Hakim Nor Hakimah Ab, Kounishi Toshiki, Alam A H M Khurshid, Tsukahara Toshifumi, Suzuki Hitoshi
Center for Nano Materials Technology, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1211, JapanGraduate School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1211, Japan.
Genes Cells. 2010 Mar;15(3):255-67. doi: 10.1111/j.1365-2443.2009.01378.x. Epub 2010 Feb 7.
Mef2c protein is one of the MADS-box type transcription factors involved in muscular differentiation and synaptic formation. Previously, it has been reported that the Mef2c gene is responsible for three alternative splicing regulations. Here, we investigated the alternative splicing variants of Mef2c during neural differentiation of P19 cells and during cardio muscular differentiation of P19 clone 6 (P19CL6). We detected that two Mef2c mRNA isoforms, using exon α1 with and without the γ region at exon 10, are mainly produced in immature P19 cells. Remarkably, Mef2c isoforms containing exon β specifically appeared in the neural cell stage. Because most transcripts contain exon β in the neural cell stage and in the brain, this suggests that the alternative splicing of exon β is highly regulated. Among known regulators, Fox-1 was specifically expressed in the neural cell stage in correlation with Mef2c exon β. Fox-1 promoted exon β inclusion in transfection experiments using Mef2cβ minigene. Moreover, we found that the promotion required RNA-binding activity of Fox-1 and GCAUG sequence located in adjacent intron of exon β. Taken together, our results suggest that Fox-1, expressed specifically in the neural cell stage, promoted Mef2c exon β inclusion via the GCAUG.
Mef2c蛋白是参与肌肉分化和突触形成的MADS盒型转录因子之一。此前有报道称，Mef2c基因负责三种可变剪接调控。在此，我们研究了P19细胞神经分化过程以及P19克隆6（P19CL6）心肌分化过程中Mef2c的可变剪接变体。我们检测到，在未成熟的P19细胞中主要产生两种Mef2c mRNA异构体，它们使用第10外显子带有或不带有γ区域的α1外显子。值得注意的是，含有β外显子的Mef2c异构体特异性出现在神经细胞阶段。由于大多数转录本在神经细胞阶段和大脑中都含有β外显子，这表明β外显子的可变剪接受到高度调控。在已知的调控因子中，Fox-1在神经细胞阶段与Mef2cβ外显子相关特异性表达。在使用Mef2cβ小基因的转染实验中，Fox-1促进了β外显子的包含。此外，我们发现这种促进作用需要Fox-1的RNA结合活性以及位于β外显子相邻内含子中的GCAUG序列。综上所述，我们的结果表明，在神经细胞阶段特异性表达的Fox-1通过GCAUG促进了Mef2cβ外显子的包含。
