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.
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