptase kit. Quantification of snRNAs was carried out by realtime PCR experiments on Applied Biosystems 7500 fast real-time PCR system using SYBR Green. The same primers were used in both reverse transcription and real-time PCR. The primers used were: U1 forward, 5'-GAT ACC ATG ATC ACG AAG GTG GTT-3', U1 reverse,

Statistical analysis
Statistical analysis of data was performed using Microsoft Excel 2010 software (Microsoft, Redmond, WA). Student's t test was conducted to evaluate differences between groups. A probability of less than 0.05 was considered to be statistically significant.

Results
The testis of SMA mice expresses high level of SMN2 full-length mRNA and SMN protein The SMA mice we used are deficient for mouse smn and express human SMN2 [30] and show pathological changes in the spinal cord and skeletal muscles similar to those of SMA patients. We examined the SMN2 mRNA splicing and SMN protein expression in several tissues of type 3 SMA mice, including brain, spinal cord, heart, liver, lung, stomach, kidney, spleen, intestine, muscle, tail, testis in male mice, and ovary in female mice. Among the tissues we examined, we found that the testis expressed high level of SMN2 full-length (FL) mRNA ( Fig. 1A and see S1 Fig. for qRT-PCR result) and SMN protein (Fig. 1B). According to the result, the ovary of SMA mice expressed high level of SMN2 truncated mRNA (Fig. 1A), indicating that the special splicing pattern in the testis is not germ cell-related. Thus, our results indicate a testis-specific mechanism of SMN2 mRNA splicing in SMA mice.
The levels of SMN2 full-length mRNA and protein decrease during testis cell primary culture To investigate the testis-specific mechanism of SMN2 mRNA splicing, we detected SMN2 mRNA splicing in three different mouse testis cell lines, including GC-1, TM3 and TM4, with SMN2 minigene transfection. GC-1 spermatogonia, TM3 Leydig cells and TM4 Sertoli cells are three major cell types in the testis. The result showed that SMN2 exon 7 inclusion is not promoted in mouse testis cell lines (S2 Fig.). We supposed that the special SMN2 mRNA splicing pattern in the testis is physiologically dependent. Therefore, ex vivo cultured mouse testis cell lines were unable to express high level of SMN2 full-length mRNA.
To solve the problem, we established primary cultures of testis cells from SMA mice and analyzed them for SMN2 exon 7 splicing. We found that SMA primary testis cells after a 2-hour culture still expressed high level of SMN2 full-length mRNA. However, the level decreased after longer cultures ( Fig. 2A and see S3 Fig. for qRT-PCR result). In addition, SMN protein level was high in 2-hour cultured cells and decreased dramatically in 96-hour cultured cells, consistent with the result of SMN2 exon 7 splicing (Fig. 2B). We also explored the function of SMN protein by measuring snRNP complex assembly. According to previous studies, SMN protein is part of the SMN complex and one of the functions of SMN complex is to participate in snRNP assembly [34]. The result showed that snRNP assembly decreased in 96-hour cultured cells compared with 2-hour cultured cells (Fig. 2C), indicating that decreased SMN expression was accompanied with the decrease of SMN functions. Therefore, our results showed that the levels of SMN2 full-length mRNA and protein decreased during testis cell primary culture, indicating the factor that promotes SMN2 exon 7 inclusion in the testis gradually disappears in the cell culture system.
The levels of Tra2-β1 and ASF/SF2 decrease during testis cell primary culture, which are correlated with SMN2 exon 7 splicing To investigate the mechanism of SMN2 full-length mRNA downregulation in the primary culture cell system, we compared the protein levels of relevant splicing factors, including ASF/SF2, hnRNP A1, Tra2-β1, SRp30c, and hnRNP Q, in 2-hour and 96-hour cultured cells. The protein levels of Tra2-β1 and ASF/SF2 were high in 2-hour cultured cells and decreased dramatically after 96 hours (Fig. 3A), consistent with the result of SMN2 exon 7 splicing. The mRNA levels of Tra2-β1 and ASF/SF2 also decreased gradually during testis cell primary culture (Fig. 3B). We also observed that the protein level of hnRNP A1 was low in 96-hour cultures cells compared with 2-hour cultured cells (Fig. 3A). However, previous studies showed that hnRNP A1 binds to the splicing silencer in SMN2 exon7 and acts as a splicing repressor to cause SMN2 exon 7 exclusion [10,35]. Thus, the downregulation of hnRNP A1 cannot explain the result of increased SMN2 exon 7 exclusion. Therefore, our results show that the changes in the levels of Tra2-β1 and ASF/SF2 during testis cell primary culture are correlated with SMN2 full-length mRNA downregulation.

