This is a proposal to test the hypothesis that Fragile-X mental retardation results from a failure of FMRP to bind specific RNAs, and to identify those RNAs. The foundation of this proposal is our finding of sequence and structure-specific targets for the FMRP RGG box and KH2 domain, the latter of which is associated with severe disease in a patient with a missense mutation (I304N) in this RNA-binding domain. We have related the KH2 RNA target, termed a kissing complex RNA, to disease by demonstrating that it competes FMRP off of brain polyribosomes, suggesting that this RNA motif is used by FMRP to effect translational regulation of specific mRNAs. Three aspects of FMRP sequence-specific RNA binding will be definitively addressed here. First, FMRP RNA targets will be identified using several methods: bioinformatic screens complemented by structural studies, microarray analysis of coimmunoprecipitating RNAs, microarray analysis of RNAs whose distribution on polyribosomes is shifted in the absence of FMRP, and cross-linking IP (CLIP) studies. Second, to help validate these new RNA targets in vivo, we will generate three new mouse models of Fragile-X mental retardation. Third, these new model systems will be used in conjunction with the existing FMR1-null mouse to validate identified RNA targets of FMRP in mouse brain. Taken together, these studies will address the hypothesis that mental retardation associated with the I304N mutation, and likely the Fragile-X syndrome more generally, may relate to a crucial role for RNAs harboring the kissing complex motif as targets for FMRP translational regulation.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD040647-09
Application #
7799774
Study Section
Synapses, Cytoskeleton and Trafficking Study Section (SYN)
Program Officer
Urv, Tiina K
Project Start
2001-04-15
Project End
2012-03-31
Budget Start
2010-04-01
Budget End
2012-03-31
Support Year
9
Fiscal Year
2010
Total Cost
$278,617
Indirect Cost
Name
Rockefeller University
Department
Internal Medicine/Medicine
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
Moore, Michael J; Zhang, Chaolin; Gantman, Emily Conn et al. (2016) Erratum: Mapping Argonaute and conventional RNA-binding protein interactions with RNA at single-nucleotide resolution using HITS-CLIP and CIMS analysis. Nat Protoc 11:616
Vasilyev, Nikita; Polonskaia, Anna; Darnell, Jennifer C et al. (2015) Crystal structure reveals specific recognition of a G-quadruplex RNA by a ?-turn in the RGG motif of FMRP. Proc Natl Acad Sci U S A 112:E5391-400
Berry-Kravis, Elizabeth; Levin, Rebecca; Shah, Haroon et al. (2015) Cholesterol levels in fragile X syndrome. Am J Med Genet A 167A:379-84
Moore, Michael J; Zhang, Chaolin; Gantman, Emily Conn et al. (2014) Mapping Argonaute and conventional RNA-binding protein interactions with RNA at single-nucleotide resolution using HITS-CLIP and CIMS analysis. Nat Protoc 9:263-93
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
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

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