Complexity and graded regulation of neuronal cell-type–specific alternative splicing revealed by single-cell RNA sequencing

Significance Alternative splicing (AS) is extensively used in the mammalian brain, but its contribution to the molecular and cellular diversity across neuronal cell types remains poorly understood. Through systematic and integrative analysis of over 100 transcriptomically defined cortical neuronal types, we found neuronal subclass-specific splicing-regulatory programs consist of overlapping alternative exons showing differential splicing at multiple hierarchical levels. Evidence is provided that this graded AS regulation is controlled by unique combinations of RNA-binding proteins (RBPs). Importantly, these RBPs also contribute to splicing dynamics across neuronal cell types that do not conform to the hierarchical taxonomy established based on transcriptional profiles, suggesting that the graded AS regulation may provide a molecular mechanism orthogonal to transcriptional regulation in specifying neuronal identity and function. The enormous cellular diversity in the mammalian brain, which is highly prototypical and organized in a hierarchical manner, is dictated by cell-type–specific gene-regulatory programs at the molecular level. Although prevalent in the brain, the contribution of alternative splicing (AS) to the molecular diversity across neuronal cell types is just starting to emerge. Here, we systematically investigated AS regulation across over 100 transcriptomically defined neuronal types of the adult mouse cortex using deep single-cell RNA-sequencing data. We found distinct splicing programs between glutamatergic and GABAergic neurons and between subclasses within each neuronal class. These programs consist of overlapping sets of alternative exons showing differential splicing at multiple hierarchical levels. Using an integrative approach, our analysis suggests that RNA-binding proteins (RBPs) Celf1/2, Mbnl2, and Khdrbs3 are preferentially expressed and more active in glutamatergic neurons, while Elavl2 and Qk are preferentially expressed and more active in GABAergic neurons. Importantly, these and additional RBPs also contribute to differential splicing between neuronal subclasses at multiple hierarchical levels, and some RBPs contribute to splicing dynamics that do not conform to the hierarchical structure defined by the transcriptional profiles. Thus, our results suggest graded regulation of AS across neuronal cell types, which may provide a molecular mechanism to specify neuronal identity and function that are orthogonal to established classifications based on transcriptional regulation.