Identification and Characterization of a Minimal Functional Splicing Regulatory Protein

Abstract

In higher eukaryotes, alternative splicing of a single gene transcript into multiple final mRNA isoforms contributes significantly towards the diversity of cellular protein composition. The process of alternative splicing is regulated in part by RNA binding proteins which bind within and adjacent to regulated exons, influencing the location and assembly of a functional spliceosome. Splice variants have been identified in many neurodegenerative diseases and cancers, underscoring the importance of alternative splicing. Detailed mechanistic and atomic understanding of how splice variants are generated will provide new targets for therapeutic intervention. The Polypyrimidine Tract Binding Protein 1 (PTBP1) is a well characterized RNA binding protein with roles in alternative splicing regulation, mRNA localization, and IRES‐mediated translation initiation. Studies highlight that PTBP1 binds preferentially to either upstream or downstream sites of target exons to promote their exclusion or inclusion from the spliced transcript. PTBP1 contains four RNA Recognition Motifs (RRMs) joined via three linker regions that bind to pyrimidine rich sequences with varying affinity and structural preferences. How the complete structure of PTBP1 binds to its target RNA and the role of each RRM during exon inclusion/exclusion is unclear and hinges on obtaining atomic structures of full‐length PTBP1 bound to a target RNA molecule. Attempts to crystallize RNA‐bound PTBP1 have been hindered by the flexible linker regions between RRMs 1 and 2, (linker 1, 42 amino acids), and RRMs 2 and 3 (linker 2, 81 amino acids). The aim of this study is to identify and characterize a minimal functional PTBP1. A series of PTBP1 mutants were created with deletions in the linker 1 (Δ19, Δ29, Δ37, Δ40) as well as the linker 2 (Δ21, Δ44, Δ53, Δ68). Mutants were tested in cell culture for protein expression and splicing repression activity using an alternatively spliced reporter gene in mouse neuroblastoma cells (N2A). Western blots indicate that the mutants are well expressed and highlight that deletions in the linker regions do not interfere with the proper folding of the protein. Splicing assays reveal that the shortest construct (linker 1Δ40 and linker 2 Δ68) maintains splicing repression of the reporter gene cassette exon similar to full length PTBP1. We are currently constructing an in vitro expression plasmid of the mutant to express and purify recombinant protein to begin crystallization trials.