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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

The levels of SMN2 full-length mRNA and protein decrease during testis cell primary culture.(A) Total RNA was isolated from primary testis cells cultured for different time periods (2 hours, 48 hours, 96 hours and 24 days) and subjected to RT-PCR to amplify SMN2 FL and Δ7 mRNAs. For comparison, total RNA isolated directly from the testis and liver was also analyzed. A representative result of three independent experiments was shown. The result showed that primary testis cells after a 2-hour culture still expressed high level of SMN2 FL mRNA. However, the level decreased after longer cultures. (B) Proteins extracted from primary testis cells cultured for 2 hours and 96 hours were subjected to Western blotting to detect SMN protein. The result showed that 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. (C) snRNP complexes were isolated from primary testis cells cultured for 2 hours and 96 hours by anti-Sm antibodies. Various snRNAs were then extracted and quantitated by real-time PCR. The result showed that the levels of snRNP complexes decreased in 96-hour cultured cells compared with 2-hour cultured cells. Error bars represent standard deviation. (*) P < 0.05, compared with 2-hour cultured cells.
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pone.0120721.g002: The levels of SMN2 full-length mRNA and protein decrease during testis cell primary culture.(A) Total RNA was isolated from primary testis cells cultured for different time periods (2 hours, 48 hours, 96 hours and 24 days) and subjected to RT-PCR to amplify SMN2 FL and Δ7 mRNAs. For comparison, total RNA isolated directly from the testis and liver was also analyzed. A representative result of three independent experiments was shown. The result showed that primary testis cells after a 2-hour culture still expressed high level of SMN2 FL mRNA. However, the level decreased after longer cultures. (B) Proteins extracted from primary testis cells cultured for 2 hours and 96 hours were subjected to Western blotting to detect SMN protein. The result showed that 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. (C) snRNP complexes were isolated from primary testis cells cultured for 2 hours and 96 hours by anti-Sm antibodies. Various snRNAs were then extracted and quantitated by real-time PCR. The result showed that the levels of snRNP complexes decreased in 96-hour cultured cells compared with 2-hour cultured cells. Error bars represent standard deviation. (*) P < 0.05, compared with 2-hour cultured cells.

Mentions: 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.


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)

The levels of SMN2 full-length mRNA and protein decrease during testis cell primary culture.(A) Total RNA was isolated from primary testis cells cultured for different time periods (2 hours, 48 hours, 96 hours and 24 days) and subjected to RT-PCR to amplify SMN2 FL and Δ7 mRNAs. For comparison, total RNA isolated directly from the testis and liver was also analyzed. A representative result of three independent experiments was shown. The result showed that primary testis cells after a 2-hour culture still expressed high level of SMN2 FL mRNA. However, the level decreased after longer cultures. (B) Proteins extracted from primary testis cells cultured for 2 hours and 96 hours were subjected to Western blotting to detect SMN protein. The result showed that 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. (C) snRNP complexes were isolated from primary testis cells cultured for 2 hours and 96 hours by anti-Sm antibodies. Various snRNAs were then extracted and quantitated by real-time PCR. The result showed that the levels of snRNP complexes decreased in 96-hour cultured cells compared with 2-hour cultured cells. Error bars represent standard deviation. (*) P < 0.05, compared with 2-hour cultured cells.
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pone.0120721.g002: The levels of SMN2 full-length mRNA and protein decrease during testis cell primary culture.(A) Total RNA was isolated from primary testis cells cultured for different time periods (2 hours, 48 hours, 96 hours and 24 days) and subjected to RT-PCR to amplify SMN2 FL and Δ7 mRNAs. For comparison, total RNA isolated directly from the testis and liver was also analyzed. A representative result of three independent experiments was shown. The result showed that primary testis cells after a 2-hour culture still expressed high level of SMN2 FL mRNA. However, the level decreased after longer cultures. (B) Proteins extracted from primary testis cells cultured for 2 hours and 96 hours were subjected to Western blotting to detect SMN protein. The result showed that 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. (C) snRNP complexes were isolated from primary testis cells cultured for 2 hours and 96 hours by anti-Sm antibodies. Various snRNAs were then extracted and quantitated by real-time PCR. The result showed that the levels of snRNP complexes decreased in 96-hour cultured cells compared with 2-hour cultured cells. Error bars represent standard deviation. (*) P < 0.05, compared with 2-hour cultured cells.
Mentions: 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.

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