UCLA

Splicing is an essential step in gene regulation to produce mature mRNA from a messenger RNA precursor. Through splicing, exons are assembled to generate a mature mRNA that can be translated into a desired protein by the cell. In mammals, splicing is extensively regulated by splicing factors, resulting in diverse exon arrangements and alternative splicing patterns. To know how splicing factors control genes through alternative splicing, it is critical to understand how they recognize their RNA and exon targets. In this study, we investigated targets of a family of strong splicing regulators, Polypyrimidine tract-binding (PTB) proteins. PTB proteins bind to RNA using their RNA binding domains (RBDs) and regulate splicing of exons. Among PTB proteins, PTBP1 is the most abundant and well-studied gene. Each RBD of PTBP1 binds to short, degenerate pyrimidine sequences allowing PTBP1 regulate a wide variety of targets. This variation makes it difficult to evaluate PTBP1 binding events and thus to identify PTBP1 target exons. We developed computational models that predict the binding and splicing targets of PTBP1. Models identify many previously unrecognized PTBP1 binding sites and novel exon targets. Encouraged by predictive PTBP1 models, we expanded these models to PTBP2, a neuronal paralog of PTBP1. PTBP1 is expressed in non-neuronal and neuronal progenitor cells down-regulating PTBP2. Upon neuronal differentiation, expression of PTBP1 decreases, and PTBP2 is up regulated. To understand why cells require switching between such similar genes, we compared targets for PTBP1 and PTBP2 on a global scale. We were able to assess redundancy and divergence of their binding and splicing codes. With the advance of highthrouput technologies, there are on going efforts to profile PTB-regulated transcriptomes in different tissues and cellular contexts. Integration of multiple datasets will highlight and prioritize direct targets of PTB proteins for future study. To this end, we integrated in vivo targets of PTBP2 from mouse mutant studies and identified potential direct targets. Our approaches can be applied to other splicing factors, and would be especially useful for studying multi-RBD proteins. We expect this study provides a general framework for the analysis of splicing factor binding and identification of functional targets.