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 sequence (ISS) elements flanking the alternatively spliced alpha-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 alpha-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% alpha-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.
Bruno
Ivone G
IG
1Department of Endocrine Neoplasia and Hormonal Disorders, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.
Jin
Wei
W
Cote
Gilbert J
GJ
D017398:Alternative Splicing / Q000235:genetics*; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000032:analysis; D002470:Cell Survival; D005091:Exons / Q000235:genetics; D005909:Glioblastoma / Q000235:genetics; D006801:Humans; D007438:Introns / Q000235:genetics*; D020320:Oligoribonucleotides, Antisense / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics*; D051496:Receptor, Fibroblast Growth Factor, Type 1; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics*; D038621:Regulatory Sequences, Ribonucleic Acid / Q000235:genetics*; D014407:Tumor Cells, Cultured
D020320:Oligoribonucleotides, Antisense; D012333:RNA, Messenger; D017468:Receptors, Fibroblast Growth Factor; D038621:Regulatory Sequences, Ribonucleic Acid; C496348:FGFR1 protein, human; D020794:Receptor Protein-Tyrosine Kinases; D051496:Receptor, Fibroblast Growth Factor, Type 1; C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases