Identification and Characterization of a Minimal Functional Splicing Regulatory Protein, PTBP1

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In higher eukaryotes, alternative splicing of a single gene transcript into multiple final spliced mRNA contributes significantly towards the diversity of cellular proteins. The process of alternative splicing is regulated in part by RNA binding proteins that bind to RNA adjacent to regulated exons and influence the assembly of a functional spliceosome at adjacent splice sites. Aberrant alternative splicing has been identified in many diseases such as Alzheimer's disease, muscular dystrophy, and ovarian cancer, underscoring the importance of alternative splicing. Consequently, detailed mechanistic and molecular understanding of how splice variants are generated will provide new targets for therapeutic interventions. 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. PTBP1 binds preferentially to either upstream or downstream sites of target exons to promote their exclusion or inclusion from the spliced transcript. PTBP1 is composed of four RNA Recognition Motifs (RRMs) that are joined by three linker peptides. Each RRM can bind to different pyrimidine rich sequences with varying affinity and structural preferences. The complete atomic structure of PTBP1 bound to a target RNA and the interactions between RRM's while bound to RNA is unknown. Attempts to crystallize RNA‐bound PTBP1 have been hindered by the flexible linker regions between RRM1 and 2 (linker 1, 42 aa's) and RRM 2 and 3 (linker 2, 81 aa's). In this study, we aim to identify and characterize a functional minimal‐linker PTBP1 mutant for structure studies via x‐ray crystallography. To this end, a series of PTBP1 mutants were generated using 2‐step PCR with deletions in both linker 1 (Δ19, Δ29, Δ37, Δ40) and 2 regions (Δ21, Δ44, Δ53, Δ68). Mutants were tested for protein expression and splicing repression activity in vivo using mouse Neuro‐2A cells. Western blots indicated that each mutant is well expressed compared to full‐length PTBP1. Splicing assays on three different PTBP1‐regulated test exons reveal that the minimal construct (PTBP1 L1Δ40 ‐ L2Δ68) maintains splicing repression activity comparable to full‐length PTBP1. We have sub cloned PTBP1 L1Δ40 ‐ L2Δ68 into an E. coli expression vector and performed large‐scale protein purification using nickel‐affinity chromatography and dialysis. Purified protein samples indicate that overexpressed PTBP1 L1Δ40 ‐ L2Δ68 is soluble and can be purified in high concentrations for use in x‐ray crystallography. In the future, we intend to further optimize the purification protocol and incorporate size exclusion to increase sample purity.