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

Ainhoa Martínez-Pizarro, Maja Dembic, Belén Pérez, Brage S Andresen, Lourdes R Desviat

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Abstract

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.

Original languageEnglish
Article numbere1007360
JournalPLOS Genetics
Volume14
Issue number4
Number of pages17
ISSN1553-7390
DOIs
Publication statusPublished - 2018

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phenylalanine 4-monooxygenase
Phenylalanine Hydroxylase
RNA Splice Sites
exons
defect
mutation
Mutation
gene
genes
U1 Small Nuclear Ribonucleoproteins
phenylketonuria
small nuclear RNA
therapeutics
RNA
inhibitor
amino acid metabolism
amino acid
metabolism
Therapeutic Uses
point mutation

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@article{c90df068a7774e068e0bb0cbf28462f6,
title = "Intronic PAH gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5' splice site",
abstract = "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.",
author = "Ainhoa Mart{\'i}nez-Pizarro and Maja Dembic and Bel{\'e}n P{\'e}rez and Andresen, {Brage S} and Desviat, {Lourdes R}",
year = "2018",
doi = "10.1371/journal.pgen.1007360",
language = "English",
volume = "14",
journal = "P L o S Genetics",
issn = "1553-7390",
publisher = "Public Library of Science",
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Intronic PAH gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5' splice site. / Martínez-Pizarro, Ainhoa; Dembic, Maja; Pérez, Belén; Andresen, Brage S; Desviat, Lourdes R.

In: PLOS Genetics, Vol. 14, No. 4, e1007360, 2018.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Intronic PAH gene mutations cause a splicing defect by a novel mechanism involving U1snRNP binding downstream of the 5' splice site

AU - Martínez-Pizarro, Ainhoa

AU - Dembic, Maja

AU - Pérez, Belén

AU - Andresen, Brage S

AU - Desviat, Lourdes R

PY - 2018

Y1 - 2018

N2 - 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.

AB - 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.

U2 - 10.1371/journal.pgen.1007360

DO - 10.1371/journal.pgen.1007360

M3 - Journal article

VL - 14

JO - P L o S Genetics

JF - P L o S Genetics

SN - 1553-7390

IS - 4

M1 - e1007360

ER -