Poster abstracts
Poster number 80 submitted by Megan Pino
5’ splice-site variants in KIF5A selectively result in exon 27 exclusion, contributing to ALS
Megan G. Pino, MS (Neuroscience Graduate Program), Nicholas J. Hall (Neurology), Chathuri Pathirage (Chemistry), Karin Musier-Forsyth, PhD (Chemistry), Arthur Burghes, PhD (Biological Chemistry and Pharmacology, Molecular Genetics), Stephen J. Kolb, MD, PhD (Neurology, Biological Chemistry and Pharmacology)
Abstract:
Kinesin Family Member 5A (KIF5A) is a gene that encodes one of the heavy chain regions of kinesin-1, a motor protein involved in cellular transport along microtubules.1 Single nucleotide variants in the cargo-binding domain of KIF5A have been linked to amyotrophic lateral sclerosis (ALS), a rapidly progressive and fatal neuromuscular disease that primarily affects motor neurons.2-5 However, the mechanisms by which KIF5A variants contribute to ALS remain unknown. Approximately one-third of all disease-associated genetic mutations exhibit disruption of the correct pattern of pre-mRNA splicing,6,7 and 12 of the 14 KIF5A variants identified are located near exon 27 splice-site junctions. Thus, we hypothesized that mis-splicing of the penultimate exon 27 results in aberrant KIF5A RNA and protein, resulting in a novel C-terminus that gains a toxic function and causes ALS. To test this, we developed an in vitro splicing assay to detect full-length (exon 27 inclusion) and mis-spliced (exon 27 exclusion) KIF5A mRNA isoforms. We performed ddPCR in multiplexed reactions with a normalizer assay to quantify expression and calculate the rate of exon 27 inclusion for each KIF5A variant. First in HEK 293 cells transfected with KIF5A variant plasmids, we showed that 5' splice-site (5'ss) variants in KIF5A selectively result in exon 27 exclusion from mRNA. We then confirmed this result in CRISPR-Cas9 genetically edited human motor neurons, a disease-relevant cell type. Further, in an in vivo mouse model of one 5'ss variant, we observed a decrease in Kif5a protein expression in addition to RNA mis-splicing, implicating disrupted transcription and translation in KIF5A-linked ALS pathogenesis. We believe this is due to disruption of consensus 5'ss sequences where crucial ribonuclear proteins (RNPs) - such as U1 RNP - bind. We are now performing Western blot quantification and pulse-chase experiments to quantify KIF5A protein levels and the rate of protein degradation, respectively. In parallel, we are performing in vitro RNA structure probing utilizing SHAPE-MaP to generate secondary structures at single-nucleotide resolution and identify essential splicing domains that could potentially be targeted with RNA-based therapeutics.8,9 We will present updated findings at the symposium.
References:
1. NCBI KIF5A Gene. https://www.ncbi.nlm.nih.gov/gene/3798
2. NINDS ALS Fact Sheet https://www.ninds.nih.gov/Disorders/Patient-Caregiver-Education/Fact-Sheets/Amyotrophic-Lateral-Sclerosis-ALS-Fact-Sheet
3. Brenner D et al. 2018. https://doi.org/10.1093/brain/awx370
4. Nicolas A et al. 2018. https://doi.org/10.1016/j.neuron.2018.02.027
5. He J et al. 2020. https://doi.org/10.1136/jnnp-2019-320483
6. Cáceres JF & Kornblihtt AR 2002. https://doi.org/10.1016/s0168-9525(01)02626-9
7. Montes M et al. 2019. https://doi.org/10.1016/j.tig.2018.10.002
8. Merino EJ et al. 2005. https://doi.org/10.1021/ja043822v
9. Smola MJ et al. 2015. https://doi.org/10.1038/nprot.2015.103
Keywords: Kinesin Family Member 5A (KIF5A), Amyotrophic lateral sclerosis (ALS), RNA splicing and processing mechanisms