SRC Kinase Isoforms Regulate mRNA Splicing during Neural Development

Abstract

Alternative mRNA splicing generates transcriptomic diversity to direct tissue-specific functions. There is a high level of alternative splicing in the brain during embryonic development, but the master regulators of this process are poorly understood. One key splicing event 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 were unknown. In the current study, we screened for N1-SRC SH3 domain interactors, surprisingly finding no unique targets compared with the C-SRC SH3 domain, but rather a subset of binding partners, 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 (RBPs). To investigate whether N1-SRC-dependent regulation of splicing underpins its role in neurogenesis, we undertook long- and short-read RNA-seq 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 are in their glycine-rich C-termini, and junctions contain putative binding sites for SFPQ/NONO and FUS RBPs. Both SFPQ and FUS are SRC substrates, suggesting 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.