The testis expresses the highest level of Tra2-β1 among the tissues examined
To confirm the importance of Tra2-β1 and ASF/SF2 in testis-specific SMN2 splicing, we examined mRNA and protein levels of Tra2-β1 and ASF/SF2 in several tissues of type 3 SMA mice. The result showed that the testis expressed the highest level of Tra2-β1 mRNA and protein ( Fig. 4A and 4B). In contrast, the expression levels of ASF/SF2 mRNA and protein were not the highest in the testis (Fig. 4A and 4B). The results support that the high level of Tra2-β1 in the testis plays an important role in SMN2 exon 7 inclusion.

Overexpression of Tra2-β1, but not ASF/SF2, increases SMN2 exon 7 inclusion in primary testis cells and spinal cord neurons of SMA mice
To clarify whether Tra2-β1 contributes to SMN2 exon 7 inclusion in the testis, we overexpressed Tra2-β1 or ASF/SF2 in 96-hour cultured primary testis cells. Because of the low transfection efficiency, the testis primary cells were transfected with overexpression plasmids and SMN2 minigene plasmid. Then we evaluated the effect of Tra2-β1 or ASF/SF2 overexpression by detecting SMN2 minigene splicing. The result showed that overexpression of Tra2-β1 enhanced SMN2 minigene exon 7 inclusion (Fig. 5A). However, overexpression of ASF/SF2 had no effect on SMN2 minigene exon 7 inclusion (Fig. 5A), indicating Tra2-β1 is the factor that plays a crucial role in SMN2 exon 7 inclusion in the testis of SMA mice. To further investigate the effect of Tra2-β1 in neuron cells of SMA mice, we also established primary neurons from The levels of Tra2-β1 and ASF/SF2 decrease during testis cell primary culture, which is correlated with SMN2 exon 7 splicing. (A) Proteins extracted from primary testis cells cultured for 2 hours and 96 hours were subjected to Western blotting to detect Tra2-β1, ASF/SF2, hnRNP A1, SRp30c, and hnRNP Q. A representative result of two independent experiments was shown. The result showed that the levels of Tra2-β1, ASF/SF2, and hnRNP A1 decreased dramatically from 2-hour to 96-hour cultures. (B) Total RNA was isolated from primary testis cells cultured for different time periods and subjected to qRT-PCR to detect Tra2-β1 and ASF/SF2 mRNA expression levels. The result showed that the mRNA levels of Tra2-β1 and ASF/SF2 decreased after longer cultures, which was consistent with the protein expression. Error bars represent standard deviation. (*) P < 0.01, compared with 2-hour cultured cells. spinal cords of SMA mouse embryos and overexpressed Tra2-β1 or ASF/SF2 in primary neurons. The result showed that overexpression of Tra2-β1, but not ASF/SF2, also enhanced SMN2 minigene exon 7 inclusion in spinal cord neurons of SMA mice (Fig. 5B).

Knockdown of Tra2-β1 decreases SMN2 exon 7 inclusion in primary testis cells of SMA mice
To further prove the role of Tra2-β1 in testis SMN2 exon 7 splicing, we knocked down Tra2-β1 expression by siRNA in primary testis cells of SMA mice. To achieve a higher transfection efficiency, we cultured the primary testis cells for 72 hours to allow cell attachment and then transfected them with Tra2-β1 siRNA or negative control (NC) siRNA. Although the level of Tra2-β1 has decreased in 72-hour cultured cells, further knockdown of Tra2-β1 and comparison of its effects with NC siRNA can still allow us to prove the role of Tra2-β1 in testis SMN2 exon 7 splicing. Knockdown of Tra2-β1 mRNA was verified by quantitative RT-PCR. Then, we found that siRNA knockdown of Tra2-β1 decreased SMN2 exon 7 inclusion compared with NC siRNA (Fig. 6), indicating that downregulation of Tra2-β1 promotes SMN2 exon 7 exclusion in primary testis cells of SMA mice. In conclusion, our results indicate that high expression level of Tra2-β1 is responsible for increased SMN2 exon 7 inclusion in the testis of SMA mice.

