Correction of aberrant FGFR1 alternative RNA splicing through targeting of intronic regulatory elements

Alternative RNA splicing is now known to be pervasive throughout the genome and a target of human disease. We evaluated if targeting intronic splicing regulatory sequences with antisense oligonucleotides could be used to correct aberrant exon skipping. As a model, we targeted the intronic silencing...

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Bibliographic Details
Published in:Human molecular genetics 2004-10, Vol.13 (20), p.2409-2420
Main Authors: Bruno, Ivone G., Jin, Wei, Cote, Gilbert J.
Format: Article
Language:English
Subjects:
Alternative Splicing - genetics
Biological and medical sciences
Caspase 3
Caspase 7
Caspases - analysis
Cell Survival
Exons - genetics
Fundamental and applied biological sciences. Psychology
Genetics of eukaryotes. Biological and molecular evolution
Glioblastoma - genetics
Humans
Introns - genetics
Molecular and cellular biology
Oligoribonucleotides, Antisense - genetics
Receptor Protein-Tyrosine Kinases - genetics
Receptor, Fibroblast Growth Factor, Type 1
Receptors, Fibroblast Growth Factor - genetics
Regulatory Sequences, Ribonucleic Acid - genetics
RNA, Messenger - analysis
RNA, Messenger - metabolism
Tumor Cells, Cultured
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Summary:Alternative RNA splicing is now known to be pervasive throughout the genome and a target of human disease. We evaluated if targeting intronic splicing regulatory sequences with antisense oligonucleotides could be used to correct aberrant exon skipping. As a model, we targeted the intronic silencing sequence (ISS) elements flanking the alternatively spliced α-exon of the endogenous fibroblast growth factor receptor 1 (FGFR1) gene, which is aberrantly skipped in human glioblastoma. Antisense morpholino oligonucleotides targeting either upstream or downstream ISS elements increased α-exon inclusion from 10% up to 70% in vivo. The effect was dose dependent, sequence specific and reproducible in several human cell lines, but did not necessarily correlate with blocking of protein association in vitro. Simultaneous targeting of the ISS elements had no additive effect, suggesting that splicing regulation occurred through a shared mechanism. Broad applicability of this approach was demonstrated by similar targeting of the ISS elements of the human hnRNPA1 gene. The correction of FGFR1 gene splicing to >90% α-exon inclusion in glioblastoma cells had no discernable effect on cell growth in culture, but was associated with an increase in unstimulated caspase-3 and -7 activity. The ability to manipulate endogenously expressed mRNA variants allows exploration of their functional relevance under normal and diseased physiological states.
ISSN:0964-6906
1460-2083
DOI:10.1093/hmg/ddh272