Laboratory for Familial Dysautonomia Research, Department of Biological Sciences, Fordham University, Bronx, NY 10458, USA.
FEBS J. 2013 Aug;280(15):3632-46. doi: 10.1111/febs.12355. Epub 2013 Jun 18.
The ability to modulate the production of the wild-type transcript in cells bearing the splice-altering familial dysautonomia (FD) causing mutation in the IKBKAP gene prompted a study of the impact of a panel of pharmaceuticals on the splicing of this transcript, which revealed the ability of the cardiac glycoside digoxin to increase the production of the wild-type, exon-20-containing, IKBKAP-encoded transcript and the full-length IκB-kinase-complex-associated protein in FD-derived cells. Characterization of the cis elements and trans factors involved in the digoxin-mediated effect on splicing reveals that this response is dependent on an SRSF3 binding site(s) located in the intron 5' of the alternatively spliced exon and that digoxin mediates its effect by suppressing the level of the SRSF3 protein. Characterization of the digoxin-mediated effect on the RNA splicing process was facilitated by the identification of several RNA splicing events in which digoxin treatment mediates the enhanced inclusion of exonic sequence. Moreover, we demonstrate the ability of digoxin to impact the splicing process in neuronal cells, a cell type profoundly impacted by FD. This study represents the first demonstration that digoxin possesses splice-altering capabilities that are capable of reversing the impact of the FD-causing mutation. These findings support the clinical evaluation of the impact of digoxin on the FD patient population.
能够调节携带 IKBKAP 基因中导致家族性自主神经机能异常 splicing 改变突变的细胞中野生型转录本的产生，促使我们研究了一组药物对该转录本剪接的影响，这揭示了心脏糖苷地高辛能够增加野生型、包含外显子 20 的、由 IKBKAP 编码的转录本和全长 IκB-激酶复合物相关蛋白在 FD 衍生细胞中的产生。对涉及地高辛介导的剪接作用的顺式元件和反式因子的特征分析表明，这种反应依赖于位于选择性剪接外显子 5' 内含子中的 SRSF3 结合位点(s)，并且地高辛通过抑制 SRSF3 蛋白的水平来发挥其作用。通过鉴定几种 RNA 剪接事件，其中地高辛处理介导外显子序列的增强包含，从而促进了对地高辛介导的 RNA 剪接过程的作用的特征分析。此外，我们证明了地高辛在神经元细胞中影响剪接过程的能力，神经元细胞是受 FD 严重影响的细胞类型。这项研究首次证明地高辛具有改变剪接的能力，能够逆转 FD 致病突变的影响。这些发现支持对地高辛对 FD 患者人群影响的临床评估。
