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An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects.
Hum Mol Genet. 2012 Jun 01; 21(11):2389-98.HM

Abstract

A significant proportion of disease-causing mutations affect precursor-mRNA splicing, inducing skipping of the exon from the mature transcript. Using F9 exon 5, CFTR exon 12 and SMN2 exon 7 models, we characterized natural mutations associated to exon skipping in Haemophilia B, cystic fibrosis and spinal muscular atrophy (SMA), respectively, and the therapeutic splicing rescue by using U1 small nuclear RNA (snRNA). In minigene expression systems, loading of U1 snRNA by complementarity to the normal or mutated donor splice sites (5'ss) corrected the exon skipping caused by mutations at the polypyrimidine tract of the acceptor splice site, at the consensus 5'ss or at exonic regulatory elements. To improve specificity and reduce potential off-target effects, we developed U1 snRNA variants targeting non-conserved intronic sequences downstream of the 5'ss. For each gene system, we identified an exon-specific U1 snRNA (ExSpeU1) able to rescue splicing impaired by the different types of mutations. Through splicing-competent cDNA constructs, we demonstrated that the ExSpeU1-mediated splicing correction of several F9 mutations results in complete restoration of secreted functional factor IX levels. Furthermore, two ExSpeU1s for SMA improved SMN exon 7 splicing in the chromosomal context of normal cells. We propose ExSpeU1s as a novel therapeutic strategy to correct, in several human disorders, different types of splicing mutations associated with defective exon definition.

Authors+Show Affiliations

Human Molecular Genetics, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

22362925

Citation

Fernandez Alanis, Eugenio, et al. "An Exon-specific U1 Small Nuclear RNA (snRNA) Strategy to Correct Splicing Defects." Human Molecular Genetics, vol. 21, no. 11, 2012, pp. 2389-98.
Fernandez Alanis E, Pinotti M, Dal Mas A, et al. An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects. Hum Mol Genet. 2012;21(11):2389-98.
Fernandez Alanis, E., Pinotti, M., Dal Mas, A., Balestra, D., Cavallari, N., Rogalska, M. E., Bernardi, F., & Pagani, F. (2012). An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects. Human Molecular Genetics, 21(11), 2389-98. https://doi.org/10.1093/hmg/dds045
Fernandez Alanis E, et al. An Exon-specific U1 Small Nuclear RNA (snRNA) Strategy to Correct Splicing Defects. Hum Mol Genet. 2012 Jun 1;21(11):2389-98. PubMed PMID: 22362925.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects. AU - Fernandez Alanis,Eugenio, AU - Pinotti,Mirko, AU - Dal Mas,Andrea, AU - Balestra,Dario, AU - Cavallari,Nicola, AU - Rogalska,Malgorzata E, AU - Bernardi,Francesco, AU - Pagani,Franco, Y1 - 2012/02/23/ PY - 2012/2/25/entrez PY - 2012/3/1/pubmed PY - 2012/9/22/medline SP - 2389 EP - 98 JF - Human molecular genetics JO - Hum. Mol. Genet. VL - 21 IS - 11 N2 - A significant proportion of disease-causing mutations affect precursor-mRNA splicing, inducing skipping of the exon from the mature transcript. Using F9 exon 5, CFTR exon 12 and SMN2 exon 7 models, we characterized natural mutations associated to exon skipping in Haemophilia B, cystic fibrosis and spinal muscular atrophy (SMA), respectively, and the therapeutic splicing rescue by using U1 small nuclear RNA (snRNA). In minigene expression systems, loading of U1 snRNA by complementarity to the normal or mutated donor splice sites (5'ss) corrected the exon skipping caused by mutations at the polypyrimidine tract of the acceptor splice site, at the consensus 5'ss or at exonic regulatory elements. To improve specificity and reduce potential off-target effects, we developed U1 snRNA variants targeting non-conserved intronic sequences downstream of the 5'ss. For each gene system, we identified an exon-specific U1 snRNA (ExSpeU1) able to rescue splicing impaired by the different types of mutations. Through splicing-competent cDNA constructs, we demonstrated that the ExSpeU1-mediated splicing correction of several F9 mutations results in complete restoration of secreted functional factor IX levels. Furthermore, two ExSpeU1s for SMA improved SMN exon 7 splicing in the chromosomal context of normal cells. We propose ExSpeU1s as a novel therapeutic strategy to correct, in several human disorders, different types of splicing mutations associated with defective exon definition. SN - 1460-2083 UR - https://www.unboundmedicine.com/medline/citation/22362925/An_exon_specific_U1_small_nuclear_RNA__snRNA__strategy_to_correct_splicing_defects_ L2 - https://academic.oup.com/hmg/article-lookup/doi/10.1093/hmg/dds045 DB - PRIME DP - Unbound Medicine ER -