Srrm234, but not canonical SR and hnRNP proteins, drive inclusion of Dscam exon 9 variable exons

Alternative splicing of pre-mRNA is a major mechanism to diversify protein functionality in metazoans from a limited number of genes. The Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam) gene, which is important for neuronal wiring and phagocytosis of bacteria, can generate up to 38,016 isoforms by mutually exclusive alternative splicing in four clusters of variable exons. However, it is not understood how a specific exon is chosen from the many variables and how variable exons are prevented from being spliced together. A main role in the regulation of Dscam alternative splicing has been attributed to RNA binding proteins (RBPs), but how they impact on exon selection is not well understood. Serine-arginine rich (SR) proteins and hnRNP proteins are the two main types of RBPs with major roles in exon definition and splice site selection. Here, we analyzed the role of SR and hnRNP proteins in Dscam exon 9 alternative splicing in mutant Drosophila melanogaster embryos because of their essential function for development. Strikingly, loss or overexpression of canonical SR and hnRNP proteins even when multiple proteins are depleted together, does not affect Dscam alternative exon selection very dramatically. Conversely, noncanonical SR protein Serine–arginine repetitive matrix 2/3/4 (Srrm234) is a main determinant of exon inclusion in the Dscam exon 9 cluster. Since long-range base-pairings are absent in the exon 9 cluster, our data argue for a small complement of regulatory factors as main determinants of exon inclusion in the Dscam exon 9 cluster.