Published online 6 August 2004
Nucleic Acids Research, Vol. 32 No. 14 © Oxford University Press 2004; all rights reserved
hnRNP H binding at the 5' splice site correlates with the pathological effect of two intronic mutations in the NF-1 and TSHß genes
1 International Centre for Genetic Engineering and Biotechnology (ICGEB), 34012 Trieste, Italy, 2 Department of Medical Genetics, Addenbrooke's Hospital, Box 134, Hills Road, Cambridge CB2 2QQ, UK and 3 Department of Physiology and Pathology, University of Trieste, Via A.Fleming 22, Trieste 34127, Italy
* To whom correspondence should be addressed at Padriciano 99, 34012 Trieste, Italy. Tel: +39 040 3757337; Fax: +39 040 3757361; Email: baralle{at}icgeb.org
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
Received May 31, 2004; Revised and Accepted July 21, 2004
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 5' splice sites. Characteristic protein patterns were observed by pulldown and band-shift/super-shift analysis. Here, we show that hnRNP H binds specifically to the wild-type 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 TSHß 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.
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