CHARACTERIZING THE TISSUE-SPECIFIC SPLICING ACTIVITY AND LOCALIZATION PROPERTIES OF RNA BINDING PROTEINS

RNA Binding Proteins regulate in part alternative pre-mRNA splicing and, in turn, gene expression patterns. Alternative splicing is regulated by cis-elements on the pre-mRNA and trans-acting RNA binding proteins (RBPs). Polypyrimidine tract binding proteins PTBP1 and PTBP2 are paralogous RNA binding proteins that share 74% amino acid sequence identity and have a similar domain organization of four structured RNA-recognition motifs connected by unstructured linker regions and an N-terminal region. The differences in PTBP1 and PTBP2 expression regulate pre-mRNA splicing of gene transcripts necessary for neuronal development, but how these paralogous proteins exert different splicing outcomes is not well understood. Previous studies revealed that PTBP2 is distinctly post-translationally phosphorylated in the unstructured N-terminal, Linker 1, and Linker 2 regions that share less sequence identity with PTBP1. In this study, we aimed to determine the role of these linker regions in the differential splicing activity of PTBP1 and PTBP2. To this end, we created hybrid PTBP1-PTBP2 proteins that had linker regions of PTBP1 inserted to an otherwise PTBP2 protein and assayed the constructs for splicing activity on exons regulated differentially by the paralogs. Our results support a role for PTBP2 linker regions in dictating its neuronal specific splicing activity. We also aimed to characterize regions that dictate PTBP1 cellular localization. PTBP1 is nuclear at steady state. PTBP1 contains an N-terminal nuclear import and export sequence. Yet, deletion of the RRM2 region abolishes PTBP1 nuclear export suggesting the presence of additional regions, protein-protein, protein-RNA interactions required for export. To this end, we aimed to identify and characterize five potential nuclear export sequences in the RRM2 region. These studies have the potential to reveal a novel mode of cellular localization for RNA binding proteins using both a N-terminal export sequence and an auxiliary motif in an RRM.