Complexity and evolution of splicing-regulatory networks Project Summary Alternative splicing (AS) generates multiple transcript isoforms from single genes and contributes critically to the molecular, cellular and phenotypic complexity of mammals. This process is tightly regulated by RNA-binding proteins (RBPs) which recognize specific regulatory elements in their target transcripts. A long-standing hypothesis from the evolutionary perspective is that changes of AS regulation due to mutations in cis-regulatory sequences provide a major driving force of speciation in mammals, including closely related species such as human and Chimpanzee. Indeed, divergent AS events are pervasive in different mammalian species, as well as in human populations as evident from widespread splicing quantitative trait loci (sQTLs). Despite remarkable progress in studies of splicing-regulatory networks over the past decade, our understanding of the splicing code remains very incomplete, leaving critical questions such as 1) which evolutionary splicing changes in different species or in human populations have functional implications? 2) what are the underlying mutations/genetic variations that led to the divergent splicing patterns? Building on our previous work on splicing-regulatory networks, this research program aims to address these questions. We will develop computational methods and experimental model systems to identify AS events under adaptive selection in specific lineages and map mutations leading to changes in splicing-regulatory elements that underlie splicing divergence. Insights learned from evolutionary changes that represent nature’s experiments will be leveraged to develop a more predictive splicing code. In these studies, computational and experimental approaches including new technology development are closely integrated by our multidisciplinary team. If successful, this study will provide tremendous insights into the contribution of AS evolution to potential phenotypic differences among different mammalian species, to health and disease in humans, and to fundamental understanding of RNA splicing regulation.