An engineered RNA binding protein with improved splicing regulation

• Published in: Nucleic acids research 2018-04, Vol.46 (6), p.3152-3168
• Main Authors: Hale, Melissa A, Richardson, Jared I, Day, Ryan C, McConnell, Ona L, Arboleda, Juan, Wang, Eric T, Berglund, J Andrew
• Format: Article
• Language: English
• Subjects: Alternative Splicing; Base Sequence; Binding Sites - genetics; HEK293 Cells; HeLa Cells; Humans; Myotonic Dystrophy - genetics; Myotonic Dystrophy - therapy; Nucleotide Motifs - genetics; Protein Engineering - methods; RNA - genetics; RNA - metabolism; RNA and RNA-protein complexes; RNA Precursors - genetics; RNA Precursors - metabolism; RNA-Binding Motifs - genetics; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Zinc Fingers - genetics
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkx1304

Abstract The muscleblind-like (MBNL) family of proteins are key developmental regulators of alternative splicing. Sequestration of MBNL proteins by expanded CUG/CCUG repeat RNA transcripts is a major pathogenic mechanism in the neuromuscular disorder myotonic dystrophy (DM). MBNL1 contains four zinc finger (ZF) motifs that form two tandem RNA binding domains (ZF1-2 and ZF3-4) which each bind YGCY RNA motifs. In an effort to determine the differences in function between these domains, we designed and characterized synthetic MBNL proteins with duplicate ZF1-2 or ZF3-4 domains, referred to as MBNL-AA and MBNL-BB, respectively. Analysis of splicing regulation revealed that MBNL-AA had up to 5-fold increased splicing activity while MBNL-BB had 4-fold decreased activity compared to a MBNL protein with the canonical arrangement of zinc finger domains. RNA binding analysis revealed that the variations in splicing activity are due to differences in RNA binding specificities between the two ZF domains rather than binding affinity. Our findings indicate that ZF1-2 drives splicing regulation via recognition of YGCY RNA motifs while ZF3-4 acts as a general RNA binding domain. Our studies suggest that synthetic MBNL proteins with improved or altered splicing activity have the potential to be used as both tools for investigating splicing regulation and protein therapeutics for DM and other microsatellite diseases.