Mutational Analysis Establishes a Critical Role for the N Terminus of Fragile X Mental Retardation Protein FMRP

Abstract

Fragile X syndrome is the most common form of heritable mental retardation caused by the loss of function of the fragile X mental retardation protein FMRP. FMRP is a multidomain, RNA-binding protein involved in RNA transport and/or translational regulation. However, the binding specificity between FMRP and its various partners including interacting proteins and mRNA targets is essentially unknown. Previous work demonstrated that dFMRP, the Drosophila homolog of human FMRP, is structurally and functionally conserved with its mammalian counterparts. Here, we perform a forward genetic screen and isolate 26 missense mutations at 13 amino acid residues in the dFMRP coding dfmr1. Interestingly, all missense mutations identified affect highly conserved residues in the N terminal of dFMRP. Loss-and gain-of-function analyses reveal altered axonal and synaptic elaborations in mutants. Yeast two-hybrid assays and in vivo analyses of interaction with CYFIP (cytoplasmic FMR1 interacting protein) in the nervous system demonstrate that some of the mutations disrupt specific protein-protein interactions. Thus, our mutational analyses establish that the N terminus of FMRP is critical for its neuronal function.

DrosophilaTubulin-specific chaperone E functions at neuromuscular synapses and is required for microtubule network formation

“…We have been using Drosophila as a model system to unravel the molecular pathogenesis of Fragile X syndrome (Reeve et al, 2008;Zhang et al, 2001;Zhang and Broadie, 2005), the most common form of inherited mental retardation. Fragile X mental retardation protein (FMRP, encoded by FMR1) plays an important role at synapses (Penagarikano et al, 2007;Zhang and Broadie, 2005).…”

The Fragile X Mental Retardation Protein in Circadian Rhythmicity and Memory Consolidation

“…Phenotypically, null dfmr1 mutants accurately mimic a host of FraX clinical symptoms including irregular circadian motor activity (137, 139, 141, 142), impaired social interaction (e.g. courtship ritual (137, 143)), reduced learning and severe loss of protein synthesis-dependent long-term memory (32), overgrown and overbranched neuronal structures in numerous neural circuits (15, 83, 88, 89, 92, 137-139, 144-147), and compromised neuronal and synaptic function (36, 83, 88). …”

“…These apparent pathfinding and axon guidance defects suggest that dfmr1 mutant LN v s may fail to recognize, or respond to, local instructive cues required to stop neurite extension and establish appropriate synaptic contacts with target neurons. Mechanistically, mutational analysis shows that the dFMRP N-terminal region interacts with the small GTPase Rac1 interactor CYFIP (93, 179), cooperatively regulating axonal projection and synaptic development in these LN v s clock neurons (147). Misregulation of the actin-binding protein Chickadee is also directly linked to architectural defects in the circadian LN v s, as decreasing Chickadee levels suppresses the dfmr1 null phenotype (89).…”

Molecular and Genetic Analysis of the Drosophila Model of Fragile X Syndrome

“…Comparable screens cannot practically be done in the mouse FXS model. Unfortunately, this vital research avenue has yet to be fully explored, and the few limited Drosophila screens to date have focused on phenotypes arising from dFMRP overexpression (Reeve et al, 2008; Yao et al, 2010; Zarnescu et al, 2005). In the eye, excess dFMRP produces a degenerative rough eye phenotype, and an enhancer/suppressor screen identified both classes of genetic interactors.…”

“…Additionally, ubiquitous overexpression of dFMRP causes lethality, allowing a simple search for viable suppressors. Perhaps not surprisingly, the majority of suppressors were mapped to point mutations in dfmr1 itself, and this has provided the basis for a detailed structure-function analysis of the dFMRP N-terminus, whose function was previously unknown (Reeve et al, 2008). Also, a non-genetic, small chemical screen has been used to identify pharmacological suppressors of dfmr1 (Chang et al, 2008).…”