Validation of trans-acting elements that promote exon 7 skipping of SMN2 in SMN2-GFP stable cell line.
Spinal muscular atrophy is a genetic disease in which the SMN1 gene is deleted. The SMN2 gene exists in all of the patients. Alternative splicing of these two genes are different. More than 90% of exon 7 included form is produced from SMN1 pre-mRNA, whereas only ∼20% of exon 7 included form is produced from SMN2 pre-mRNA. Only exon 7 inclusion form produces functional protein. Exon 7 skipped SMN isoform is unstable. Here we constructed a GFP reporter system that recapitulates the alternative splicing of SMN1 and SMN2 pre-mRNA. We designed a system in which GFP protein is expressed only when exon 7 of is included in alternative splicing. The stable cell that expresses SMN1-GFP produces ∼4 times more GFP protein than the stable cell line that expresses SMN2-GFP; as demonstrated by microscopy, FACS analysis and immunoblotting. In addition the ratio of exon 7 inclusion and skipping of SMN1-GFP and SMN2-GFP pre-mRNA was similar to endogenous SMN1 and SMN2 pre-mRNA as shown in RT-PCR. Furthermore the knockdown with hnRNP A1 shRNA, a known protein which promotes exon 7 skipping of SMN2, induces exon 7 inclusion of exon 7 in SMN2-GFP pre-mRNA in SMN2-GFP cell line. We conclude that we have established the stable cell lines that recapitulate alternative splicing of the SMN1 and SMN2 genes. The stable cell line can be used to identify the trans-acting elements with siRNA.
School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea.
Sunghee
S
Cho
Heegyum
H
Moon
Xiaoming
X
Yang
Jianhua
J
Zhou
Hye-Ran
HR
Kim
Myung-Geun
MG
Shin
Tiing Jen
TJ
Loh
Xuexiu
X
Zheng
Haihong
H
Shen
D017398:Alternative Splicing; D002460:Cell Line; D005091:Exons; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D009134:Muscular Atrophy, Spinal / Q000235:genetics; D014176:Protein Biosynthesis; D012322:RNA Precursors; D050436:Regulatory Elements, Transcriptional; D055533:Survival of Motor Neuron 1 Protein / Q000235:genetics; D055540:Survival of Motor Neuron 2 Protein / Q000235:genetics
D012322:RNA Precursors; C526471:SMN1 protein, human; C526477:SMN2 protein, human; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein; D049452:Green Fluorescent Proteins