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Alternative splicing of pre-mRNA is a major mechanism to diversify protein functionality in metazoans from a limited number of genes. In the Drosophila Down Syndrome Cell Adhesion Molecule (Dscam) important for neuronal wiring up to 38,016 isoforms can be generated 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, 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 RNA binding proteins 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 embryos because of their essential function for development. Strikingly, Dscam alternative exon selection is very robust against loss or overexpression of canonical SR and hnRNP proteins even when multiple proteins are depleted together. Conversely, non-canonical SR protein Serine-arginine repetitive matrix 234 (Srrm234) is a main determinant of exon inclusion in 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. performed experiments. A. T-M and M.I. generated and validated the Srrm234 ∆ N allele. R.A. performed bioinformatic analysis. P.U. and M.S. wrote the manuscript. All authors read and approved the final manuscript. References Anko ML, Morales L, Henry I, Beyer A, Neugebauer KM. 2010. Global analysis reveals SRp20and SRp75-specific mRNPs in cycling and neural cells. Nat Struct Mol Biol 17: 962-970. Anko ML, Muller-McNicoll M, Brandl H, Curk T, Gorup C, Henry I, Ule J, Neugebauer KM. 2012. The RNA-binding landscapes of two SR proteins reveal unique functions and binding to diverse RNA classes. Genome Biol 13: R17. Appocher C, Mohagheghi F, Cappelli S, Stuani C, Romano M, Feiguin F, Buratti E. 2017. Major hnRNP proteins act as general TDP-43 functional modifiers both in Drosophila and human neuronal cells. Nucleic Acids Res 45: 8026-8045. Ustaoglu et al. 20 Bessonov S, Anokhina M, Will CL, Urlaub H, Luhrmann R. 2008. Isolation of an active step I spliceosome and composition of its RNP core. Nature 452: 846-850. Best A, Dalgliesh C, Kheirollahi-Kouhestani M, Danilenko M, Ehrmann I, Tyson-Capper A, Elliott DJ. 2014. Tra2 protein biology and mechanisms of splicing control. Biochem Soc Trans 42: 1152-1158. Black DL. 2003. Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem 72: 291-336. Blanchette M, Green RE, MacArthur S, Brooks AN, Brenner SE, Eisen MB, Rio DC. 2009. Genome-wide analysis of alternative pre-mRNA splicing and RNA-binding specificities of the Drosophila hnRNP A/B family members. Mol Cell 33: 438-449....