School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea.
Biochem Biophys Res Commun. 2012 Jul 6;423(3):531-5. doi: 10.1016/j.bbrc.2012.05.161. Epub 2012 Jun 7.
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.
脊髓性肌萎缩症是一种遗传性疾病，其中 SMN1 基因缺失。所有患者都存在 SMN2 基因。这两个基因的选择性剪接不同。SMN1 前体 mRNA 产生超过 90%的包含外显子 7 的形式，而 SMN2 前体 mRNA 仅产生约 20%的包含外显子 7 的形式。只有包含外显子 7 的形式才能产生有功能的蛋白。外显子 7 跳过的 SMN 异构体不稳定。我们构建了一个 GFP 报告系统，该系统再现了 SMN1 和 SMN2 前体 mRNA 的选择性剪接。我们设计了一个系统，只有当外显子 7 包含在选择性剪接中时才表达 GFP 蛋白。表达 SMN1-GFP 的稳定细胞产生的 GFP 蛋白比表达 SMN2-GFP 的稳定细胞系多约 4 倍；这通过显微镜、FACS 分析和免疫印迹证明。此外，SMN1-GFP 和 SMN2-GFP 前体 mRNA 的外显子 7 包含和跳过的比例与内源性 SMN1 和 SMN2 前体 mRNA 相似，如 RT-PCR 所示。此外，hnRNP A1 shRNA 的敲低，一种已知促进 SMN2 外显子 7 跳过的蛋白，可诱导 SMN2-GFP 前体 mRNA 中外显子 7 的包含，在 SMN2-GFP 细胞系中。我们得出结论，我们已经建立了能够再现 SMN1 和 SMN2 基因选择性剪接的稳定细胞系。稳定细胞系可用于使用 siRNA 鉴定反式作用元件。
