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High expression level of Tra2-β1 is responsible for increased SMN2 exon 7 inclusion in the testis of SMA mice.

Chen YC, Chang JG, Jong YJ, Liu TY, Yuo CY - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Overexpression of Tra2-β1, but not ASF/SF2, increases SMN2 exon 7 inclusion in primary testis cells and spinal cord neurons of SMA mice.Primary testis cells (A) and primary spinal cord neurons (B) of SMA mice were co-transfected with SMN2 minigene plasmid and Tra2-β1 overexpression plasmid, ASF/SF2 overexpression plasmid or blank vector as control for 48 hours. Total RNA was isolated from transfected cells and then subjected to RT-PCR to amplify SMN2 minigene FL and Δ7 mRNAs. The result showed that overexpression of Tra2-β1, but not ASF/SF2, remarkably increased SMN2 exon 7 inclusion in both primary testis cells and primary spinal cord neurons of SMA mice. Error bars represent standard deviation. (*) P < 0.05, compared with the vector control.
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pone.0120721.g005: Overexpression of Tra2-β1, but not ASF/SF2, increases SMN2 exon 7 inclusion in primary testis cells and spinal cord neurons of SMA mice.Primary testis cells (A) and primary spinal cord neurons (B) of SMA mice were co-transfected with SMN2 minigene plasmid and Tra2-β1 overexpression plasmid, ASF/SF2 overexpression plasmid or blank vector as control for 48 hours. Total RNA was isolated from transfected cells and then subjected to RT-PCR to amplify SMN2 minigene FL and Δ7 mRNAs. The result showed that overexpression of Tra2-β1, but not ASF/SF2, remarkably increased SMN2 exon 7 inclusion in both primary testis cells and primary spinal cord neurons of SMA mice. Error bars represent standard deviation. (*) P < 0.05, compared with the vector control.

Mentions: 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 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).


High expression level of Tra2-β1 is responsible for increased SMN2 exon 7 inclusion in the testis of SMA mice.

Chen YC, Chang JG, Jong YJ, Liu TY, Yuo CY - PLoS ONE (2015)

Overexpression of Tra2-β1, but not ASF/SF2, increases SMN2 exon 7 inclusion in primary testis cells and spinal cord neurons of SMA mice.Primary testis cells (A) and primary spinal cord neurons (B) of SMA mice were co-transfected with SMN2 minigene plasmid and Tra2-β1 overexpression plasmid, ASF/SF2 overexpression plasmid or blank vector as control for 48 hours. Total RNA was isolated from transfected cells and then subjected to RT-PCR to amplify SMN2 minigene FL and Δ7 mRNAs. The result showed that overexpression of Tra2-β1, but not ASF/SF2, remarkably increased SMN2 exon 7 inclusion in both primary testis cells and primary spinal cord neurons of SMA mice. Error bars represent standard deviation. (*) P < 0.05, compared with the vector control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4363149&req=5

pone.0120721.g005: Overexpression of Tra2-β1, but not ASF/SF2, increases SMN2 exon 7 inclusion in primary testis cells and spinal cord neurons of SMA mice.Primary testis cells (A) and primary spinal cord neurons (B) of SMA mice were co-transfected with SMN2 minigene plasmid and Tra2-β1 overexpression plasmid, ASF/SF2 overexpression plasmid or blank vector as control for 48 hours. Total RNA was isolated from transfected cells and then subjected to RT-PCR to amplify SMN2 minigene FL and Δ7 mRNAs. The result showed that overexpression of Tra2-β1, but not ASF/SF2, remarkably increased SMN2 exon 7 inclusion in both primary testis cells and primary spinal cord neurons of SMA mice. Error bars represent standard deviation. (*) P < 0.05, compared with the vector control.
Mentions: 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 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).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus