Fragile X syndrome (FXS) is a genetic disease that accounts for a majority of the cases of inherited intellectual disability in humans. FXS results from the silencing of the fragile X mental retardation 1 (FMR1) gene which is critical for proper brain development. In the healthy brain, the FMR1 gene expresses high levels of fragile X mental retardation protein (FMRP) which has been shown to be an important nuclear shuttling protein that also partakes in translational regulation of messenger RNAs (mRNAs). Although the root cause of this disorder is relatively well understood, the exact mechanism of its development is not. Because the FXS phenotype results from a lack of FMRP, a translational regulator, we have decided to study its possible mRNA targets to better understand the pathogenesis of the disorder. One way to select these mRNAs is to search for those with a high probability of forming G-quadruplex secondary structures, which are known to be targeted by the arginine-glycine-glycine (RGG) domain of FMRP. We have selected the cyclin-dependent kinase 5, regulatory subunit 2 (CDK5R2) mRNA for study because in silico energy modeling has predicted a high probability of G-quadruplex formation, and this mRNA has been shown to play a vital role in early brain development. Circular dichroism spectroscopy, 1H-nuclear magnetic resonance spectroscopy, UV spectroscopy, thermal denaturation experiments, and polyacrylamide gel electrophoresis were utilized to confirm the presence of and characterize G-quadruplex structures in the selected sequence. Electromobility shift assays and fluorescence spectroscopy experiments were performed to demonstrate the binding of FMRP to CDK5R2 mRNA, illustrating that FMRP may perform in vivo regulation of the mRNA in healthy (non-FXS) individuals, and therefore implicating it in the development of FXS.

Biochemical analysis of G-quadruplex secondary structures in CDK5R2 mRNA and of their role in fragile X syndrome

249th National Meeting (2015)

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