Generation of hypomorphic model mice utilizing an mRNA instability element

我们通过定位克隆技术鉴定了与舒米亚白内障大鼠（SCR）白内障形成相关的基因。羊毛甾醇合成酶（Lss）基因发生突变。SCR的白内障发病受Lss位点上不同突变（Lss^l）和多态等位基因（Lss^s）的特定组合的独特调控。Lss^s是一个亚型等位基因，在第4外显子上有G到a核苷酸的替换。G到A核苷酸的替换也导致了Lss mRNA转录本的不稳定性。当一个荧光素酶报告基因连接到含有mRNA不稳定元件的Lss^s的外显子4序列，并在COS细胞中表达时，荧光素酶活性显著降低。这一结果表明，mRNA不稳定元件的功能与它所在的基因无关。然后，我们研究了外显子剪接增强子（ESE）元件通过G到A的替换而改变的可能性是导致Lss mRNA前体不稳定的原因。通过计算机程序ESEfinder的分析，在Lss^s等位基因中发现了一个新的SRp40结合基序，得分为4.15。SRp40在选择性剪接中的作用已经被研究过，并被证明能够以一种底物特异性的方式选择近端和远端5'剪接位点。结果表明，如果SRp40剪接因子结合到Lss^s mRNA前体的不相关位点，则Lss^s mRNA前体变得不稳定。

We identified the genes associated with cataract formation in Shumiya cataract rat (SCR) by positional cloning. Mutations of the gene for lanosterol synthase (Lss) were identified. Cataract onset in SCR was uniquely regulated by a specific combination of different mutant (Lss^l) and polymorphic alleles (Lss^s) on the Lss locus. Lss^s was a hypomorphic allele with a G to A nucleotide substitution in exon 4. The G to A nucleotide substitution also caused instability of the Lss mRNA transcripts.When a luciferase reporter gene was linked to the exon 4 sequence of Lss^s, which contain the mRNA instability element and expressed in the COS cells, luciferase activity was significantly reduced. This result indicated that the mRNA instability element functions regardless of the gene, in which it is located. We then examined the possibility that the change of an exonic splicing enhancer (ESE) element by the G to A substitution is the cause of instability of the Lss^s mRNA precursor. Analysis by the computer program ESEfinder identified a novel SRp40 binding motif, with a score of 4.15, in the Lss^s allele. SRp40 has previously been studied for its role in alternative splicing and has been shown to select both proximal and distal 5' splice sites in a substrate-specific manner. It was suggested that the Lss^s mRNA precursor becomes unstable if SRp40 splicing factor is bound to irrelevant sites in the precursor.