Structure/function studies of RBPs in neurologic disease

描述(由申请人提供)：这项计划的长期目标 应用程序是严格地证明RNA结合在 副肿瘤性眼阵挛-肌阵挛共济失调(POMA)抗原Nova的功能 在神经元内部。我们的目标是描述新星中的结构域，称为 能够与高亲和力RNA结合并服从于 能够与这些Nova KH结构域共结晶的靶，通过使用 RNA选择方法的一部分。将生长Nova KH域-RNA共晶体，并 通过X射线衍射法测定了它们的结构。这些结构将使我们能够 预测和设计Nova Kb结构域中的特定突变 我们将使用先前建立的检测方法来测试这些突变体的活性。 异源转基因神经母细胞瘤细胞中的系统。从这些 大脑。 转基因进入(先前产生的)NOVA缺失的小鼠。这些研究将允许 美国将测试RNA结合在生成过程中起关键作用的假设 NOVA缺失小鼠的表型，包括运动神经元变性 脑干和脊髓，这些组织特别受到 患有POMA综合征的患者。

DESCRIPTION (provided by applicant): 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.