Fragile X mental retardation syndrome is the most common form of inherited mental retardation, affecting ~1 in 4000 males and ~ 1 in 8000 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 fifteen years, 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 believed to be 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 have the potential to form G quadruplex structures. This project focuses on the study of one such mRNA for which there is strong evidence in the literature that it is a relevant in vivo FMRP target, namely the microtubule associated protein 1B (MAP1B) mRNA. This proposal has the following specific aims: 1. Investigation of a regulatory switch for FMRP function: from translation repressor to translation activator of G quadruplex forming mRNA. We proposed a model according to which the variation of the FMRP concentration in response to a neurotransmitter stimulation event acts as a switch for the protein function from repressor to activator of translation of its G quadruplex forming mRNA targets. This model will be tested by using recombinant FMRP ISO1 in an in vitro translation assay of a reporter gene that contains the MAP1B RNA G quadruplex structure in its 5'-untranslated region (UTR). 2. Investigation of the role played by protein post-translational modifications (phosphorylation and arginine methylation) in modulating the FMRP translation regulator function. We will phosphorylate and arginine methylate FMRP ISO1 that has been expressed in bacteria devoid of posttranslational modifications and will quantify its binding properties to the G quadruplex forming MAP1B RNA, as well as its ability to regulate the translation of a reporter gene that has this G quadruplex structure in its 5'-UTR. 3. Characterization of different FMRP isoforms interactions with the G quadruplex forming MAP1B mRNA. We will perform a comparative analysis of the FMRP isoforms ISO1, ISO2 and ISO3 interactions with the G quadruplex forming MAP1B RNA to determine at the molecular level to what extent naturally occurring sequence modifications near the RGG box (occurring in the ISO2 and ISO3 isoforms) affect the FMRP interactions with its specific RNA targets. PUBLIC HEALTH REVELANCE: This is a detailed study of the FMRP-RNA interactions at the molecular level, whose results will contribute to our understanding of the mechanisms by which FMRP achieves its function of translation regulator. The identification of such mechanisms and of the regulatory signals that modulate them could in turn facilitate the design and analysis of synthetic chemical compounds that mimic the protein function. In addition, this study of the FMRP interactions with the G quadruplex forming MAP1B mRNA is valuable beyond the fragile X syndrome context, in that will provide information about the role of the RNA G quadruplex structure in the regulation of gene expression at the translational level.
This is a detailed study of the FMRP-RNA interactions at the molecular level, whose results will contribute to our understanding of the mechanisms by which FMRP achieves its function of translation regulator. The identification of such mechanisms and of the regulatory signals that modulate them could in turn facilitate the design and analysis of synthetic chemical compounds that mimic the protein function. In addition, this study of the FMRP interactions with the G quadruplex forming MAP1B mRNA is valuable beyond the fragile X syndrome context, in that will provide information about the role of the RNA G quadruplex structure in the regulation of gene expression at the translational level.
