Exploring hidden determinants of splicing with genome-targeted proximity labeling

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PROJECT SUMMARY/ABSTRACT The vast majority of human genes are multi-exonic and undergo alternative splicing to generate a several- fold increase in the variety of transcripts and proteins that are produced. In diseases like cancer, splicing is often globally aberrant and highly distinct from normal tissues, with increased levels of intron retention, alternative splicing and usage of de novo splicing junctions. A fundamental unanswered question in RNA biology is how the many layers that comprise the splicing code are interpreted to produce a given splicing outcome, and how these processes become dysregulated in human diseases. The extensive crosstalk between processes occurring on the DNA and those that act on the RNA suggest the chromatin state in particular may participate in orchestrating alternative splicing. We have observed that random genomic integration of a splicing reporter construct results in populations of cells corresponding to each possible splicing outcome, suggesting the genomic context at the site of integration exerts a powerful influence on splicing. However, due primarily to technological limitations, much remains unknown about the precise nature of the regulatory interplay between chromatin and RNA splicing and the potential factors that may be involved. The goal of this proposal is to address the current challenges impeding de novo discovery and develop unbiased experimental approaches to reveal hidden determinants of splicing regulation at the chromatin level. Our approach is designed to directly connect splicing outcome with unbiased proteomic profiling of the associated genomic region, in order to identify all of the potential chromatin features that are involved in enforcing a particular pattern of splicing. We also propose to engineer new systems to functionally validate a direct role for the epigenome in splicing regulation and to modulate splicing at will. Our findings have implications for both epigenetics and RNA biology, and in particular, how their interactions influence the various processes involved in gene expression. It also has the potential to shed insight into the causes and consequences of the widespread epigenetic and splicing dysregulation observed in human cancer. Importantly, our approach is not limited to the study of splicing and will also enable unbiased discovery of the mechanisms involved in other fundamental processes.