Understanding how two related RNA binding proteins determine different splicing outcomes (742.2)

Abstract

The process of alternative splicing is tightly regulated by RNA binding proteins that bind to corresponding cis‐acting elements and influence the assembly of spliceosomal components at adjacent splice sites. Many of these RNA binding proteins occur as gene families, with family members generally sharing high amino acid sequence identity and domain structure yet targeting overlapping but distinct sets of exons. PTBP1 (polypyrimidine tract binding protein 1) and PTBP2 (polypyrimidine tract binding protein 2) are paralogous RNA binding proteins. PTBP1 is well characterized as a splicing repressor. PTBP2, shares a similar domain structure and 74% primary structure identity to PTBP1, yet displays different splicing regulatory properties, with some exons affected by both proteins and other exons more strongly affected by PTBP1. The two proteins have non‐overlapping expression patterns, where PTBP1 is expressed in neuronal progenitor cells but is replaced by PTBP2 during neuronal differentiation. This mediates a switch to a neuronal splicing program with altered isoform expression and altered activity for many proteins. PTBP1 and PTBP2 have very similar RNA recognition properties and how they might exhibit different splicing regulatory properties is not understood. We created a series of hybrid proteins between PTBP1 and PTBP2. We assayed their in vitro RNA binding properties, their ability to repress neuronal exons in vitro and in vivo. We identified interacting partners for the two proteins via Flag‐IP and mass spectroscopy. We identified regions in PTBP1 that can confer splicing repression activity on PTBP2 for certain exons. Some of these regions were previously identified as sites of protein interaction on PTBP1. Together our results indicate that the differential targeting of the two proteins to different exons is largely determined by different protein‐protein interactions.