hnRNP H binding at the 5′ splice site correlates with the pathological effect of two intronic mutations in the NF-1 and TSHβ genes
hnRNP H binding at the 5′ splice site correlates with the pathological effect of two intronic mutations in the NF-1 and TSHβ genes
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DOI：
10.1093/nar/gkh752
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发表时间：
2004-08-01
影响因子：
14.9
通讯作者：
Baralle, FE
中科院分区：
文献类型：
--
作者：
Buratti, E;Baralle, M;Baralle, FE
我们最近报道了NF-1外显子3供体位点的致病取代（+5G > C），该取代产生其跳变。我们现在详细研究了剪接机制，分析了51个剪接位点上的rna -蛋白复合物。通过下拉和带移/超移分析观察特征蛋白模式。在这里，我们发现hnRNP H特异性结合NF-1外显子3的野生型GGGgu供体序列。损耗分析表明，该蛋白限制U1小核核糖核蛋白（U1snRNA）进入供体位点。在这种情况下，+5G > C突变同时取消了U1snRNP碱基配对和5‘剪接位点（5’s）功能。然而，突变体中的外显子识别可以通过破坏hnRNP H的结合来恢复，这表明该蛋白增强了+5G > C取代的效果。值得注意的是，在TSHbeta外显子2的5's处发现了第二个致病的+5G > a替换，其中包含GGgu供体序列。因此，为什么类似的核苷酸取代可以是中性的或非常破坏剪接功能的原因可以通过特定结合签名的存在来解释，这取决于局部环境。
We have recently reported a disease-causing substitution (+5G > C) at the donor site of NF-1 exon 3 that produces its skipping. We have now studied in detail the splicing mechanism involved in analyzing RNA-protein complexes at several 51 splice sites. Characteristic protein patterns were observed by pull-down and band-shift/super-shift analysis. Here, we show that hnRNP H binds specifically to the wildtype GGGgu donor sequence of the NF-1 exon 3. Depletion analyses shows that this protein restricts the accessibility of U1 small nuclear ribonucleoprotein (U1snRNA) to the donor site. In this context, the +5G > C mutation abolishes both U1snRNP base pairing and the 5' splice site (5'ss) function. However, exon recognition in the mutant can be rescued by disrupting the binding of hnRNP H, demonstrating that this protein enhances the effects of the +5G > C substitution. Significantly, a similar situation was found for a second disease-causing +5G > A substitution in the 5'ss of TSHbeta exon 2, which harbors a GGgu donor sequence. Thus, the reason why similar nucleotide substitutions can be either neutral or very disruptive of splicing function can be explained by the presence of specific binding signatures depending on local contexts.