Gainesville, Florida, United States
Alternative splicing influences many aspects of metabolism from cellular fate to human disease. In this joint position, I examined alternative splicing from two very different aspects. Project I I developed a method to facilitate RNA structure studies on low abundance RNAs in cells. By enriching for targets of interest before the reverse transcription step, I am able to improve the data sensitivity and quality. I was interested in examining an autoregulated alternative splicing event on an endogenous pre-mRNA target. Combining this approach with SHAPE-MaP profiling in an engineered cell line containing an inducible RNA binding protein of interest, I demonstrated an unexpected alteration in access to the branchpoint upon protein induction. This technique is simple, compatible with all structure probing chemistries and readout approaches and will broaden the accessible targets for RNA structure interrogation in cellular contexts. Project II Our lab generated a CRISPR-Cas9 knockin mouse model of myotonic dystrophy type I (DM1), by inserting a CTG repeat expansion into the orthologous position in the DMPK 3' UTR, thereby placing spatial and temporal expression under the control of the endogenous locus. I constructed, ran and analyzed RNAseq libraries from adult mouse tissues as well as a primary cell model of muscle differentiation. In addition to the expected splicing alterations in myotubes, these analyses revealed early defects in splicing within the choroid plexus, a neural tissue that was not previously appreciated to be an affected tissue in DM1.
Established methodologies in the lab to interrogate nucleotide identity in selenoprotein mRNAs such as RNAse H directed positional RNA cleavage, 1-D and 2-D thin layer chromotography, and anti-nucleotide immunoprecipitation based approaches.
Focused on regulation of Selenoprotein S, which is involved in ER-associated degradation (ERAD) and resolution of inflammation. Demonstrated that expression of the selenocysteine-containing form was controlled by multiple mechanisms, such as alternative 3' UTR usage, as well as regulatory RNA sequence elements embedded in the 3' UTR.
Translational recoding of the UGA stop codon to selenocysteine is a requirement for human development and health and requires an intricate molecular interplay to occur. Mutations in one component, SECIS-Binding Protein 2 (SBP2) resulted in the first documented cause of inherited thyroid hormone dysfunction. My studies demonstrated that a point mutation in the RNA binding region altered RNA binding activity sufficiently to disrupt recoding of a subset of targets including Deiodinase 2, which is required for thyroid hormone interconversion.