hnRNP H binding at the 5′ splice site correlates with the pathological effect of two intronic mutations in the NF-1 and TSHβ genes

被引:61
作者
Buratti, E
Baralle, M
De Conti, L
Baralle, D
Romano, M
Ayala, YM
Baralle, FE
机构
[1] Int Ctr Genet Engn & Biotechnol, I-34012 Trieste, Italy
[2] Addenbrookes Hosp, Dept Med Genet, Cambridge CB2 2QQ, England
[3] Univ Trieste, Dept Physiol & Pathol, I-34127 Trieste, Italy
关键词
D O I
10.1093/nar/gkh752
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:4224 / 4236
页数:13
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