Presentation Title

Identification and Characterization of a Minimal Functional Splicing Regulatory Protein

Faculty Mentor

Niroshika Keppetipola (5)

Start Date

23-11-2019 8:45 AM

End Date

23-11-2019 9:30 AM

Location

60

Session

poster 2

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Alternative splicing contributes significantly towards the diversity of cellular proteins. The process of alternative splicing is regulated in part by RNA binding proteins that influence the assembly of a functional spliceosome. Splice variants have been identified in many neurodegenerative diseases and cancer, underscoring the importance of alternative splicing. Understanding how splice variants are generated will provide new targets for therapeutic intervention. 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 contains four RNA binding domains (RBDs) joined via three linker regions which bind to pyrimidine rich sequences with varying affinity and structural preferences (Oberstrass et al., 2005). How 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 PTBP1 bound to a target RNA molecule.

Attempts to crystallize RNA-bound PTBP1 are hindered by the flexible N-terminal (57 aa’s) and two linker regions (Linker 1, 42 aa’s; Linker 2, 81 aa’s). Previously, we characterized a minimal functional PTBP1 protein (PTBmin) with deletions in Linker 1 (L1Δ40) and Linker 2 (L2Δ71) regions. In this study, we generated an N-terminal deletion mutant (Δ50) of that minimal functional mutant (ΔN-PTBmin), cloned it into a bacterial expression plasmid, and assayed the mutant for protein expression in vitro. Our data highlights the mutant protein as soluble and well-expressed. We obtained 7.25 mgs of protein from a 500-ml bacterial culture. Our results indicate that the ΔN-PTBmin binds with high affinity to target RNA sequences. We performed a crystallization trial using 4 high-throughput, sparse matrix screens to test 384 known, minimally-redundant crystal-forming conditions via the sitting drop method. The JCSG+ and the 1+2 screens yielded promising results. We are currently optimizing these conditions. Keywords: alternative splicing, RNA binding proteins, recombinant DNA technology, x-ray crystallography

This document is currently not available here.

Share

COinS
 
Nov 23rd, 8:45 AM Nov 23rd, 9:30 AM

Identification and Characterization of a Minimal Functional Splicing Regulatory Protein

60

Alternative splicing contributes significantly towards the diversity of cellular proteins. The process of alternative splicing is regulated in part by RNA binding proteins that influence the assembly of a functional spliceosome. Splice variants have been identified in many neurodegenerative diseases and cancer, underscoring the importance of alternative splicing. Understanding how splice variants are generated will provide new targets for therapeutic intervention. 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 contains four RNA binding domains (RBDs) joined via three linker regions which bind to pyrimidine rich sequences with varying affinity and structural preferences (Oberstrass et al., 2005). How 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 PTBP1 bound to a target RNA molecule.

Attempts to crystallize RNA-bound PTBP1 are hindered by the flexible N-terminal (57 aa’s) and two linker regions (Linker 1, 42 aa’s; Linker 2, 81 aa’s). Previously, we characterized a minimal functional PTBP1 protein (PTBmin) with deletions in Linker 1 (L1Δ40) and Linker 2 (L2Δ71) regions. In this study, we generated an N-terminal deletion mutant (Δ50) of that minimal functional mutant (ΔN-PTBmin), cloned it into a bacterial expression plasmid, and assayed the mutant for protein expression in vitro. Our data highlights the mutant protein as soluble and well-expressed. We obtained 7.25 mgs of protein from a 500-ml bacterial culture. Our results indicate that the ΔN-PTBmin binds with high affinity to target RNA sequences. We performed a crystallization trial using 4 high-throughput, sparse matrix screens to test 384 known, minimally-redundant crystal-forming conditions via the sitting drop method. The JCSG+ and the 1+2 screens yielded promising results. We are currently optimizing these conditions. Keywords: alternative splicing, RNA binding proteins, recombinant DNA technology, x-ray crystallography