The long-term objective of this application is to rigorously demonstrate the role RNA binding plays in the function of the paraneoplastic opsoclonus-myoclonus ataxia (POMA) antigen Nova within neurons.
We aim to characterize the structural domains in Nova, called KH domains that are capable of high-affinity RNA binding and amenable to analysis by X-ray crystallography. We then aim to identify high-affinity RNA targets capable of cocrystallizing with these Nova KH domains, through the use of RNA selection methods. Nova KH domain-RNA cocrystals will be grown and the structures determined through X-ray diffraction. These structures will allow us to predict and engineer specific mutations in the Nova KB domains that either abrogate RNA binding completely or abrogate sequence-specific RNA recognition. We will test the activity of these mutants using previously established assay systems in heterologous transfected neuroblastoma cells. From these experiments, we will choose """"""""designer"""""""" mutants for in vivo expression in mouse brain. These experimental models will be generated by crossing mutant Nova transgenes into (previously generated) Nova-null mice. These studies will allow us to test the hypothesis that RNA binding plays a critical role in generating the phenotype of Nova-null mice, which includes motor neuron degeneration in the brainstem and spinal cord, tissues that are specifically affected in patients with the POMA syndrome.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS040955-01A1
Application #
6399213
Study Section
Special Emphasis Panel (ZRG1-MDCN-3 (02))
Program Officer
Gwinn, Katrina
Project Start
2001-09-30
Project End
2006-08-31
Budget Start
2001-09-30
Budget End
2002-08-31
Support Year
1
Fiscal Year
2001
Total Cost
$629,157
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
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Licatalosi, Donny D; Yano, Masato; Fak, John J et al. (2012) Ptbp2 represses adult-specific splicing to regulate the generation of neuronal precursors in the embryonic brain. Genes Dev 26:1626-42
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, 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
Racca, Claudia; Gardiol, Alejandra; Eom, Taesun et al. (2010) The Neuronal Splicing Factor Nova Co-Localizes with Target RNAs in the Dendrite. Front Neural Circuits 4:5
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
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
Ruggiu, Matteo; Herbst, Ruth; Kim, Natalie et al. (2009) Rescuing Z+ agrin splicing in Nova null mice restores synapse formation and unmasks a physiologic defect in motor neuron firing. Proc Natl Acad Sci U S A 106:3513-8
Licatalosi, Donny D; Mele, Aldo; Fak, John J et al. (2008) HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456:464-9
Jensen, Kirk B; Darnell, Robert B (2008) CLIP: crosslinking and immunoprecipitation of in vivo RNA targets of RNA-binding proteins. Methods Mol Biol 488:85-98

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