Poster abstracts
Poster number 35 submitted by Tasnin Akter Nila
Probing the secondary structure of KIF5A pre-mRNA in wild-type and disease-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), 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 disease with no cure and limited treatment options. Point mutations in a motor protein, KIF5A, have been associated with ALS. KIF5A is the heavy chain subunit of KIF5, or Kinesin 1, and contains three domains: the N-terminal motor domain, a central stalk domain, and a C-terminal domain. Numerous ALS-associated KIF5A single-nucleotide variants (SNVs) are clustered in the C-terminal domain. SNVs proximal to exon 27 result in heterogeneous site-specific effects on pre-mRNA splicing. Whereas 5' splice-site (5'ss) SNVs induce exon 27 exclusion through alternative splicing, 3' splice-site (3'ss) SNVs include exon 27. Based on the observed differences in exon 27 exclusion vs inclusion, we hypothesize that significant structural differences among wild-type (WT) and 5'ss disease-associated mutant pre-mRNAs result in alternative splicing that leads to defects in KIF5A protein function. To investigate the pre-mRNA secondary structures, both in vitro and in human neuronal cells, the nanopore dimethylsulfate mutational profiling (Nano-DMS-MaP) approach will be used. For in vitro probing studies, we transcribed a 1400-nt region of human KIF5A exons 26-28 to generate WT and mutant pre-mRNAs. ALS-associated point mutations at the 3'ss (c.2993-1G>A) and 5'ss (c.3020+1G>A) of exon 27 were chosen as representative mutants because these SNVs demonstrate near-normal and significantly decreased mRNA expression, respectively. These samples have been probed with DMS and submitted for nanopore sequencing analysis. To observe the structural differences in the cellular context, we will probe KIF5A pre-mRNAs from previously established WT human iPSC-derived lower motor neurons and homozygous cell lines representing SNVs at the 3'ss (c.2993-1G>A) and 5'ss (c.3020+1G>A). Overall, this study will elucidate the role that RNA structure plays in alternative splicing caused by ALS-associated point mutations and may lead to future therapeutic interventions.
Keywords: ALS, RNA secondary structure, DMS probing