Alternative splicing (AS) is one crucial step of gene expression that must be tightly regulated during neurodevelopment. However, the precise timing of developmental splicing switches and the underlying regulatory mechanisms are poorly understood. Here we systematically analyze the temporal regulation of AS in a large number of transcriptome profiles of developing mouse cortices, in vivo purified neuronal subtypes, and neurons differentiated in vitro. Our analysis reveals early-switch and late-switch exons in genes with distinct functions, and these switches accurately define neuronal maturation stages. Integrative modeling suggests that these switches are under direct and combinatorial regulation by distinct sets of neuronal RNA-binding proteins including Nova, Rbfox, Mbnl, and Ptbp. Surprisingly, various neuronal subtypes in the sensory systems lack Nova and/or Rbfox expression. These neurons retain the “immature” splicing program in early-switch exons, affecting numerous synaptic genes. These results provide new insights into the organization and regulation of the neurodevelopmental transcriptome.

Summary RNA-binding proteins (RBPs) regulate post-transcriptional gene expression by recognizing short and degenerate sequence motifs in their target transcripts, but precisely defining their binding specificity remains challenging. Crosslinking and immunoprecipitation (CLIP) allows for mapping of the exact protein-RNA crosslink sites, which frequently reside at specific positions in RBP motifs, at single-nucleotide resolution. Here, we have developed a computational method, named mCross, to jointly model RBP binding specificity while precisely registering the crosslinking position in motif sites. We applied mCross to 112 RBPs using ENCODE eCLIP data and validated the reliability of the discovered motifs by genome-wide analysis of allelic binding sites. Our analyses revealed that the prototypical SR protein SRSF1 recognizes clusters of GGA half sites in addition to its canonical GGAGGA motif. Therefore, SRSF1 regulates splicing of a much larger repertoire of transcripts than previously appreciated, including HNRNPD and HNRNPDL, which are involved in multivalent protein assemblies and phase separation.

Neuronal maturation requires dramatic morphological and functional changes, but the molecular mechanisms governing this process are not well understood. Here, we studied the role of Rbfox1, Rbfox2, and Rbfox3 proteins, a family of tissue-specific splicing regulators mutated in multiple neurodevelopmental disorders. We generated Rbfox triple knockout (tKO) ventral spinal neurons to define a comprehensive network of alternative exons under Rbfox regulation and to investigate their functional importance in the developing neurons. Rbfox tKO neurons exhibit defects in alternative splicing of many cytoskeletal, membrane, and synaptic proteins, and display immature electrophysiological activity. The axon initial segment (AIS), a subcellular structure important for action potential initiation, is diminished upon Rbfox depletion. We identified an Rbfox-regulated splicing switch in ankyrin G, the AIS "interaction hub" protein, that regulates ankyrin G-beta spectrin affinity and AIS assembly. Our data show that the Rbfox-regulated splicing program plays a crucial role in structural and functional maturation of postmitotic neurons.

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