Modulating the exon 7 skipping or inclusion of SMN gene by pH changes   Graduate Institute of Medicine, Faculty of Biomedi...
<ul><li>An autosomal recessive disease characterized  </li></ul><ul><li>by degeneration of motor neuron in anterior  </li>...
<ul><li>Gene structure of SMN :   </li></ul>J Med Genet 1999; 36: 1-8 S urvival  M otor  N euron (SMN) <ul><li>The disease...
SMN 1 and SMN 2 are highly homologous , only 5 base differences (three are in 3’ ends , two are in exon 7 ESE site and exo...
<ul><li>More than 95% of SMA patients show deletions  </li></ul><ul><li>in the telomeric SMN copy (SMN1) </li></ul><ul><li...
RT-PCR analysis of SMN expression in human lymphoblast cell lines cultured in different pH media (I) Exon 7 inclusion was ...
The exon 7 splicing change occurred after the cells were cultured over 2 hrs RT-PCR analysis of SMN expression in human ly...
Western blotting analysis of SMN expression in human lymphoblast cell lines cultured in different pH media Intact SMN prot...
Gem in SMA I fibroblast cultured in pH7.0 medium
 
21.0  +  1.7  pH 7 4.7  +  0.6  b   pH 6 Total number of nuclear gems  per 100 cells (mean  +  SD)   pH of media  a a   Pr...
Lower pH (pH6.0) Decrease   1.Cytoplasmic  SRp20  2.phosphorylation from  ASF/SF2  in both cytoplasmic and nuclear fractio...
<ul><li>The amount of exon 7-containing SMN protein has  </li></ul><ul><li>been shown to be an inverse indicator of diseas...
<ul><li>In this study, we find that increase extracellular pH change  </li></ul><ul><li>results in  SMN2  exon7 inclusion ...
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Modulating the exon 7 skipping or inclusion of SMN gene by pH ...

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  • Spinal muscular atrophy, or SMA, is an autosomal recessive disease characterized by degeneration of motorneuron in spinal cord, which leads to muscular paralysis and atrophy. The incidence is one in six to ten thousand live births, and carrier frequency of one in fifty. SMA can be categorized into three types according to the age of onset and its severity, type one is the most severity, type three is mild.
  • The disease gene of SMA is survival motor neuron 1 gene which located at chromosome 5q, a second ctntromic copy, SMN2, also locates at the same region.
  • SMN1 and SMN2 are highly homologus, only 5 bases differences, the difference base in exon 7 results in different alternative splicing pattern, most transcripts of SMN1 are full-length include exon 7, but most transcripts of SMN2 are with out exon 7
  • Several studies have shown micronviromnent pH may be an important elemnt for the regulation of RNA altrnative splicing in vivo, we cultures the cell lines in different pH condition to check the change of exon 7 splicing of SMN2 gene.
  • RT-PCE analysis of SMN expression in human lymphoblast cell lines cultured in different pH media show that exon 7 inclusion is increase vary with pH increase. The exon 7 of SMN2 gene is skipping when the cells cultured in the media below pH7.0, and increase inclusion when medium above pH7.0
  • The mechanism of alternative splicing after extracellular pH change has been suggested that it is through the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 3/6-p38 pathway
  • The amount of exon 7-containing SMN protein has been shown to be an inverse indicator of disease severity in SMA patients and mice. Therefore, the drug increasing the expression of intact SMN protein may have clinically therapeutic effects on SMA patients
  • Modulating the exon 7 skipping or inclusion of SMN gene by pH ...

