Simultaneous assessment of the effects of exonic mutations on RNA splicing and protein functions |
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Authors: | Ho Po-yi Huang Miao-Zeng Fwu Victor Tramjay Lin Sheng-Chieh Hsiao Kwang-Jen Su Tsung-Sheng |
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Affiliation: | aFaculty of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan;bDepartment of Medical Research and Education, Taipei Veterans General Hospital, No. 201, Sec. 2, Shih-pai Road, Taipei 11217, Taiwan;cInstitute of Microbiology & Immunology, National Yang-Ming University, Taipei, Taiwan |
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Abstract: | 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. |
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Keywords: | Phenylalanine hydroxylase Phenylketonuria Exon splicing enhancer Intron-inclusive cDNA (Intinc) expression system |
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