Regulatory paradigm of Dscam1 stochastic alternative splicing through conserved long-range RNA structures

Pancrustacean Dscam1 genes encode 2 000–120 000 distinct isoforms via mutually exclusive splicing; however, the underlying regulatory mechanisms are not fully understood. Here, we revealed a regulatory paradigm of stochastic Dscam1 alternative splicing mediated by conserved long-range RNA structures over 450 million years of evolution. By integrating comparative genomic analysis with mutational evidence, we defined two distinct, evolutionarily conserved RNA structures—the balancer RNA structure and a multi-subunit RNA architecture—that drive the stochastic selection of variable exon 6. The former balances stochastic selection of variable exon 6 isoforms via RNA structural spatial compensation, while the latter acts as a temporal splicing rheostat to counteract the ‘‘window of opportunity’’ for the splicing of 5′-proximal exon 6 variants. Genetic analyses further indicate that these RNA structural elements are essential for proper development and neural wiring. Thus, we proposed a regulatory model in which multiplexed RNA secondary structures coordinate temporally and spatially to orchestrate the stochastic selection of variable exon 6 isoforms. Our findings reveal a new regulatory paradigm of alternative splicing through RNA structural dynamics in cellular processes.