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

Robert J Ontiveros, Luis Hernandez, Haylena Nguyen, Adrian Lino Hernandez Lopez, Archana Shankar, Enoch Kim, Niroshika M Keppetipola

Biochemistry 2020 Dec 22

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Polypyrimidine tract binding protein 1 (PTBP1) is a well-studied RNA binding protein that serves as an important model for understanding molecular mechanisms underlying alternative splicing regulation. PTBP1 has four RNA binding domains (RBDs) connected via linker regions. Additionally, PTBP1 has an N-terminal unstructured region that contains nuclear import and export sequences. Each RBD can bind to pyrimidine rich elements with high affinity to mediate splicing activity. Studies support a variety of models for how PTBP1 can mediate splicing regulation on target exons. Obtaining a detailed atomic view hinges on determining a crystal structure of PTBP1 bound to a target RNA transcript. Here, we created a minimal functional PTBP1 with deletions in both linker 1 and linker 2 regions and assayed for activity on certain regulated exons, including the c-Src N1 exon. We show that for a subset of PTBP1-regulated exons the linker regions are not necessary for splicing repression activity. Gel mobility shift assays reveal the linker deletion mutant binds with 12-fold higher affinity to a target RNA sequence compared to wild-type PTBP1. A minimal PTBP1 that also contains an N-terminal region deletion binds to a target RNA with an affinity higher than that of wild-type PTBP1. Moreover, this minimal protein oligomerizes readily to form a distinct higher-order complex previously shown to be required for mediating splicing repression. This minimal functional PTBP1 protein can serve as a candidate for future structure studies to understand the mechanism of splicing repression for certain regulated exons.

Citation

Robert J Ontiveros, Luis Hernandez, Haylena Nguyen, Adrian Lino Hernandez Lopez, Archana Shankar, Enoch Kim, Niroshika M Keppetipola. Identification and Characterization of a Minimal Functional Splicing Regulatory Protein, PTBP1. Biochemistry. 2020 Dec 22;59(50):4766-4774