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外显子特异性U1可纠正由影响双功能剪接调控元件的同义替换导致的SPINK5外显子11跳跃。

Exon-Specific U1s Correct SPINK5 Exon 11 Skipping Caused by a Synonymous Substitution that Affects a Bifunctional Splicing Regulatory Element.

作者信息

Dal Mas Andrea, Fortugno Paola, Donadon Irving, Levati Lauretta, Castiglia Daniele, Pagani Franco

机构信息

International Centre for Genetic Engineering and Biotechnology (ICGEB), Human Molecular Genetics, Trieste, Italy.

出版信息

Hum Mutat. 2015 May;36(5):504-12. doi: 10.1002/humu.22762. Epub 2015 Mar 19.

Abstract

The c.891C>T synonymous transition in SPINK5 induces exon 11 (E11) skipping and causes Netherton syndrome (NS). Using a specific RNA-protein interaction assay followed by mass spectrometry analysis along with silencing and overexpression of splicing factors, we showed that this mutation affects an exonic bifunctional splicing regulatory element composed by two partially overlapping silencer and enhancer sequences, recognized by hnRNPA1 and Tra2β splicing factors, respectively. The C-to-T substitution concomitantly increases hnRNPA1 and weakens Tra2β-binding sites, leading to pathological E11 skipping. In hybrid minigenes, exon-specific U1 small nuclear RNAs (ExSpe U1s) that target by complementarity intronic sequences downstream of the donor splice site rescued the E11 skipping defect caused by the c.891C>T mutation. ExSpe U1 lentiviral-mediated transduction of primary NS keratinocytes from a patient bearing the mutation recovered the correct full-length SPINK5 mRNA and the corresponding functional lympho-epithelial Kazal-type related inhibitor protein in a dose-dependent manner. This study documents the reliability of a mutation-specific, ExSpe U1-based, splicing therapy for a relatively large subset of European NS patients. Usage of ExSpe U1 may represent a general approach for correction of splicing defects affecting skin disease genes.

摘要

SPINK5基因中c.891C>T同义突变导致外显子11(E11)跳跃,引发Netherton综合征(NS)。通过特定的RNA-蛋白质相互作用检测,随后进行质谱分析,并结合剪接因子的沉默和过表达实验,我们发现该突变影响了一个外显子双功能剪接调控元件,该元件由两个部分重叠的沉默子和增强子序列组成,分别被hnRNPA1和Tra2β剪接因子识别。C到T的替换同时增加了hnRNPA1的结合位点并削弱了Tra2β的结合位点,导致病理性的E11跳跃。在杂交微型基因中,与供体剪接位点下游内含子序列互补靶向的外显子特异性U1小核RNA(ExSpe U1s)挽救了由c.891C>T突变引起的E11跳跃缺陷。ExSpe U1慢病毒介导的对携带该突变患者的原发性NS角质形成细胞的转导,以剂量依赖的方式恢复了正确的全长SPINK5 mRNA和相应的功能性淋巴细胞上皮Kazal型相关抑制蛋白。这项研究证明了针对相对较大比例的欧洲NS患者,基于ExSpe U1的突变特异性剪接疗法的可靠性。ExSpe U1的使用可能代表了一种纠正影响皮肤病基因的剪接缺陷的通用方法。

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