The role of neural specific Src splice variants in neuronal specification and differentiation

C-Src is a ubiquitously expressed non-receptor tyrosine kinase that functions in cell growth, adhesion, movement and differentiation. N1-Src is an alternatively spliced variant of c-Src, evolutionarily conserved in vertebrates, with expression enriched in neural tissues. N1-Src is formed by the insertion of a 15-18 base pair microexon, depending on species, in the SH3 domain of Src. Expression of n-Srcs is correlated with a favourable prognosis in neuroblastoma, a rare childhood cancer, leading to spontaneous differentiation and resolution of the cancer. Xenopus models, and mammalian cell culture, were used to investigate the role for n1-Src in early vertebrate development. It was found that n1-Src acts between the neural prepattern and proneural genes during the hierarchy of transcriptional activation and repression. The activity of the mitogen activated kinase pathway is regulated by n1-Src, and it is potentially through this modulation that n1-Src directs the transcriptional programme required for neural development. RNA-Seq was utilised to examine the transcriptional programme downstream of n1-Src. Gene Ontology analysis identified that transcripts significantly downregulated following n1-src knockdown are required for development of the nervous system and the neural crest. In situ hybridisation and RT-PCR analysis confirmed that n1-src expression is required for neural crest induction. Transcripts significantly upregulated following n1-src knockdown are enriched for Gene Ontology terms spanning RNA processing and splicing. The requirement for n1-Src in regulating splicing was investigated using Illumina short read and Nanopore long read RNA-Seq technology, which revealed that n1-Src regulates the alternative splicing of a variety of mRNA processing and splicing factors. This study shows that n1-Src is essential for neuronal differentiation and neural crest development, and it potentially drives these processes through splicing machinery regulation. Splicing regulation by n1-Src could be partly responsible for the favourable prognosis in some neuroblastoma patients as aberrant alternative splicing has been associated with cancers.