Species-specific alternative splicing has been recognized for many years, but its regulation has remained enigmatic and its functionality is still being discussed. Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key player in learning and memory in vertebrates and highly conserved across evolution. In mammals, CaMKII is encoded by four homologous genes and alternative splicing leads to multiple protein isoforms. Here, I characterize Camk2β alternative splicing and reveal several primate-specific splice isoforms, which are generated through exclusion of exon 16. These isoforms show altered kinetic properties, changes in substrate specificity and differential intracellular localization. Furthermore, I show that primate-specific Camk2β alternative splicing is cis-regulated, as a human minigene maintains exon skipping in different trans-acting environments. I demonstrate that weakening of the branch point during evolution is sufficient for primate-specific Camk2β exon 16 skipping, thus providing a paradigm for cis-directed species-specific alternative splicing regulation. Building on this observation, we used CRISPR/Cas9 and introduced the human intronic cis-acting element, containing the identified branch point sequence, into the mouse genome. This indeed results in a human-like Camk2β splicing pattern in the brain of mutant mice. Electrophysical characterization of CA1 neurons of mutant mice reveals a strong impairment of long-term potentiation (LTP), thus connecting species-specific alternative splicing with a fundamental function in learning and memory. The mutant mice exhibit various behavioural alterations, highlighting the importance of a correct balance of CaMKII isoforms for normal brain function. By studying species-specific Camk2β alternative splicing on a molecular, cellular and organismal level, this work contributes to the understanding of species-specific splicing regulation and reveals a role of alternative splicing in generating diverse, functional proteomes and species-specific functionality.
