The role of poison exons in neurodevelopment

Exons in the genome that lead to the introduction of premature truncation (stop) codon (PTC) and mark these transcripts as targets for nonsense-mediated decay (NMD) are called poison exons (PEs). These PEs have recently been shown to be used throughout mouse and human neurodevelopment where they are alternatively spliced over the course of development. Moreover, genetic variants that perturb the splicing of PEs have been associated with neurodevelopmental disorders, including epilepsy and malformations of cortical development. However, the full complement of PEs that are alternatively spliced throughout human development are not known. Therefore, in Aim 1 of this study we will use long-read sequencing in induced pluripotent stem cell (iPSC)-derived neuronal progenitor cells (NPCs) and neurons, treated with cycloheximide to inhibit NMD, to identify PEs. Moreover, while recent efforts have elucidated the use of PEs during neurodevelopment, we have very limited understanding of their function. In Aim 2 we will adapt the recently described paired guide RNAs for alternative exon removal (pgFARM) approach to delete candidate PEs in iPSC-derived neuronal progenitor cells (NPCs) and determine the effects on proliferation of these cells. Our preliminary analysis suggests that many PEs that are included in transcripts in NPCs (NPC-PEs), encode for genes required for mature neuronal function, including ion transport and synaptic vesicle assembly and release. We hypothesize that deletion of NPC-PEs will lead to the ectopic expression of these genes, exit of the cell-cycle and premature neuronal differentiation of NPCs. This exploratory, high-risk, high-reward study is not intended to identify all PEs that are alternatively spliced throughout development, rather we aim to determine the efficacy of using long-read sequencing where NMD is inhibited to identify PEs. Moreover, we aim to develop a high-throughput assay for determining the role of these PEs in early stages of neurodevelopment. Collectively, the outcomes of this study will provide the platforms and experimental paradigms for (1) identifying PEs used in human neurodevelopment (2) provide a framework for the interpretation of intronic genetic variants identified by genome sequencing in individuals with neurodevelopmental and neuropsychiatric disorders (3) determining the function of these PEs in development. We intend that this data will be used to pursue future individual or program level funding applications.