The molecular mechanism underlying the differential subcellular distribution of dystrophin Dp71 isoforms

Background. Dystrophin Dp71 is a versatile protein that plays a critical role in regulating diverse processes, including cell adhesion, nuclear architecture, neuronal differentiation, and synapse organization. Moreover, Dp71 is involved in cognitive, retinal vascular and cancer diseases. This multifunctional role is believed to result from the alternative splicing it undergoes. As previously outlined, the splicing of exon 78 appears to influence the localization of Dp71 isoforms. Specifically, Dp71d, containing exon 78, shows nuclear localization, while Dp71f, lacking exon 78 and instead having 31 hydrophobic residues (founder sequence), shows cytoplasmic localization. We delineate the mechanism dictating the differential localization between Dp71 isoforms, using various reporter vectors, in combination with protein-protein interaction assays, site-directed mutagenesis on nuclear export signal (NES), knockdown experiments on importin α2 and CRM1 nuclear transporters, and molecular dynamic simulations. Results. We demonstrate that both Dp71d and Dp71f translocate to the nucleus via importin α2/β1, interact with nuclear proteins, and are exported through a shared NES in exon 77 (NES1). However, we identified and characterized a second NES (NES2) located at the founder sequence, that binds to the exportin CRM1 with higher affinity, and thereby drives efficient nuclear export of Dp71f. Conclusions. Alternative splicing at exon 78, generates an NES unique to Dp71f, which dictates isoform localization and function of Dp71. As a result, Dp71f is efficiently exported and becomes predominantly cytoplasmic, whereas Dp71d, without this NES, is exported less efficiently and remains longer in the nucleus.