| Summary: | 碩士 === 國立陽明大學 === 遺傳學研究所 === 95 === During pre-mRNA splicing, exons are first recognized which lead to the definition of the boundaries of intron. In addition to classical 5’ and 3’splice sites, auxiliary cis-elements on the exons and introns participate in exon recognition. These are known as exon and intron splicing enhancers (ESEs and ISEs) and exon and intron splicing silencers (ESSs and ISSs). Splicing signals are the frequent target of mutations in genetic diseases. Because ESEs and ESSs are embedded in protein-coding sequences, some coding sequence alterations that have been classified as nonsense or missense mutations may have actually inactivated an ESE which results in partial or complete exon skipping. Such effect can therefore markedly affect the structure or amounts of the protein product.
We are interested in knowing how a mutation exerts its deleterious effects on gene expression. We use a human genetic disease, phenylketonuria, as a study model. Classical phenylketonuria is an inborn error of metabolism resulting from a deficiency of phenylalanine hydroxylase (PAH). Conventionally, if one wants to know whether a disease-associated change is truly disease-causing mutation and to assess the severity of a mutation, one normally employs cDNA expression systems by placing both the mutant and wild-type cDNAs into plasmid vectors and introducing them into host cells to assay the PAH enzyme activity. However, such expression systems only examine the effects of nucleotide changes in the context of cDNA, not that of genomic DNA. Therefore any effects of apparent missense, nonsense or silent substitutions upon RNA splicing in vivo will not be observed.
To correct such shortage, in this study, we develop a PAH minigene expression system that can assay the effect of a mutation acting at levels of RNA and protein. We generate an expression plasmid that in addition to cDNA sequence, also includes sequences of flanking introns of a particular exon. Thus, when placing a point mutation into such an exon and transfecting the expression plasmid into COS-1 cells, the resulting PAH activity would reflect the combination effect acting at levels of RNA and protein. If a mutation disrupts one of a few crucial elements for exon recognition, the extent of exon inclusion can markedly be affected. In such situation, lower PAH activity would be observed compared to that using conventional cDNA expression system. Furthermore, to confirm exon skipping or aberrant splicing indeed occurs, PAH RNA structure is analyzed by S1 nuclease mapping. We took PAH exon 9 as our study target after evaluating favorable informations such as the chance of harboring ESE element, availability of patient data and the mutation density of a given exon. To study, a total of 25 naturally occurred mutations, including 20 missense, 3 deletion and 2 silence/polymorphism mutations, were introduced individually into cDNA and minigene expression plasmids. Our results show when harboring c.922C>T(L308F),c.937G>A (A313T), c.943G>T(D315Y) or c.963C>T (L321L), an increase in exon 9 skipping is observed. Thus, these missense mutation probably cause a splicing defect affecting the recognition of PAH exon 9. On the other hand, an enhancement of exon 9 recognition was found when harboring c.922C>T(L308F)，c.932T>C (L311P) or c.935G>A (G312V) mutation. These missense mutations may have disrupted the ESS element(s) or have introduced a new ESE sequence.
Using PAH exon 9 as a model, this study shows that many naturally occurred missense mutations in addition to affect protein structure have their effect on exon recognition. Thus, establishing an appropriate functional assay, such as the one developed in this study, is important to understand the nature of pathogenic mutations.
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