Date of Award
Doctor of Philosophy (PhD)
Darnell Robert Laboratory
Fragile X Syndrome presents with a clinical picture of moderate to severe mental retardation and behavioral abnormalities including autistic features resulting from the loss of function of a RNA-binding protein, Fragile X mental retardation protein (FMRP). This work is devoted to the understanding of functional roles of FMRP in normal neuronal development and in the pathogenesis of the Fragile X Syndrome. Particularly it focuses on the study of FMRP RNA binding properties, using both mouse models and biochemical analyses. The disease is usually caused by a triplet repeat expansion in the 5’UTR of the FMR1 gene leading to loss of transcription of FMR1 mRNA, but one severely affected patient has an isoleucine to asparagine point mutation in one of the RNA-binding domains of FMRP, hnRNP K homology (KH-type) domain 2. This I304N mutation has previously been shown to abrogate RNA binding. We generated and analyzed mouse models harboring the I304N mutation. The mutant protein retains some normal activities, as it is competent to bind both protein partners such as FXR1P and FXR2P and, via its RGG-domain, to G-quartet RNA, confirming it is not a completely functionless denatured protein. However, I304N protein is defective in RNA binding and is dissociated from polyribosomes. Moreover, electrophysiologic and behavioral deficits of the I304N mouse reveals that the mutation confers an FMRP-null-like phenotype on the mouse. These observations support the suggestion that a key function of FMRP in mediating normal cognition is sequence-specific RNA binding, and heightens the interest in identifying FMRP-RNA interactions. To find mRNAs bound by FMRP in mouse brain, we have used UV crosslinking and immunoprecipitation (CLIP), a new methodology that has several advantages over conventional methods for identification of bona fide in vivo RNA ligands for RNA binding proteins. We obtained a defined set of FMRP RNA targets, including previously validated microtubule associated protein 1b (Mtap1b). These RNA targets encode proteins of coherent biological functions related to cytoskeletal organization and synaptic transmission, suggesting FMRP may regulate cytoskeletal dynamics, dendritic and axonal functions that all converge at the developing synapse. Therefore, loss of FMRP KH2 domain specific RNA binding and proper regulation of RNA metabolism contributes to the synaptic dysfunction underlying the pathogenesis of cognitive and behavioral deficits observed in Fragile X Syndrome.
Zang, Julie B., "Identification of Loss of Specific FMRP-RNA Interactions as a Cause of Fragile X Syndrome" (2008). Student Theses and Dissertations. 206.