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研究生(外文):Cheng-Yi Cheng
論文名稱(外文):Analysis of the Splicing Regulation of the Human Phenylalanine Hydroxylase Gene
指導教授(外文):Tsung-Sheng Su
外文關鍵詞:phenylalanine hydroxylaseexon skipping
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人類苯丙胺酸羥化酶(phenylalanine hydroxylase, PAH)具有肝臟表現的專一性,是負責將體內的苯丙胺酸(phenylalanine)代謝轉化為酪胺酸(tyrosine)。一旦發生缺失即會導致苯酮尿症的產生。
研究指出原生型PAH mRNA的外顯子11在進行剪接時會發生被略過的狀況,而當外顯子11上具有c.1197A>T (V399V) silent mutation時,會導致mRNA剪接異常,使得外顯子11完全的被略過。為了探究其原因,本研究利用S1 nuclease mapping的方法確定肝臟細胞中內生性原生型PAH mRNA有相同的現象後,我們針對外顯子11剪接位的序列進行分析,然後以定點突變的方式,改善位在內含子10的剪接接受者序列。之後將這樣的minigene質體送入人類肝癌細胞株,HuH-7,取得RNA,以RT-PCR的方式分析其RNA剪接的狀況。結果發現,PAH外顯子11的剪接錯誤和剪接位的不理想有關。而且改善之剪接位足以彌補c.1197A>T突變所造成之剪接錯誤。
由於剪接位的不理想,我們預期在外顯子11或內含子10區域,可能有輔助剪接位使用的剪接加強子存在,因此利用bioinformatics的方式,進行剪接加強子SR protein結合序列比對,企圖找出其位置。我們選擇了四個預測的位置進行分析。以定點突變的方式,破壞其與SR蛋白質的結合位,發現經由這樣的改變後,有部分確會造成外顯子11被略過的情況加劇,但並非所有預測的位置皆是如此,顯示bioinformatics的預測並非全然可靠。
Human phenylalanine hydroxylase (PAH) is a liver-specific enzyme, which involved in the rate-limiting step of phenylalanine catabolism. Deficiency of PAH activity results in phenylketonuria, an autosomal recessive disorder.
Study has shown that PAH exon 11 skipping occurs in a fraction of PAH mRNA when illegitimate PAH RNA from lymphoblasts was analyzed by RT-PCR. And, exon 11 is totally skipped when the exon 11 mutation, c.1197A>T, occurs. By S1 nuclease mapping, we confirmed that exon 11 skipping indeed occurs in RNA isolated from liver cells. To study the mechanism of exon 11 skipping, we compared sequence of splice site to that of consensus, and found that the splicing acceptor site of intron 10 has poor pyrimidine content. When we modified such site in the minigene to become optimal by site-directed mutagenesis, we found that exon 11 recognition improved greatly. Thus, the poor exon 11 recognition in the human PAH gene may be mainly due to the sub-optimal splice acceptor site in intron 10.
Because of weak splice site, one would predict the existence of splicing enhancer on exon 11 and/or intron 10 to facilitate exon 11 recognition. Two strategies were used to search for such sequences: (1) to correlate between SR protein score matrices and PAH exon 11 splicing; (2) to evaluate naturally occurring missense, nonsense, frameshift and silent mutations on PAH exon 11 recognition. The nucleotide substitutions of predicted SR protein binding site or sequences correspond to the nature mutations were introduced into minigene, RNA splicing pattern was then analyzed by RT-PCR. Our results show the lack of the predictive capacity of SR protein score matrices on the PAH exon 11 recognition. On the other hand, several nature mutations induce changes in the splicing pattern with either positive or negative effect on exon 11 recognition. The results suggest that human PAH exon 11 recognition may act through multiple sequence motifs.
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