Modulating the exon 7 Modulating the exon 7 skipping or inclusion of skipping or inclusion of

SMN gene by pH changesSMN 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

An autosomal recessive disease characterized by degeneration of motor neuron in anterior horn cells of spinal cord

Spinal Muscular Atrophy (SMA)

Categorized into three types according to the age of onset and its severity: type I, severe; type II, intermediate; type III, mild

Incidence of 1/6,000~10,000 live births, and carrier frequency of 1 in 50

Gene structure of SMN ：

J Med Genet 1999;36:1-8

Survival Motor Neuron (SMN)

The disease gene of SMA located at chromosome 5q13

SMN 1 or SMNTelSMN 2 or SMNCen

Cell. 1995 Jan 13;80(1):155-65.

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)

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

The exon 7 splicing change occurred after the cells were cultured over 2 hrs

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

RT-PCR analysis of SMN expression in human lymphoblast cell lines cultured in different pH media (II)

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

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

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

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

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

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