Intronic PAH gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5’ splice site

Phenylketonuria (PKU), one of the most common inherited diseases of amino acid metabolism, is caused by mutations in the phenylalanine hydroxylase (PAH) gene. Recently, PAH exon 11 was identified as a vulnerable exon due to a weak 3’ splice site, with different exonic mutations affecting exon 11 spl...

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Detalles Bibliográficos
Autores: Martínez Pizarro, Ainhoa, Dembic, Maja, Pérez González, María Belén, Andresen, Brage S., Ruiz Desviat, Lourdes
Tipo de recurso: artículo
Fecha de publicación:2018
País:España
Institución:Universidad Autónoma de Madrid
Repositorio:Biblos-e Archivo. Repositorio Institucional de la UAM
Idioma:inglés
OAI Identifier:oai:repositorio.uam.es:10486/709212
Acceso en línea:http://hdl.handle.net/10486/709212
https://dx.doi.org/10.1371/journal.pgen.1007360
Access Level:acceso abierto
Palabra clave:Base Sequence
Computer Simulation
Exons
Hep G2 Cells
Humans
Introns
Mutation
Phenylalanine Hydroxylase
Phenylketonurias
Ribonucleoprotein
U1 Small Nuclear
RNA Splice Sites
RNA Splicing
RNA, Small Nuclear
Biología y Biomedicina / Biología
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spelling Intronic PAH gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5’ splice siteMartínez Pizarro, AinhoaDembic, MajaPérez González, María BelénAndresen, Brage S.Ruiz Desviat, LourdesBase SequenceComputer SimulationExonsHep G2 CellsHumansIntronsMutationPhenylalanine HydroxylasePhenylketonuriasRibonucleoproteinU1 Small NuclearRNA Splice SitesRNA SplicingRNA, Small NuclearBiología y Biomedicina / BiologíaPhenylketonuria (PKU), one of the most common inherited diseases of amino acid metabolism, is caused by mutations in the phenylalanine hydroxylase (PAH) gene. Recently, PAH exon 11 was identified as a vulnerable exon due to a weak 3’ splice site, with different exonic mutations affecting exon 11 splicing through disruption of exonic splicing regulatory elements. In this study, we report a novel intron 11 regulatory element, which is involved in exon 11 splicing, as revealed by the investigated pathogenic effect of variants c.1199+17G>A and c.1199+20G>C, identified in PKU patients. Both mutations cause exon 11 skipping in a minigene system. RNA binding assays indicate that binding of U1snRNP70 to this intronic region is disrupted, concomitant with a slightly increased binding of inhibitors hnRNPA1/2. We have investigated the effect of deletions and point mutations, as well as overexpression of adapted U1snRNA to show that this splicing regulatory motif is important for regulation of correct splicing at the natural 5’ splice site. The results indicate that U1snRNP binding downstream of the natural 5’ splice site determines efficient exon 11 splicing, thus providing a basis for development of therapeutic strategies to correct PAH exon 11 splicing mutations. In this work, we expand the functional effects of non-canonical intronic U1 snRNP binding by showing that it may enhance exon definition and that, consequently, intronic mutations may cause exon skipping by a novel mechanism, where they disrupt stimulatory U1 snRNP binding close to the 5’ splice site. Notably, our results provide further understanding of the reported therapeutic effect of exon specific U1 snRNA for splicing mutations in diseaseThis work was supported by Fundación Ramón Areces (http://www.fundacionareces.es/fundacionareces/, Grant XVII CN to LRD), European Cooperation in Science And Technology (http://www.cost.eu/, Action BM1207 to LRD), Natur og Univers, Det Frie Forskningsråd (https://dff.dk/ Grant 4181-00515 to BSA) and Novo Nordisk Fonden (DK) (http://novonordiskfonden.dk/en, Grant 61310-0128 to BSA). Centro de Biología Molecular Severo Ochoa receives an institutional grant from Fundación Ramón Areces (http://www.fundacionareces.es/fundacionareces). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors thank Diana Gallego for her help with some experiments.Public Library of ScienceDepartamento de Biología MolecularFacultad de Ciencias20182018-04-23research articlehttp://purl.org/coar/resource_type/c_2df8fbb1VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10486/709212https://dx.doi.org/10.1371/journal.pgen.1007360reponame:Biblos-e Archivo. Repositorio Institucional de la UAMinstname:Universidad Autónoma de MadridInglésengopen accesshttp://purl.org/coar/access_right/c_abf2info:eu-repo/semantics/openAccessoai:repositorio.uam.es:10486/7092122026-06-23T12:46:27Z
dc.title.none.fl_str_mv Intronic PAH gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5’ splice site