    1. 1. Modulating the exon 7 skipping or inclusion of SMN gene by pH changes Graduate Institute of Medicine, Faculty of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan, Department of Medical Research and Department of Laboratory Medicine, Division of Pediatric Neurology, Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan Yi-Ching Chen , Chung-Yee Yuo, Yuh-Jyh Jong, Hui-Hua Lin, Ya-Sian Chang and Jan-Gowth Chang
    2. 2. <ul><li>An autosomal recessive disease characterized </li></ul><ul><li>by degeneration of motor neuron in anterior </li></ul><ul><li>horn cells of spinal cord </li></ul>S pinal M uscular A trophy (SMA) <ul><li>Categorized into three types according to the </li></ul><ul><li>age of onset and its severity: type I, severe; </li></ul><ul><li>type II, intermediate; type III, mild </li></ul><ul><li>Incidence of 1/6,000~10,000 live births, and </li></ul><ul><li>carrier frequency of 1 in 50 </li></ul>
    3. 3. <ul><li>Gene structure of SMN : </li></ul>J Med Genet 1999; 36: 1-8 S urvival M otor N euron (SMN) <ul><li>The disease gene of SMA located at </li></ul><ul><li>chromosome 5q13 </li></ul>Cell. 1995 Jan 13;80(1):155-65. SMN 1 or SMN Tel SMN 2 or SMN Cen
    4. 4. SMN 1 and SMN 2 are highly homologous , only 5 base differences (three are in 3’ ends , two are in exon 7 ESE site and exon 8), results in different alternative splicing pattern
    5. 5. <ul><li>More than 95% of SMA patients show deletions </li></ul><ul><li>in the telomeric SMN copy (SMN1) </li></ul><ul><li>The severity of SMA is reversely correlated with </li></ul><ul><li>the amount of intact SMN protein in the spinal cord </li></ul>Nat Genet. 1997 Jul;16(3):265-9. <ul><li>SMN is expressed in all tissues and localizes to </li></ul><ul><li>both the cytoplasm and nucleus. In the nucleus it </li></ul><ul><li>is concentrated in dot-like structures, called gems, </li></ul><ul><li>which are often associated with coiled bodies </li></ul><ul><li>In cells and tissues from SMA patients the number </li></ul><ul><li>of gems is reduced with the most severe, type I, </li></ul><ul><li>patients showing very few or no gems </li></ul>
    6. 6. RT-PCR analysis of SMN expression in human lymphoblast cell lines cultured in different pH media (I) Exon 7 inclusion was increased after cultured at pH 7.0, 7.5, and 8.0 FL/  7 0.04 0.02 0.83 1.12 0.93 3.57 3.99 6.87 5.51 5.50
    7. 7. The exon 7 splicing change occurred after the cells were cultured over 2 hrs RT-PCR analysis of SMN expression in human lymphoblast cell lines cultured in different pH media (II) FL/  7 0.8 1.2 0. 3 1.1 0.6 1.2 0.9 3.6 0.3 4.7 0.3 1.8 0.8
    8. 8. Western blotting analysis of SMN expression in human lymphoblast cell lines cultured in different pH media Intact SMN protein increased when cell was cultured in pH 7.0 medium
    9. 9. Gem in SMA I fibroblast cultured in pH7.0 medium
    10. 11. 21.0 + 1.7 pH 7 4.7 + 0.6 b pH 6 Total number of nuclear gems per 100 cells (mean + SD) pH of media a a Primary fibroblasts from SMA patient were grown in pH 6 or pH 7 media for 72 hours. b Significant difference between cells cultured in pH 6 and pH 7 media, P < 0.001. SMA fibroblasts cultured in pH 6 media have less nuclear gems than those cultured in pH 7 media
    11. 12. Lower pH (pH6.0) Decrease 1.Cytoplasmic SRp20 2.phosphorylation from ASF/SF2 in both cytoplasmic and nuclear fraction Increase hnRNPA1 in nuclear fraction Higher pH (pH7.0) Increase 1. SRp20 in nuclear fraction 2. Sam68 in nuclear fraction
    12. 13. <ul><li>The amount of exon 7-containing SMN protein has </li></ul><ul><li>been shown to be an inverse indicator of disease </li></ul><ul><li>severity in SMA patients and mice </li></ul><ul><li>Extracellular pH change have impact on the transcript of </li></ul><ul><li>SMN2 gene in SMA cell lines: </li></ul><ul><li>extracellular pH exon 7-containing transcripts </li></ul><ul><li>extracellular pH exon 7-containing transcripts </li></ul>
    13. 14. <ul><li>In this study, we find that increase extracellular pH change </li></ul><ul><li>results in SMN2 exon7 inclusion which may due to </li></ul><ul><li>decrease the amount of hnRNPA1 in the nucleus. </li></ul><ul><li>hnRNPA1 has been shown to act in a general manner to </li></ul><ul><li>block splicing of exon7, the decrease of hnRNPA1 will favor </li></ul><ul><li>the SMN2 exon7 inclusion </li></ul><ul><li>The mechanism of alternative splicing after extracellular </li></ul><ul><li>pH change has been suggested that it is through the </li></ul><ul><li>mitogen-activated protein kinase/extracellular signal- </li></ul><ul><li>regulated kinase kinase 3/6-p38 pathway </li></ul>
    14. 15. Thank you for attention!

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