The loss of expression of the fragile X mental retardation protein (FMRP) leads to fragile X syndrome. Fragile X syndrome is the most prevalent inheritable mental retardation. FMRP has two types of RNA binding domains, two K-homology domains and an arginine-glycine-glycine box domain, and is proposed to act as a translation regulator of specific mRNA. Despite extensive research, the mechanism by which FMRP loss leads to the fragile X syndrome remains unclear. Thus, there is high interest to produce sufficient quantities of pure recombinant FMRP for biochemical and biophysical studies of the protein function. However, the recombinant bacterial expression of FMRP has had limited success, and subsequent recombinant eukaryotic and in vitro systems may produce FMRP which is posttranslationally modified, as phosphorylation and arginine methylation have been shown to occur on FMRP. In this study, we have successfully isolated the conditions for recombinant expression, purification and dialysis of full-length FMRP using Escherichia coli, with a high yield. The expression of FMRP using E. coli renders the protein devoid of the posttranslational modifications of phosphorylation and arginine methylation, allowing for the further study of the direct effects of these modifications individually and simultaneously. Additionally, FMRP has been shown to undergo alternative splicing, with one of the splicing sites in close proximity to the FMRP domain shown to be involved in binding G quadruplex mRNA with high affinity and specificity. We have analyzed how naturally occurring truncations in the FMRP sequence affect its RNA binding affinity, by applying the expression, purification and dialysis process to the second and third longest FMRP isoforms, followed by subsequent analysis of the G quadruplex mRNA binding properties by fluorescence spectroscopy. Our results show that as FMRP gets truncated by alternative splicing, its mRNA binding affinity increases. To test a model we proposed for FMRP translation regulation activity, we developed a luciferase reporter gene construct that contains the G quadruplex structure in the mRNA 5���-untranslated region. Using luminescence spectroscopy to analyze luciferase translation, we showed that low levels of full-length FMRP reduces luciferase translation, and as the concentration of full-length FMRP increases the luciferase translation increases. / Bayer School of Natural and Environmental Sciences / Chemistry and Biochemistry; / PhD; / Dissertation;

Thesis (Ph.D. in Clinical Science) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 59-67). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Reduced activity of Fragile X mental retardation protein (FMRP) in brain neurons results in the most common form of heritable mental retardation in humans, Fragile X Syndrome (FXS). FMRP is a selective RNA-binding protein that is implicated in the translational regulation of specific mRNAs in neurons. Although very few direct targets of FMRP have been identified and verified in vivo, FXS is thought to result from the aberrant regulation of potentially hundreds of mRNAs causing defects in neuron morphology and synapse function. Identifying additional targets will be important for elucidating the mechanism of FMRP regulation as well as the etiology of FXS. Drosophila melanogaster offers a unique and powerful system for studying the function of FMRP. Flies with loss of FMRP activity have neuronal and behavioral defects similar to those observed in humans with FXS. Importantly, FMRP regulates common target mRNAs in neurons in both mice and flies. Here, I will describe our discovery of a previously unknown requirement for Drosophila FMRP (dFMRP) during the cleavage stage of early embryonic development. First, we identified a requirement for dFMRP for proper cleavage furrow formation and found that dFMRP functions to regulate the expression of specific target mRNAs during the cleavage stage. Among these is trailer hitch (tral) mRNA, which encodes a translational regulator as well, and represents a new in vivo target of dFMRP translational regulation. In addition, I have identified twenty-eight proteins that change in expression in the absence of dFMRP using a comparative proteomics based screen for dFMRP targets. One of these is the Chaperonin containing tcp-1 complex (CCT), a previously unidentified target, which I found is itself also required for cleavage furrow formation. Finally, we have identified a new dFMRP protein-binding partner, Caprin, and found that together dFMRP and Caprin are required for the proper timing of the MBT. This set of work has led to a better understanding of the mechanism of dFMRP-dependent regulation of cellular morphogenesis in early embryos and has the potential to lead to a better understanding of the etiology of FXS. / text

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Sept. 10, 2009). Includes bibliographical references.