Branch point evolution controls species-specific alternative splicing and regulates long term potentiation

Regulation and functionality of species-specific alternative splicing has remained enigmatic for many years. Calcium/calmodulin-dependent protein kinase IIbeta (CaMKIIbeta) is expressed in several splice variants and plays a key role in learning and memory. Here, we identify and characterize several primate-specific CAMK2B splice isoforms, which show altered kinetic properties and changes in substrate specificity. Furthermore, we demonstrate that primate-specific Camk2beta alternative splicing is achieved through branch point weakening during evolution. We show that reducing branch point and splice site strength during evolution globally renders constitutive exons alternative, thus providing a paradigm for cis-directed species-specific alternative splicing regulation. Using CRISPR/Cas9 we introduced the weaker human branch point into the mouse genome, resulting in human-like CAMK2B splicing in the brain of mutant mice. We observe a strong impairment of long-term potentiation in CA3-CA1 synapses of mutant mice, thus connecting branch point-controlled, species-specific alternative splicing with a fundamental function in learning and memory.多年来，物种特异性可变剪接的调控机制与功能始终是未解之谜。钙/钙调蛋白依赖性蛋白激酶IIβ（CaMKIIbeta）存在多种剪接变体，在学习记忆过程中发挥关键作用。本研究鉴定并表征了数种灵长类特异性的CAMK2B剪接异构体，这类异构体的动力学特性与底物特异性均发生改变。此外，本研究证实，灵长类特异性的Camk2beta可变剪接是通过进化过程中剪接分支点（branch point）的弱化实现的。研究表明，进化过程中剪接分支点与剪接位点强度的整体弱化，可使组成型外显子转变为可变外显子，从而为顺式调控的物种特异性可变剪接调控机制提供了研究范式。本研究借助CRISPR/Cas9技术将弱化的人类剪接分支点引入小鼠基因组，使突变小鼠脑内的CAMK2B剪接模式呈现人类特征。研究人员观察到突变小鼠CA3-CA1突触的长时程增强作用出现显著损伤，从而将受剪接分支点调控的物种特异性可变剪接与学习记忆的核心功能联系起来。