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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 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.
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pone.0120721.g003: 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.

Mentions: 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.


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 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.
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Related In: Results  -  Collection

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pone.0120721.g003: 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.
Mentions: 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.

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