N1-SRC regulates mRNA splicing during neural development

Alternative mRNA splicing generates transcriptomic diversity to direct tissue-specific functions. There is a high level of alternative splicing in the brain, particularly in development, but the master regulators are poorly understood. A key splicing event early in neuronal differentiation is the inclusion of a microexon in the SH3 domain of the ubiquitous tyrosine kinase, C-SRC, to yield the constitutively active, neural-specific N1-SRC kinase. We previously demonstrated that specific inhibition of N1-SRC in developing Xenopus embryos inhibits neurogenesis, but the targets and mode of action of N1-SRC are unknown. In the current study we screened for N1-SRC SH3 domain interactors, surprisingly finding no unique targets compared to C-SRC, but rather a subset of low affinity binders, enriched in splicing regulators. Analysis of public phosphoproteomic data revealed that SRC-dependent phosphorylation of the splicing machinery is widespread and enriched in RNA binding proteins. To investigate whether N1-SRC-dependent regulation of splicing underpins its role in neurogenesis, we undertook long and short read RNAseq analysis of N1-SRC knockdown Xenopus embryos. We observed an upregulation of splicing factor expression and aberrant splicing of splicing regulators, principally HNRNPA1 and TRA2A. The affected splice junctions in both genes were in their glycine rich C-termini and enriched in SFPQ/NONO and FUS binding sites. These RNA binding proteins are SRC substrates and suggest a mechanism by which N1-SRC knockdown leads to mis-splicing of HNRNPA1 and TRA2A. Thus, the neuronal splicing of C-SRC to generate N1-SRC regulates the alternative splicing landscape during neurogenesis. Overall design: We undertook triplicate RNA-seq analysis on standard control morpholino and n1-src splice blocking slice blocking morpholino injected Xenopus laevis embryos at neurula stage 16.