Combined Computational-Experimental Analyses of CFTR Exon Strength Uncover Predictability of Exon-Skipping Level
Combined Computational-Experimental Analyses of CFTR Exon Strength Uncover Predictability of Exon-Skipping Level
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DOI：
10.1002/humu.22300
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发表时间：
2013-06-01
期刊：
影响因子：
3.9
通讯作者：
Hinzpeter, Alexandre
中科院分区：
文献类型：
--
作者：
Aissat, Abdel;de Becdelievre, Alix;Hinzpeter, Alexandre
随着基因中核苷酸序列变异数量的增加，目前的挑战是将其分类为致病或中性。这些未知临床意义的变异可以改变多个过程，从基因转录到RNA剪接或蛋白质功能。使用一种方法结合几个在硅片工具，我们确定了一些外显子呈现较弱的剪接基序比其他外显子在囊性纤维化跨膜传导调节（CFTR）基因。这些外显子表现出更高的基础跳跃率比外显子窝藏没有可识别的弱剪接信号，使用minigene测定。然后，我们在三个不同的外显子中筛选了19个所描述的突变，并鉴定了外显子跳跃取代。这些取代在具有一个或多个弱剪接基序的外显子中诱导更高的跳跃水平。事实上，这一水平保持在2%以下的外显子具有强剪接基序，并可以达到40%的外显子具有至少一个弱基序。进一步的分析揭示了外显子3内的功能性外显子剪接增强子，其与SR蛋白SF 2/ASF相关，并且其破坏诱导外显子跳跃。在两个鼻上皮细胞刷拭样品中，在体内证实了外显子跳跃。我们的方法，指出外显子与一些剪接信号的弱点，将有助于现场剪接突变的临床相关性。
With the increased number of identified nucleotide sequence variations in genes, the current challenge is to classify them as disease causing or neutral. These variants of unknown clinical significance can alter multiple processes, from gene transcription to RNA splicing or protein function. Using an approach combining several in silico tools, we identified some exons presenting weaker splicing motifs than other exons in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. These exons exhibit higher rates of basal skipping than exons harboring no identifiable weak splicing signals using minigene assays. We then screened 19 described mutations in three different exons, and identified exon-skipping substitutions. These substitutions induced higher skipping levels in exons having one or more weak splicing motifs. Indeed, this level remained under 2% for exons with strong splicing motifs and could reach 40% for exons having at least one weak motif. Further analysis revealed a functional exon splicing enhancer within exon 3 that was associated with the SR protein SF2/ASF and whose disruption induced exon skipping. Exon skipping was confirmed in vivo in two nasal epithelial cell brushing samples. Our approach, which point out exons with some splicing signals weaknesses, will help spot splicing mutations of clinical relevance.