Poster abstracts
Poster number 147 submitted by Tasnin Nila
ALS-Associated KIF5A Mutations Alter Pre-mRNA Secondary Structure Revealed by Nanopore DMS Mutational Profiling
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, and 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, and 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 motor neuron 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 cause 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 27 inclusion, and a 5′ss mutation (c.3020+1G>A) that results in exon 27 exclusion. The pre-mRNAs were treated with DMS, which modifies single-stranded or otherwise accessible adenine and cytosine residues. This allows us to infer RNA structural features from mutation patterns after reverse transcription and nanopore sequencing. Consistent with our hypothesis, bioinformatics analysis confirmed increased DMS reactivity in treated samples and revealed distinct base-pairing interactions. Structural modeling showed the formation of a short hairpin near the mutation site, which is absent in both the WT and 3′ss mutant. These findings support a model in which ALS-associated splice-site mutations can alter local KIF5A pre-mRNA structure and potentially influence splice-site recognition. We are currently investigating U1 snRNP binding to the KIF5A 5′ss to determine whether the altered structure of the 5′ss mutant reduces splice-site accessibility and U1 snRNP recruitment relative to WT and the 3′ss mutant. Future studies in 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