Simultaneous assessment of the effects of exonic mutations on RNA splicing and protein functions
Simultaneous assessment of the effects of exonic mutations on RNA splicing and protein functions
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DOI：
10.1016/j.bbrc.2008.06.072
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发表时间：
2008-09-05
影响因子：
3.1
通讯作者：
Su, Tsung-Sheng
中科院分区：
文献类型：
--
作者：
Ho, Po-yi;Huang, Miao-Zeng;Su, Tsung-Sheng
为了同时评估外显子突变对RNA剪接和蛋白质功能的影响，我们在这里报告了一个包含内含子的cDNA（Intinc）表达系统。作为测试模型，在由PAH cDNA组成的Intinc表达质粒中检查了苯丙氨酸羟化酶（PAH）基因外显子9中的24个突变，其中外显子9两侧是其真实内含子。当从Intinc质粒转染细胞的PAH酶活性进行了比较，一个标准的cDNA表达系统，五个突变导致PAH活动的显着相对差异，归因于改变外显子9的mRNA水平。其中两个突变可能通过剪接位点修饰影响外显子识别，其余三个突变影响实验验证的外显子剪接增强子（ESE）基序。Intinc表达系统不仅可以更好地将突变基因型与疾病表型联系起来，而且有助于进一步了解突变有害作用的分子机制。(C)2008年爱思唯尔公司All rights reserved.
To simultaneously assess the effects of exonic mutations on RNA splicing and protein functions, we report here an intron-inclusive cDNA (Intinc) expression system. As a test model, twenty-four mutations in exon 9 of the phenylalanine hydroxylase (PAH) gene were examined in an Intinc expression plasmid composed of the PAH cDNA with the exon 9 flanked by its authentic introns. When the PAH enzyme activities from the Intinc plasmid-transfected cells were compared to those of a standard cDNA expression system, five mutations resulted in significant relative differences in PAH activities attributed to altered exon 9-inclusive mRNA levels. Two of the mutations affected exon recognition probably through splice site modifications and the remaining three affected experimentally verified exon splicing enhancer (ESE) motifs. The Intinc expression system allows not only a better link between mutation genotype to disease phenotype but also contributes to further understanding of molecular mechanisms of deleterious effects of mutations. (C) 2008 Elsevier Inc. All rights reserved.