Keegan S. Krick1,2,3,4, Marissa Maroni4,5, Erica Korb4, Kristen W. Lynch2 and Elizabeth A. Heller3,4 1Cell and Molecular Biology Graduate Group, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA; 2Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA; 3Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA; 4Epigenetics Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA; 5Neuroscience Graduate Group, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA Corresponding authors: klyncpennmedicine.upenn.edu, ehellerpennmedicine.upenn.edu Abstract

Activity-dependent gene expression in neurons is well established, yet few studies have examined activity-dependent alternative splicing. Alternative splicing regulates >95% of genes and is essential to diverse neuronal functions, including synapse development and calcium channel diversity. Alternative splicing is regulated by the expression and activity of RNA-binding proteins and through changes in the local chromatin environment. To date, most analyses of activity-dependent alternative splicing are focus primarily on microexons, a small subclass of neuron-specific exons. To broaden knowledge of activity-dependent alternative splicing in neurons, we analyzed five independent RNA-seq studies to identify splicing events that consistently respond to potassium chloride (KCl) depolarization. We found that the majority of activity-dependent exons become less included upon activation, are basally constitutive, are not microexons, and reside in genes that are not differentially expressed after KCl treatment. Functionally, alternative splicing of RNA processing machinery and regulators precedes splicing of genes related to neuronal function. Given recent advances in elucidating chromatin-mediated alternative splicing in the brain, we explored the coincident regulation of histone modifications over activity-dependent exons. We found KCl-dependent changes in H3K36me3 and H4K20me1, both enriched in active gene bodies, over a subset of KCl-dependent exons, suggesting coordination of activity-dependent histone modification and alternative splicing. Together, these findings identify a diverse class of activity-dependent alternative splicing and describes the temporality and features of its regulation in cultured neurons.

Received October 1, 2024. Accepted April 7, 2025.