Fragile X mental retardation syndrome is the most common form of inherited mental retardation, affecting ~ 1 in 3600 males and ~ 1 in 6000 females. The syndrome is caused by the loss of a normal cellular protein, named the fragile X mental retardation protein (FMRP). Despite extensive research in the past two decades, the relationship between the absence of FMRP and the phenotype of the fragile X syndrome is still not fully understood. FMRP is an RNA binding protein involved in the transport and translation regulation of specific messenger RNA (mRNA) targets. Biochemical studies have determined that FMRP uses its arginine-glycine- glycine (RGG) box to bind with high affinity to RNA sequences that form G quadruplex structures. The mechanisms by which FMRP exerts its translation regulator function are not known, however it has recently been proposed that the protein works in conjunction with the microRNA pathway to regulate local protein synthesis in response to synaptic input. This proposal has the following specific aims: 1. Biochemical analysis of the miRNA-mediated translational regulator function of FMRP. We hypothesize that FMRP exerts its translation regulator function on a sub-class of its mRNA targets containing miRNA- binding sites, by altering their structures to facilitate/prevent their interactions with th miRNA-guided RISC, which will suppress/allow their translation in response to synaptic input. 2. Biochemical characterization of FMRP isoforms 2 and 3: interactions with FMR1 mRNA and translational regulator function in the context of the miRNA pathway. It has been shown that phosphorylation is essential in modulating the miRNA-mediated translation regulator function of FMRP. We will determine how the FMRP isoforms 2 and 3 interactions with the miRNA pathway are affected by their inability to be regulated through phosphorylation, due to the loss of the sites of phosphorylation from their sequence through alternative splicing. We will also determine if the production of the FMRP isoforms 2 and 3 is regulated through feedback inhibition due to their high binding affinity for the G quadruplex exonic splicing enhancer site within FMR1 mRNA. 3. Functional characterization of a C-terminus frame-shifted FMRP, which causes fragile X syndrome. There is recent evidence that the fragile X syndrome is caused in a patient in Belgium by a G insertion in the RGG box coding region of the FMR1 gene, which leads to a C-terminus frame shifted FMRP. We will determine if this C-terminus frame-shifted FMRP leads to fragile X syndrome due to the altered sequence of its RGG box, which will impair its ability to bind to G quadruplex RNA and exert its translation regulator function within or outside of the miRNA pathway context.
This is a detailed study of several FMRP isoforms interactions with the microRNA pathway and with the FMR1 mRNA, whose results will contribute to our understanding of the protein translation regulator function and of the biological functions of its isoforms. Additionally, this study will characterize the RNA binding properties and translation regulator function of a mutated FMRP protein which has been shown to cause fragile X syndrome, elucidating the molecular mechanisms of a novel cause of fragile X syndrome.
|McAninch, Damian S; Heinaman, Ashley M; Lang, Cara N et al. (2017) Fragile X mental retardation protein recognizes a G quadruplex structure within the survival motor neuron domain containing 1 mRNA 5'-UTR. Mol Biosyst 13:1448-1457|
|Bartley, Christopher M; O'Keefe, Rachel A; Blice-Baum, Anna et al. (2016) Mammalian FMRP S499 Is Phosphorylated by CK2 and Promotes Secondary Phosphorylation of FMRP. eNeuro 3:|
|Williams, Kathryn R; McAninch, Damian S; Stefanovic, Snezana et al. (2016) hnRNP-Q1 represses nascent axon growth in cortical neurons by inhibiting Gap-43 mRNA translation. Mol Biol Cell 27:518-34|
|Stefanovic, Snezana; Bassell, Gary J; Mihailescu, Mihaela Rita (2015) G quadruplex RNA structures in PSD-95 mRNA: potential regulators of miR-125a seed binding site accessibility. RNA 21:48-60|
|Mihailescu, Rita (2015) Gene expression regulation: lessons from noncoding RNAs. RNA 21:695-6|
|Stefanovic, Snezana; DeMarco, Brett A; Underwood, Ayana et al. (2015) Fragile X mental retardation protein interactions with a G quadruplex structure in the 3'-untranslated region of NR2B mRNA. Mol Biosyst 11:3222-30|
|Zhang, Yang; Gaetano, Christian M; Williams, Kathryn R et al. (2014) FMRP interacts with G-quadruplex structures in the 3'-UTR of its dendritic target Shank1 mRNA. RNA Biol 11:1364-74|
|Blice-Baum, Anna C; Mihailescu, Mihaela-Rita (2014) Biophysical characterization of G-quadruplex forming FMR1 mRNA and of its interactions with different fragile X mental retardation protein isoforms. RNA 20:103-14|