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

hnRNP H binding at the 5' splice site correlates with the pathological effect of two intronic mutations in the NF-1 and TSHbeta genes
hnRNP H binding at the 5' splice site correlates with the pathological effect of two intronic mutations in the NF-1 and TSHbeta genes
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 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.
heterogeneous-nuclear ribonucleoprotein group f-h, binding sites, u1 small nuclear, chemistry, beta subunit, protein, mutation, genetic predisposition to disease, genes, italy, heterogeneous-nuclear ribonucleoproteins, genetic, humans, thyrotropin, research support, point mutation, metabolism, messenger, neurofibromin 1, hela cells, ribonucleoproteins, research, base pairing, analysis, models, rna splice sites, exons, introns, genetics, rna, ribonucleoprotein, genetic engineering, rna splicing
0305-1048
4224-4236
Buratti, E.
bf128132-8b56-444c-a325-3b008aa1fb13
Baralle, M.
ee248bdf-c13c-44a7-ac1e-e5a3f1c552b0
De Conti, L.
06308c6d-7e11-4740-b6d0-17f64e99c63c
Baralle, D.
faac16e5-7928-4801-9811-8b3a9ea4bb91
Romano, M.
0c26ba48-a574-4f6a-90bc-7e9bb7058bd1
Ayala, Y.M.
bf9ca343-6137-4972-a473-b22b72ba4d82
Baralle, F.E.
ad4b66eb-4fd0-416a-a77a-1479be42de1b
Buratti, E.
bf128132-8b56-444c-a325-3b008aa1fb13
Baralle, M.
ee248bdf-c13c-44a7-ac1e-e5a3f1c552b0
De Conti, L.
06308c6d-7e11-4740-b6d0-17f64e99c63c
Baralle, D.
faac16e5-7928-4801-9811-8b3a9ea4bb91
Romano, M.
0c26ba48-a574-4f6a-90bc-7e9bb7058bd1
Ayala, Y.M.
bf9ca343-6137-4972-a473-b22b72ba4d82
Baralle, F.E.
ad4b66eb-4fd0-416a-a77a-1479be42de1b

Buratti, E., Baralle, M., De Conti, L., Baralle, D., Romano, M., Ayala, Y.M. and Baralle, F.E. (2004) hnRNP H binding at the 5' splice site correlates with the pathological effect of two intronic mutations in the NF-1 and TSHbeta genes. Nucleic Acids Research, 32 (14), 4224-4236. (doi:10.1093/nar/gkh752).

Record type: Article

Abstract

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 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.

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More information

Published date: August 2004
Keywords: heterogeneous-nuclear ribonucleoprotein group f-h, binding sites, u1 small nuclear, chemistry, beta subunit, protein, mutation, genetic predisposition to disease, genes, italy, heterogeneous-nuclear ribonucleoproteins, genetic, humans, thyrotropin, research support, point mutation, metabolism, messenger, neurofibromin 1, hela cells, ribonucleoproteins, research, base pairing, analysis, models, rna splice sites, exons, introns, genetics, rna, ribonucleoprotein, genetic engineering, rna splicing
Organisations: Human Genetics

Identifiers

Local EPrints ID: 48497
URI: http://eprints.soton.ac.uk/id/eprint/48497
ISSN: 0305-1048
PURE UUID: 30ca3879-14d4-4b45-9f5b-ba49759b6a3f
ORCID for D. Baralle: ORCID iD orcid.org/0000-0003-3217-4833

Catalogue record

Date deposited: 26 Sep 2007
Last modified: 17 Dec 2019 01:43

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