Discussion
We were surprised to find that the testis of SMA mice expresses high level of SMN2 full-length mRNA and SMN protein. In this study, we investigated the mechanism responsible for this special SMN2 splicing pattern in the testis of SMA mice. We found that the level of the splicing factor Tra2-β1 decreases during testis cell primary culture, which is correlated with SMN2 fulllength mRNA downregulation. Also, the testis of SMA mice expresses the highest level of Tra2-β1 among the many tissues examined. Furthermore, overexpression of Tra2-β1 in primary testis cells of SMA mice enhances SMN2 exon 7 inclusion, whereas knockdown of Tra2-β1 reduces SMN2 exon 7 inclusion. Therefore, our results indicate that high expression level of Tra2-β1 is responsible for increased SMN2 exon 7 inclusion in the testis of SMA mice. Previous studies had already shown that Tra2-β1 promoted the inclusion of SMN exon 7 and stimulated full-length SMN2 expression [11]. However, these studies used cell lines, such as human HEK-293 and mouse NIH3T3, to demonstrate the effect of Tra2-β1. In contrast, our study is the first one to show the important role of Tra2-β1 in SMN2 exon 7 splicing in an animal model. It remains to be shown whether this phenomenon also occurs in SMA patients.
In this study, we also found that overexpression of Tra2-β1 enhances SMN2 mRNA exon 7 inclusion not only in primary testis cells but also in spinal cord neurons of SMA mice. The result suggests that the expression level of Tra2-β1 may be a modifying factor of SMA disease. In support of this hypothesis, Helmken et al. showed that Tra2-β1 protein is differentially expressed between type I-III SMA patients and between affected and unaffected siblings of discordant SMA families, at least in lymphoblastoid cells [36]. Differential expression of Tra2-β1 may account for the different ratios of SMN2 full-length vs. truncated mRNA in type I-III SMA patients, which cannot be explained by different copy numbers of SMN2 gene. An analysis of 36 SMA patients and their siblings from 15 discordant families did not identify any mutation or polymorphism within the coding and promoter regions of Tra2-β1 gene [37], excluding a change on genomic level as a direct cause for differential expression of Tra2-β1 protein. Therefore, further studies on the regulatory mechanism of Tra2-β1 expression, especially in motor neurons, may reveal other SMA modifying factors that modulate full-length SMN production through regulating Tra2-β1 expression level.
Differential regulation of Tra2-β1 in testis tissue, primary testis cells, and testis cell lines is also intriguing. Identifying the mechanisms underlying this regulation may provide a way of modulating Tra2-β1 levels in motor neurons, and thus serve as a means of augmenting SMN. Previously, we suspected that high expression of Tra2-β1 in testis tissue might be induced by factors in testis microenvironment, which were not reproduced in cell culture. Thus, we treated testis cell lines with several testis microenvironmental factors, including low temperature, lactic acid, and androgens. Although we did not measure the Tra2-β1 levels directly, we found that these treatments could not increase SMN2 exon 7 inclusion in testis cell lines. The mechanism of Tra2-β1 regulation in the testis requires further investigation.
A variety of potential therapies have been developed for SMA, including small molecule drugs, antisense oligonucleotides, polypeptides and proteins, gene therapy, and stem cell therapy [38]. One kind of small molecules is histone deacetylase (HDAC) inhibitor, such as valproic acid, phenylbutyrate, trichostatin A and suberoylanilide hydroxamic acid. Previous studies showed that trichostatin A (TSA) can promote SMN2 gene transcription and increase survival rate in the SMA mouse model [39]. The mechanism of TSA activation of SMN2 gene transcription is to inhibit histone deacetylation at the promoter. However, the effects of TSA on inhibiting histone deacetylation and activating transcription are not specific to SMN2 gene. We have found that TSA can also activate Tra2-β1 gene transcription and increase SMN2 exon 7 inclusion (data not shown). In addition, we showed that overexpression of Tra2-β1, but not ASF/SF2, increases SMN2 exon 7 inclusion in spinal cord neurons of SMA mice. Therefore, our results in this study suggest that Tra2-β1 may be a potential target for SMA therapy.
Supporting Information S1 Checklist. The ARRIVE guidelines checklist for animal research. (DOCX) S1 Fig. Quantitative RT-PCR of SMN2 mRNA in tissues of SMA mice. Various tissues of type 3 SMA mice were collected. Total RNA was isolated and subjected to quantitative realtime RT-PCR to amplify SMN2 full-length (FL) and exon 7-lacking (Δ7) mRNAs independently. The expression levels of SMN2 FL and Δ7 mRNA were normalized to GAPDH separately. Then, the FL/Δ7 ratios of various tissues were shown. The result showed that the testis expressed high level of SMN2 FL mRNA. (TIF) S2 Fig. Analysis of SMN2 exon 7 splicing in testis-derived cell lines. Mouse testis cell lines, GC-1 spermatogonia, TM3 Leydig cells, and TM4 Sertoli cells, were transfected with the SMN2 minigene plasmid for 48 hours. For comparison, mouse NSC34 motor neuron cell lines were also transfected with SMN1 and SMN2 minigene plasmids. Total RNA was isolated and subjected to RT-PCR to amplify SMN1 and SMN2 minigene FL and Δ7 mRNAs. The result showed that SMN2 exon 7 inclusion was not promoted in these testis cell lines. (TIF) S3 Fig. Quantitative RT-PCR of SMN2 mRNA in primary testis cells of SMA mice. Total RNA was isolated from primary testis cells cultured for different time periods (2 hours, 48 hours, 96 hours) and subjected to quantitative real-time RT-PCR to amplify SMN2 full-length (FL) and exon 7-lacking (Δ7) mRNAs independently. The expression levels of SMN2 FL and Δ7 mRNA were normalized to GAPDH separately. Then, the FL/Δ7 ratios were shown. The result showed that SMN2 FL mRNA was high in 2-hour cultured cells and decreased in 96-hour cultured cells. (TIF)