Integrative analysis of tissue-specific alternative splicing regulation under adaptive selection

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Integrative analysis of tissue-specific alternative splicing regulation under adaptive selection PROJECT SUMMARY Tissue-specific alternative splicing (AS) generates multiple transcripts from single genes and contributes critically to the 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. While recent studies convincingly demonstrated the pervasive species difference of AS in multiple tissues, two fundamental questions remain: 1) which evolutionary splicing changes are under adaptive selection in specific lineages because of acquired fitness advantages? 2) what are the underlying mutations that led to the shifted selective regimes? These challenges are associated with the intrinsic degeneracy of the splicing code and lack of effective computational and experimental approaches for direct comparative analysis. The primary goal of this project is to overcome these limitations, so that we can identify divergent AS events under adaptive selection and the causal mutations affecting important cis-regulatory elements. To achieve this overarching goal, we formulated three specific aims. In Aim 1, we will use phylogenetic genetic methods to detect lineage- specific changes in selection intensity and adaptive evolution of AS and map AS regulatory regions experiencing lineage-specific adaptive evolution. In Aim 2, to elucidate the underlying regulatory mechanisms that led to splicing divergence, we will systematically map divergent protein-RNA interactions and reconstruct the evolutionary history in the phylogeny through a combination of CLIP footprints, perturbation of RBPs and comparative genomic analysis. In Aim 3, we will focus on MAPT alternative exon 10, which is implicated in frontotemporal dementia (FTD), and perform detailed analysis to reveal the mechanism of its divergent splicing in primates and the associated functions at the cellular level. If successful, this study will advance our understanding of the contribution of AS evolution to potential phenotypic differences among different mammalian species and the underlying mechanisms. The strategy established in this study will also provide a new and generalizable paradigm to better understand the splicing code.