isoTarget: A Genetic Method for Analyzing the Functional Diversity of Splicing Isoforms In Vivo

Protein isoforms generated by alternative splicing contribute to proteome diversity. Because of the lack of effective techniques, the isoform-specific function, expression, localization, and signaling of endogenous proteins are unknown for most genes. Here, we report a genetic method, isoTarget, for multi-purpose studies of targeted isoforms in select cells. Applying isoTarget to two isoforms of Drosophila Dscam, Dscam[TM1] and [TM2], we found that, in neurons, endogenous Dscam[TM1] is in dendrites, whereas Dscam[TM2] is in both dendrites and axons. We demonstrate that the difference in subcellular localization, rather than biochemical properties, leads to the two isoforms’ functional differences. Moreover, we show that the subcellular enrichment of functional partners results in a DLK/Wallenda-Dscam[TM2]-Dock signaling cascade in axons. We further apply isoTarget to study two isoforms of a GABA receptor to demonstrate its general applicability. isoTarget is an effective technique for studying how alternative splicing enhances proteome complexity. [Display omitted] •Translational stop cassettes allow isoform-specific manipulations of genes•isoTarget enables multi-purpose studies of a targeted isoform in select cells•The subcellular localizations of Dscam isoforms determine their cellular functions•isoTarget is generalizable for studying alternative splicing isoforms Liu et al. develop a genetic method that enables the investigation of isoform-specific function, expression, localization, and signaling of endogenous proteins in select cells. Using this method, they demonstrate that the difference in subcellular localization of two isoforms of Down syndrome cell adhesion molecule leads to functional differences between them.