mRNA Alternative Splicing Regulatory Networks in the Specification of Cortical Interneuron Subtypes

Project Summary/Abstract Alternative splicing of pre-mRNAs is extensively employed by the nervous system to expand the transcriptomic manifold. Regulated in specific cellular contexts by multiple RNA-binding proteins (RBPs), this process is a major contributor to cellular identity that acts orthogonally to transcriptional regulation and has been implicated in many neurodevelopmental disorders. Understanding and integrating the impact of alternative RNA splicing on the establishment and organizational hierarchy of interneuron subtype specification is therefore of high value, though there has been relatively little progress in this direction. To address this gap, we have designed a high throughput system to determine the role of alternative splicing regulatory networks in cortical interneuron subtype specification. We hypothesize that alternative splicing is an informative component of interneuron cell fate decisions. Using scRNA-seq datasets with in-depth and full transcript read coverage across diverse neuronal cell types, we will identify key RBPs by highly correlated expression with cell-type specific splicing patterns together with integrative analysis of RBP target networks. We will then test the role of these RBPs by CRISPR knockout in mESCs differentiated into interneuron subtypes using a dual interneuron lineage fluorescent reporter line that we have developed. Training in splicing analysis in the expert environment of the Zhang lab is an ideal complement to the trainee’s previous extensive training in interneuron biology. The proposed studies and training plan will provide a fertile basis for a productive independent research program synthesizing traditional neurodevelopment with state of the art bioinformatics. Alternative splicing and cellular diversity are known to be closely intertwined and important in mature neurons; a better understanding of the inception of these relationships will be greatly impactful to our understanding of the nervous system in health and neurodevelopmental disease.