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Modulating the exon 7
skipping or inclusion of
SMN gene by pH changes
Yi-Ching Chen, Chung-Yee Yuo, Yuh-Jyh Jong,
Hui-Hua Lin, Ya-Sian Chang and Jan-Gowth Chang
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
Spinal Muscular Atrophy (SMA)
An autosomal recessive disease characterized
by degeneration of motor neuron in anterior
horn cells of spinal cord
Incidence of 1/6,000~10,000 live births, and
carrier frequency of 1 in 50
Categorized into three types according to the
age of onset and its severity: type I, severe;
type II, intermediate; type III, mild
Survival Motor Neuron (SMN)
The disease gene of SMA located at
Cell. 1995 Jan 13;80(1):155-65.
chromosome 5q13
Gene structure of SMN：
SMN 2 or SMNCen
SMN 1 or SMNTel
J Med Genet 1999;36:1-8
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
More than 95% of SMA patients show deletions
in the telomeric SMN copy (SMN1)
The severity of SMA is reversely correlated with
the amount of intact SMN protein in the spinal cord
Nat Genet. 1997 Jul;16(3):265-9.
SMN is expressed in all tissues and localizes to
both the cytoplasm and nucleus. In the nucleus it
is concentrated in dot-like structures, called gems,
which are often associated with coiled bodies
In cells and tissues from SMA patients the number
of gems is reduced with the most severe, type I,
patients showing very few or no gems
RT-PCR analysis of SMN expression in human
lymphoblast cell lines cultured in different pH media (I)
FL/ D7
0.04 0.02 0.83 1.12 0.93
3.57 3.99 6.87 5.51 5.50
Exon 7 inclusion was increased after cultured
at pH 7.0, 7.5, and 8.0
RT-PCR analysis of SMN expression in human
lymphoblast cell lines cultured in different pH media (II)
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
The exon 7 splicing change occurred after the
cells were cultured over 2 hrs
FL/ D7
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
Gem in SMA I fibroblast
cultured in pH7.0 medium
SMA fibroblasts cultured in pH 6 media have less nuclear
gems than those cultured in pH 7 media
pH of media
a
a
Total number of nuclear gems
per 100 cells (mean + SD)
pH 6
4.7 + 0.6 b
pH 7
21.0 + 1.7
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.
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
The amount of exon 7-containing SMN protein has
been shown to be an inverse indicator of disease
severity in SMA patients and mice
Extracellular pH change have impact on the transcript of
SMN2 gene in SMA cell lines:
extracellular pH
exon 7-containing transcripts
extracellular pH
exon 7-containing transcripts
The mechanism of alternative splicing after extracellular
pH change has been suggested that it is through the
mitogen-activated protein kinase/extracellular signalregulated kinase kinase 3/6-p38 pathway
In this study, we find that increase extracellular pH change
results in SMN2 exon7 inclusion which may due to
decrease the amount of hnRNPA1 in the nucleus.
hnRNPA1 has been shown to act in a general manner to
block splicing of exon7, the decrease of hnRNPA1 will favor
the SMN2 exon7 inclusion
Thank you for attention!