Poster abstracts
Poster number 35 submitted by Tasnin Akter Nila
Probing the secondary structure of KIF5A wild-type pre-mRNA and ALS-associated mutants
Tasnin Akter Nila (Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State University, Columbus, OH ), Ashley Fox (Departments of Neurology and Biological Chemistry & Pharmacology, Center for RNA Biology, Ohio State University, Columbus, OH ), Ryan Denniston (Center for RNA Biology, Ohio State University, Columbus, OH ), Stephen Kolb (Departments of Neurology and Biological Chemistry & Pharmacology, Center for RNA Biology, Ohio State University, Columbus, OH ), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, Ohio State University, Columbus, OH )
Abstract:
Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder with no cure and limited treatment options. Mutations in the KIF5A gene, which encodes the heavy chain of the microtubule motor protein Kinesin-1, have been linked to ALS. Many ALS-associated single-nucleotide variants (SNVs) cluster in the C-terminus of KIF5A, particularly around exon 27, and produce site-specific effects on pre-mRNA splicing. Notably, 5′ splice-site (5′ss) SNVs promote exon 27 exclusion through alternative splicing, whereas 3′ splice-site (3′ss) SNVs include exon 27. We hypothesize that structural differences between wild-type (WT) and 5'ss mutant pre-mRNAs lead to alternative splicing. To investigate this, nanopore dimethylsulfate mutational profiling (Nano-DMS-MaP) was performed in vitro. A 1450-nucleotide region spanning exons 26–28 of KIF5A was transcribed to generate WT and two representative ALS-associated mutants: a 3′ss mutation (c.2993-1G>A), associated with near-normal exon inclusion, and a 5′ss mutation (c.3020+1G>A), linked to reduced exon inclusion. The pre-mRNAs were treated with DMS, which modifies single-stranded adenine and cytosine residues. These modifications induce mismatches during reverse transcription that are detected by nanopore sequencing. Bioinformatics analysis confirmed increased DMS reactivity in treated samples and revealed distinct base-pairing interactions in the 5′ss variant, particularly within intron 26. Structural modeling showed a more compact intron 26 structure and formation of a short hairpin near the mutation site in the 5′ss mutant. Future studies in human neuronal cells aim to validate these findings and clarify how RNA structural changes contribute to ALS-associated splicing defects, informing potential therapeutic strategies.
Keywords: ALS, KIF5A, Nano-DMS-Map