id_717. TARGETED BLOCKING OF GENE SPLICING CAN DYSREGULATE INTRON-EMBEDDED PRIMARY MICRORNAS AND AXONAL MORPHOGENESIS
Md Hasan Ali1, Athul R. Ramesh1, Naveen Nedunchezhian1, Wojciech Kwiatkowski1, Piotr Kopeć1, Natalia Kowalewska1, Sebastian Pęcherz1, Barbara Gnutti2, Dario Finazzi2, Savani Anbalagan1
1 Institute of Molecular Biology & Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznan, Poland
2 Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy
INTRODUCTION: Addressing whether antisense oligonucleotide (ASO)-based targeting of neuroglial genes embedded with intronic noncoding RNAs (ncRNAs) affects the expression and function of intronic ncRNAs is crucial for ASO success in clinical trials. In vitro studies are limited in their ability to assess ASO toxic effects.
AIM(S): The aim of this study was to determine whether ASOs targeting the slit3 gene in zebrafish affect slit3 intron-embedded mir-218a-1 expression or function in neurohypophyseal axonal morphogenesis.
METHOD(S): Wildtype or mir218a-1 mutant zebrafish single-cell stage embryos were injected with control ASOs or ASOs targeting slit3 splice sites or mir-218a-1-adjacent splice sites or mir-218a-1 mimics. Zebrafish larvae were imaged on confocal microscopy and neurohypophyseal axonal morphogenesis was quantified.
RESULTS: We observed that an ASO targeting the slit3 splice site but not the one targeting its translation start site, disrupts neurohypophyseal axonal morphogenesis. In addition to altered slit3 splicing, we also observed increased expression of slit3 and slit3 intron-embedded primary mir218a-1 transcripts. The ASO-induced phenotype was not observed when mature mir218a-1 was blocked by an ASO or in mir218a-1-/- mutants. The axonal phenotype was also phenocopied in samples injected with mir-218a mimic in a dose-dependent manner.
CONCLUSIONS: Our results indicate that despite ASO-induced genetic compensation, intron-retained transcripts can escape NMD machinery, become stabilized, and drive increased intronic primary microRNA expression and function. As prematurely terminated intron-retained transcripts can translocate to axons and impact neuronal function, our study warrants further validation across ncRNA classes.
FINANCIAL SUPPORT: National Science Centre grant [SONATA 2021/43/D/NZ3/01798].