International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
PLoS One. 2011;6(8):e23349. doi: 10.1371/journal.pone.0023349. Epub 2011 Aug 8.
We have previously identified an Alu-derived Intronic Splicing enhancer (ISE) in the Ataxia Teleangectasia Mutated gene (ATM) that facilitates intron pre-mRNA processing and leads to the inclusion of a cryptic exon in the final mRNA transcript. By using an RNA pull-down assay, we show here that hnRNPA1/A2, HuR and DAZAP1 splicing factors and DHX36 RNA helicase bind to the ISE. By functional studies (overexpression and siRNA experiments), we demonstrate that hnRNPA1 and DAZAP1 are indeed involved in ISE-dependent ATM cryptic exon activation, with hnRNPA1 acting negatively and DAZAP1 positively on splicing selection. On the contrary, HuR and DHX36 have no effect on ATM splicing pattern. These data suggest that splicing factors with both negative and positive effect can assemble on the intronic Alu repeats and regulate pre-mRNA splicing.
我们之前在共济失调毛细血管扩张突变基因（ATM）中鉴定出一个 Alu 衍生的内含子剪接增强子（ISE），它有助于前体 mRNA 的剪接加工，并导致最终 mRNA 转录本中包含一个隐蔽外显子。通过使用 RNA 下拉测定法，我们在这里表明 hnRNPA1/A2、HuR 和 DAZAP1 剪接因子以及 DHX36 RNA 解旋酶与 ISE 结合。通过功能研究（过表达和 siRNA 实验），我们证明 hnRNPA1 和 DAZAP1 确实参与了 ISE 依赖性 ATM 隐蔽外显子激活，hnRNPA1 对剪接选择起负作用，而 DAZAP1 起正作用。相反，HuR 和 DHX36 对 ATM 剪接模式没有影响。这些数据表明，具有正反两种作用的剪接因子可以组装在内含子 Alu 重复序列上，并调节前体 mRNA 的剪接。