title Intronic PAH gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5’ splice site
spellingShingle Intronic PAH gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5’ splice site
Martínez Pizarro, Ainhoa
Base Sequence
Computer Simulation
Exons
Hep G2 Cells
Humans
Introns
Mutation
Phenylalanine Hydroxylase
Phenylketonurias
Ribonucleoprotein
U1 Small Nuclear
RNA Splice Sites
RNA Splicing
RNA, Small Nuclear
Biología y Biomedicina / Biología
title_short Intronic PAH gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5’ splice site
title_full Intronic PAH gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5’ splice site
title_fullStr Intronic PAH gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5’ splice site
title_full_unstemmed Intronic PAH gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5’ splice site
title_sort Intronic PAH gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5’ splice site
dc.creator.none.fl_str_mv Martínez Pizarro, Ainhoa
Dembic, Maja
Pérez González, María Belén
Andresen, Brage S.
Ruiz Desviat, Lourdes
author Martínez Pizarro, Ainhoa
author_facet Martínez Pizarro, Ainhoa
Dembic, Maja
Pérez González, María Belén
Andresen, Brage S.
Ruiz Desviat, Lourdes
author_role author
author2 Dembic, Maja
Pérez González, María Belén
Andresen, Brage S.
Ruiz Desviat, Lourdes
author2_role author
author
author
author
dc.contributor.none.fl_str_mv Departamento de Biología Molecular
Facultad de Ciencias
dc.subject.none.fl_str_mv Base Sequence
Computer Simulation
Exons
Hep G2 Cells
Humans
Introns
Mutation
Phenylalanine Hydroxylase
Phenylketonurias
Ribonucleoprotein
U1 Small Nuclear
RNA Splice Sites
RNA Splicing
RNA, Small Nuclear
Biología y Biomedicina / Biología
topic Base Sequence
Computer Simulation
Exons
Hep G2 Cells
Humans
Introns
Mutation
Phenylalanine Hydroxylase
Phenylketonurias
Ribonucleoprotein
U1 Small Nuclear
RNA Splice Sites
RNA Splicing
RNA, Small Nuclear
Biología y Biomedicina / Biología
description Phenylketonuria (PKU), one of the most common inherited diseases of amino acid metabolism, is caused by mutations in the phenylalanine hydroxylase (PAH) gene. Recently, PAH exon 11 was identified as a vulnerable exon due to a weak 3’ splice site, with different exonic mutations affecting exon 11 splicing through disruption of exonic splicing regulatory elements. In this study, we report a novel intron 11 regulatory element, which is involved in exon 11 splicing, as revealed by the investigated pathogenic effect of variants c.1199+17G>A and c.1199+20G>C, identified in PKU patients. Both mutations cause exon 11 skipping in a minigene system. RNA binding assays indicate that binding of U1snRNP70 to this intronic region is disrupted, concomitant with a slightly increased binding of inhibitors hnRNPA1/2. We have investigated the effect of deletions and point mutations, as well as overexpression of adapted U1snRNA to show that this splicing regulatory motif is important for regulation of correct splicing at the natural 5’ splice site. The results indicate that U1snRNP binding downstream of the natural 5’ splice site determines efficient exon 11 splicing, thus providing a basis for development of therapeutic strategies to correct PAH exon 11 splicing mutations. In this work, we expand the functional effects of non-canonical intronic U1 snRNP binding by showing that it may enhance exon definition and that, consequently, intronic mutations may cause exon skipping by a novel mechanism, where they disrupt stimulatory U1 snRNP binding close to the 5’ splice site. Notably, our results provide further understanding of the reported therapeutic effect of exon specific U1 snRNA for splicing mutations in disease
publishDate 2018
dc.date.none.fl_str_mv 2018
2018-04-23
dc.type.none.fl_str_mv research article
http://purl.org/coar/resource_type/c_2df8fbb1
VoR
http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/10486/709212
https://dx.doi.org/10.1371/journal.pgen.1007360
url http://hdl.handle.net/10486/709212
https://dx.doi.org/10.1371/journal.pgen.1007360
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Public Library of Science
publisher.none.fl_str_mv Public Library of Science
dc.source.none.fl_str_mv reponame:Biblos-e Archivo. Repositorio Institucional de la UAM
instname:Universidad Autónoma de Madrid
instname_str Universidad Autónoma de Madrid
reponame_str Biblos-e Archivo. Repositorio Institucional de la UAM
collection Biblos-e Archivo. Repositorio Institucional de la UAM
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15.298079

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