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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 testis of SMA mice expresses high levels of SMN2 full-length mRNA and protein.Various tissues of type 3 SMA mice were collected. (A) Total RNA was isolated and subjected to RT-PCR to amplify both SMN2 full-length (FL) and exon 7-lacking (Δ7) mRNAs. A representative result of six independent experiments was shown. The result showed that the testis expressed high level of SMN2 FL mRNA. (B) Proteins were extracted and subjected to Western blotting to detect SMN protein expression. The result showed that SMA mouse testis expressed the highest level of SMN protein among the tissues examined.
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pone.0120721.g001: The testis of SMA mice expresses high levels of SMN2 full-length mRNA and protein.Various tissues of type 3 SMA mice were collected. (A) Total RNA was isolated and subjected to RT-PCR to amplify both SMN2 full-length (FL) and exon 7-lacking (Δ7) mRNAs. A representative result of six independent experiments was shown. The result showed that the testis expressed high level of SMN2 FL mRNA. (B) Proteins were extracted and subjected to Western blotting to detect SMN protein expression. The result showed that SMA mouse testis expressed the highest level of SMN protein among the tissues examined.

Mentions: 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.


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 testis of SMA mice expresses high levels of SMN2 full-length mRNA and protein.Various tissues of type 3 SMA mice were collected. (A) Total RNA was isolated and subjected to RT-PCR to amplify both SMN2 full-length (FL) and exon 7-lacking (Δ7) mRNAs. A representative result of six independent experiments was shown. The result showed that the testis expressed high level of SMN2 FL mRNA. (B) Proteins were extracted and subjected to Western blotting to detect SMN protein expression. The result showed that SMA mouse testis expressed the highest level of SMN protein among the tissues examined.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4363149&req=5

pone.0120721.g001: The testis of SMA mice expresses high levels of SMN2 full-length mRNA and protein.Various tissues of type 3 SMA mice were collected. (A) Total RNA was isolated and subjected to RT-PCR to amplify both SMN2 full-length (FL) and exon 7-lacking (Δ7) mRNAs. A representative result of six independent experiments was shown. The result showed that the testis expressed high level of SMN2 FL mRNA. (B) Proteins were extracted and subjected to Western blotting to detect SMN protein expression. The result showed that SMA mouse testis expressed the highest level of SMN protein among the tissues examined.
Mentions: 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.

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