Structure-Stabilizing RNA Modifications Prevent MBNL Binding to Toxic CUG and CCUG Repeat RNA in Myotonic Dystrophy

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dc.contributor.advisorDeRose, Victoria
dc.contributor.authorDelorimier, Elaine
dc.date.accessioned2015-08-18T23:10:08Z
dc.date.available2015-08-18T23:10:08Z
dc.date.issued2015-08-18
dc.description.abstractMyotonic dystrophy is a genetic neurodegenerative disease caused by repeat expansion mutations. Myotonic dystrophy type 1 (DM1) is caused by a CTG repeat expansion in the 3’ UTR of the dystrophia myotonia protein kinase (DMPK) gene, while myotonic dystrophy type 2 (DM2) is caused by a CCTG repeat expansion in intron 1 of the zinc finger protein nine (Znf9) gene. When expressed, these genes produce long CUG/CCUG repeat RNAs that bind and sequester a family of RNA-binding proteins known as muscleblind-like 1, 2 and 3 (MBNL1, MBNL2, MBNL3). Sequestration of these proteins plays a prominent role in pathogenicity in myotonic dystrophy. MBNL proteins regulate alternative splicing, and myotonic dystrophy symptoms are a result of mis-spliced transcripts that MBNL proteins regulate. MBNL proteins bind to a consensus sequence YGCY (Y = pyrimidine), which is found in CUG and CCUG repeats, and cellular RNA substrates that MBNL proteins bind and regulate. CUG and CCUG repeats can form A-form helices, however it is hypothesized that MBNL proteins bind to the helices when they are open and the YGCY binding site is single-stranded in nature. To evaluate this hypothesis, we used structure-stabilizing RNA modifications pseudouridine (Ψ) and 2’-O-methylation to determine if stabilization of CUG and CCUG repeat helices affected MBNL1 binding and toxicity. We also used Ψ to determine if the structure-stabilizing modification affected MBNL binding to single-stranded YGCY RNA. CUG repeats modified with Ψ or 2’-O-methyl groups exhibited enhanced structural stability and reduced affinity for MBNL1. Ψ also stabilized the structure of CCUG repeats and rigidified single-stranded YGCY RNA and inhibited MBNL1 binding to both of these RNAs. Binding data from CCUG repeats and single-stranded YGCY RNA suggest that both pyrimidines in the YGCY motif must be modified for significant inhibition. Molecular dynamics and X-ray crystallography suggest a potential water-bridging mechanism for Ψ-mediated CUG repeat stabilization. Molecular dynamics simulations suggest that Ψ increases base-stacking interactions, and reducing the flexibility of single-stranded RNA leads to reduced MBNL1 binding. Ψ modification rescued mis-splicing in a cellular DM1 model and prevented CUG repeat toxicity in zebrafish embryos. This dissertation includes previously published and unpublished coauthored material.en_US
dc.identifier.urihttps://hdl.handle.net/1794/19313
dc.language.isoen_US
dc.publisherUniversity of Oregon
dc.rightsAll Rights Reserved.
dc.subjectMuscleblinden_US
dc.subjectMyotonic dystrophyen_US
dc.subjectPseudouridineen_US
dc.titleStructure-Stabilizing RNA Modifications Prevent MBNL Binding to Toxic CUG and CCUG Repeat RNA in Myotonic Dystrophy
dc.typeElectronic Thesis or Dissertation
thesis.degree.disciplineDepartment of Chemistry and Biochemistry
thesis.degree.grantorUniversity of Oregon
thesis.degree.leveldoctoral
thesis.degree.namePh.D.

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