Medical Research Center, The Affiliated Hospital of Qingdao University, Qingdao 266555, China.
Department of Nephrology, The Affiliated Qingdao Municipal Hospital of Qingdao University, Qingdao 266071, China.
Hum Mol Genet. 2022 Nov 28;31(23):4006-4018. doi: 10.1093/hmg/ddac148.
All mutations in exon 9 of the Cullin3 gene associated with pseudohypoaldosteronism type II (PHA II) contribute to exon skipping to different degrees, but the specific molecular mechanism of this aberrant splicing is still unclear. The aims of this study were to investigate the regulatory mechanism underlying two synonymous splicing events, c.1221A > G (p. Glu407Glu) and c.1236G > A (p. Leu412Leu), and to discover a therapeutic strategy for correcting this aberrant splicing by targeting potential regulatory sites. Through a series of RNA pulldown, silver staining, western blotting, small interfering RNA knockdown, in vitro overexpression and single or double site-directed mutagenesis experiments, we first explored the pathogenesis of exon 9 skipping caused by mutations in the CUL3 gene and verified that the main splicing regulators associated with the synonymous c.1221A > G and c.1236G > A mutations were heterogeneous nuclear ribonucleoproteins. In addition, we verified that introducing another synonymous mutation, c.1224A > G (A18G), significantly rescued the abnormal splicing caused by c.1221A > G and c.1236G > A, highlighting the therapeutic potential for the treatment of PHA II.
所有与假性醛固酮增多症 II 型（PHA II）相关的 Cullin3 基因外显子 9 突变都不同程度地导致外显子跳跃，但这种异常剪接的具体分子机制仍不清楚。本研究旨在探讨两种同义剪接事件（c.1221A>G [p.Glu407Glu] 和 c.1236G>A [p.Leu412Leu]）的调控机制，并发现针对潜在调节位点的纠正这种异常剪接的治疗策略。通过一系列 RNA 下拉、银染、Western blot、小干扰 RNA 敲低、体外过表达和单或双定点诱变实验，我们首先探讨了 CUL3 基因突变导致外显子 9 跳跃的发病机制，并验证了与同义 c.1221A>G 和 c.1236G>A 突变相关的主要剪接调节剂是异质核核糖核蛋白。此外，我们验证了引入另一个同义突变 c.1224A>G（A18G）可显著挽救 c.1221A>G 和 c.1236G>A 引起的异常剪接，突出了治疗 PHA II 的治疗潜力。
