Fragile X Syndrome (FXS) is caused by a triplet repeat expansion whose abnormal methylation silences expression of the Fragile X Mental Retardation protein, FMRP. Loss of function of this neuronal RNA- binding protein results in the intellectual disability, seizures and autistic features characteristic of FXS. FMRP is widely accepted to regulate translation of specific mRNAs and many forms of synaptic plasticity in neurons are dependent on its function. A missense mutation occurring in a Fragile X patient within an RNA binding domain of FMRP is sufficient to cause the disease and abolishes FMRP's polysome association, suggesting that identifying FMRP's mRNA targets and how FMRP regulates their translation is key to understanding the molecular basis for the cognitive and behavioral changes typical of the disease. A new technique designed to capture in vivo interactions of RNA binding proteins with their RNA targets, UV crosslinking- immunoprecipitation combined with high throughput sequencing (HITS-CLIP), was used to identify FMRP- interacting mRNAs in total brain polysomes including a mixture of many neuronal subtypes in different states of development or activity. FMRP was found to regulate mRNAs encoding both pre- and postsynaptic proteins important for synaptic function, suggesting that their mis-expression in the absence of FMRP might underlie defects in synaptic plasticity. This list of 842 FMRP targets is a resource for focusing research to ameliorate symptoms of FXS by inhibiting the activity of proteins that may be overexpressed in the absence of FMRP. To identify the subset of targets of FMRP that are most relevant for phenotypic defects this proposal aims to develop and apply an innovative approach to measuring cell-specific interactions of FMRP with RNA by engineering a new mouse (cTAG) which will conditionally tag FMRP from the endogenous locus in specific cells with temporal control by breeding with inducible Cre lines so that the tag can be used for HITS-CLIP. FXS arises due to loss of FMRP in the context of normal expression of its two family members, FXR1P and FXR2P, which share some functional redundancy with FMRP. This has led to the fundamental question of whether FXS arises due to loss of a function specific to FMRP and not shared by its paralogs, or whether FXS results from decreased dosage of a family of functionally redundant proteins. This proposal will test in vivo functional redundancy of the three paralogs with regard to RNA interactions, both globally and in specific neurons with FXR1/2P cTAG mice, and test whether increasing expression of FXR1/2P in the absence of FMRP can rescue phenotype. Successful accomplishment of the proposed Aims has significance for understanding the molecular basis of synaptic function as well as the human diseases that result from its dysfunction, including Fragile X Syndrome and autism. Identification of specific, phenotype-relevant mRNA targets of FMRP not shared by its paralogs will focus attention on inhibition of this subset while evidence for redundancy will spur attempts to therapeutically upregulate the levels of the FXR1/2P proteins in FXS.
Recent data suggests that the levels of many synaptic proteins may be tightly controlled by the opposing processes of new translation and protein turnover in neurons. Alterations in this balance or in the levels of these dosage-sensitive synaptic proteins can result in altered stoichiometry of protein complexes at developing and remodeling synapses, thought to underlie several human cognitive diseases including Fragile X Syndrome, autism spectrum disorders, Angelman syndrome and some cases of non-syndromic mental retardation. Probing the molecular basis of Fragile X Syndrome, which results from the absence of a critical regulator of the translation of these synaptic proteins, is an excellent opportunity to significantly expand our understanding of the underlying cause of several human cognitive diseases.
|Darnell, Jennifer C; Klann, Eric (2013) The translation of translational control by FMRP: therapeutic targets for FXS. Nat Neurosci 16:1530-6|
|Teplova, Marianna; Malinina, Lucy; Darnell, Jennifer C et al. (2011) Protein-RNA and protein-protein recognition by dual KH1/2 domains of the neuronal splicing factor Nova-1. Structure 19:930-44|
|Darnell, J C (2011) Defects in translational regulation contributing to human cognitive and behavioral disease. Curr Opin Genet Dev 21:465-73|
|Darnell, Jennifer C; Van Driesche, Sarah J; Zhang, Chaolin et al. (2011) FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism. Cell 146:247-61|
|Phan, Anh Tuan; Kuryavyi, Vitaly; Darnell, Jennifer C et al. (2011) Structure-function studies of FMRP RGG peptide recognition of an RNA duplex-quadruplex junction. Nat Struct Mol Biol 18:796-804|
|Zang, Julie B; Nosyreva, Elena D; Spencer, Corinne M et al. (2009) A mouse model of the human Fragile X syndrome I304N mutation. PLoS Genet 5:e1000758|
|Darnell, Jennifer C; Fraser, Claire E; Mostovetsky, Olga et al. (2009) Discrimination of common and unique RNA-binding activities among Fragile X mental retardation protein paralogs. Hum Mol Genet 18:3164-77|
|Darnell, J C; Mostovetsky, O; Darnell, R B (2005) FMRP RNA targets: identification and validation. Genes Brain Behav 4:341-9|
|Darnell, Jennifer C; Fraser, Claire E; Mostovetsky, Olga et al. (2005) Kissing complex RNAs mediate interaction between the Fragile-X mental retardation protein KH2 domain and brain polyribosomes. Genes Dev 19:903-18|
|Stefani, Giovanni; Fraser, Claire E; Darnell, Jennifer C et al. (2004) Fragile X mental retardation protein is associated with translating polyribosomes in neuronal cells. J Neurosci 24:7272-6|
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