44 research outputs found

    The testis expresses the highest level of Tra2-β1 among the tissues examined.

    No full text
    <p>Various tissues of 18–20-week-old type 3 SMA mice were collected. (A) Total RNA was isolated and subjected to qRT-PCR to detect <i>Tra2-β1</i> and <i>ASF/SF2</i> mRNA levels. The result showed that among the tissues examined, the testis expressed the highest level of <i>Tra2-β1</i> mRNA, but not the highest level of <i>ASF/SF2</i> mRNA. (B) Proteins were extracted and subjected to Western blotting to detect Tra2-β1 and ASF/SF2 proteins. The result also showed that among the tissues examined, the testis expressed the highest level of Tra2-β1 protein, but not the highest level of ASF/SF2 protein.</p

    The levels of <i>SMN2</i> full-length mRNA and protein decrease during testis cell primary culture.

    No full text
    <p>(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 <i>SMN2</i> 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 <i>SMN2</i> 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 <i>SMN2</i> 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. (*) <i>P</i> < 0.05, compared with 2-hour cultured cells.</p

    Knockdown of Tra2-β1 decreases <i>SMN2</i> exon 7 inclusion in primary testis cells of SMA mice.

    No full text
    <p>Primary testis cells of SMA mice were cultured for 72 hours and then transfected with Tra2-β1 siRNA or negative control (NC) siRNA for 48 hours. Total RNA was isolated from transfected cells and then subjected to RT-PCR to amplify <i>SMN2</i> FL and Δ7 mRNAs. The results show that knockdown of Tra2-β1 promoted <i>SMN2</i> exon 7 exclusion in primary testis cells of SMA mice. Error bars represent standard deviation. (*) <i>P</i> < 0.05, compared with the siNC control.</p

    The levels of Tra2-β1 and ASF/SF2 decrease during testis cell primary culture, which is correlated with <i>SMN2</i> exon 7 splicing.

    No full text
    <p>(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 <i>Tra2-β1</i> and <i>ASF/SF2</i> mRNA expression levels. The result showed that the mRNA levels of <i>Tra2-β1</i> and <i>ASF/SF2</i> decreased after longer cultures, which was consistent with the protein expression. Error bars represent standard deviation. (*) <i>P</i> < 0.01, compared with 2-hour cultured cells.</p

    The testis of SMA mice expresses high levels of <i>SMN2</i> full-length mRNA and protein.

    No full text
    <p>Various tissues of type 3 SMA mice were collected. (A) Total RNA was isolated and subjected to RT-PCR to amplify both <i>SMN2</i> 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 <i>SMN2</i> 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.</p

    Overexpression of Tra2-β1, but not ASF/SF2, increases <i>SMN2</i> exon 7 inclusion in primary testis cells and spinal cord neurons of SMA mice.

    No full text
    <p>Primary testis cells (A) and primary spinal cord neurons (B) of SMA mice were co-transfected with <i>SMN2</i> minigene plasmid and <i>Tra2-β1</i> overexpression plasmid, <i>ASF/SF2</i> 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 <i>SMN2</i> minigene FL and Δ7 mRNAs. The result showed that overexpression of Tra2-β1, but not ASF/SF2, remarkably increased <i>SMN2</i> exon 7 inclusion in both primary testis cells and primary spinal cord neurons of SMA mice. Error bars represent standard deviation. (*) <i>P</i> < 0.05, compared with the vector control.</p

    Modulation the alternative splicing of <i>GLA</i> (IVS4+919G>A) in Fabry disease

    No full text
    <div><p>While a base substitution in intron 4 of <i>GLA</i> (IVS4+919G>A) that causes aberrant alternative splicing resulting in Fabry disease has been reported, its molecular mechanism remains unclear. Here we reported that upon IVS4+919G>A transversion, H3K36me3 was enriched across the alternatively spliced region. PSIP1, an adapter of H3K36me3, together with Hsp70 and NONO were recruited and formed a complex with SF2/ASF and SRp20, which further promoted <i>GLA</i> splicing. Amiloride, a splicing regulator in cancer cells, could reverse aberrant histone modification patterns and disrupt the association of splicing complex with <i>GLA</i>. It could also reverse aberrant <i>GLA</i> splicing in a PP1-dependant manner. Our findings revealed the alternative splicing mechanism of <i>GLA</i> (IVS4+919G>A), and a potential treatment for this specific genetic type of Fabry disease by amiloride in the future.</p></div
    corecore