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An intronic RNA element modulates Factor VIII exon-16 splicing.
Victor Tse1,2, Guillermo Chacaltana3,2, Martin Gutierrez1,2
1Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, Santa Cruz, CA, 95064, USA.
Nucleic Acids Research
|November 14, 2023
Summary
Pathogenic variants in the Factor VIII (F8) gene cause Hemophilia A (HA). We identified a novel RNA structure in intron-15 that sequesters the polypyrimidine tract, leading to aberrant splicing, and developed antisense oligonucleotides (ASOs) to rescue splicing defects.
Area of Science:
- Molecular Biology
- Genetics
- RNA Biology
Background:
- Hemophilia A (HA) is caused by pathogenic variants in the human Factor VIII (F8) gene.
- Splicing defects in F8 gene variants can lead to HA pathogenesis.
Purpose of the Study:
- To investigate the impact of HA-causing variants on F8 gene splicing.
- To elucidate the RNA structural mechanisms underlying splicing defects in F8 exon-16.
- To develop therapeutic strategies targeting aberrant splicing in HA.
Main Methods:
- Analysis of 97 HA-causing single-nucleotide variants in 11 F8 exons.
- RNA chemical probing to identify RNA structures.
- Antisense oligonucleotide (ASO) design and application to modulate RNA structure and splicing.
Main Results:
- Most F8 variants did not affect splicing, but some impacted splicing regulatory sequences.
- A three-way junction structure (TWJ-3-15) in intron-15 sequesters the polypyrimidine tract, affecting exon-16 splicing.
- ASOs targeting TWJ-3-15 and intronic splicing silencers (ISSs) partially or fully rescued splicing defects of F8 exon-16 variants.
Conclusions:
- A novel RNA structure in intron-15 sensitizes F8 exon-16 to aberrant splicing in HA.
- Targeting this structure and associated ISSs with ASOs offers a potential therapeutic approach for HA patients with specific F8 splicing mutations.
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